Assay for detecting neurodegeneration

Abstract

The present invention provides a method of measuring the amount of mono-or poly-phosphorylated p217 + tau protein in a sample. Also provided are methods of detecting or diagnosing tauopathies, methods of determining the effectiveness of treatment of tauopathies, and methods of determining whether a subject is suitable for anti-p217 + tau antibody therapy. Antibodies for use in the methods and kits comprising the antibodies are also described.

Description

Technical Field

[0001] The present invention relates to compositions and methods for detecting neurodegeneration. Specifically, the present invention relates to methods for measuring the amount of singly phosphorylated or multiply phosphorylated p217+ tau protein in a biological sample and uses thereof, as well as antibodies and kits used in these methods. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative brain disorder characterized by a progressive loss of memory, cognition, reasoning, judgment, and emotional stability, which gradually leads to severe mental decline and ultimately death. AD is a very common cause of progressive mental disorder (dementia) in the elderly and is considered the fourth most common medical cause of death in the United States. AD has been observed in ethnic groups throughout the world and represents a major current and future public health problem.

[0003] The brains of individuals with AD exhibit characteristic lesions called senile (or amyloid) plaques, amyloid angiopathy (deposition of amyloid in blood vessels), and neurofibrillary tangles. Large numbers of these lesions are generally found in several regions of the human brain that are important for memory and cognitive function in patients with AD, particularly amyloid plaques and paired helical filament neurofibrillary tangles.

[0004] Neurofibrillary tangles are mainly composed of aggregates of hyperphosphorylated tau protein. The main physiological function of tau is microtubule polymerization and stabilization. The binding of tau to microtubules occurs through ionic interactions between the positive charges of the microtubule-binding region of tau and the negative charges on the microtubule network framework (Butner and Kirschner, J Cell Biol. 115(3):717-30, 1991). The tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the main function of tau. Tau bound to the axonal microtubule network framework is in a hypophosphorylated state, while the aggregated tau in AD is hyperphosphorylated, thus providing a unique epitope different from the physiological active complex of tau (Iqbal et al., Curr Alzheimer Res. 7(8):656–664, 2010).

[0005] The development of tauopathies in AD brains follows a distinct spreading pattern. The tauopathy seeding and spreading hypothesis has been described based on the Braak stages of tauopathy development in the human brain and the spreading of tau aggregates after tau aggregate injection in preclinical tau models (Frost et al., J Biol Chem. 284:12845-52, 2009; Clavaguera et al., Nat Cell Biol. 11:909-13, 2009). It is believed that tauopathies can spread from one brain region to the next in a prion-like manner. This spreading process would involve the exocytosis of tau seeds, which can be taken up by nearby neurons and induce further tauopathies.

[0006] Tau protein fragments in neurofibrillary tangles move into cerebrospinal fluid (CSF), where the tau protein fragments can be obtained by lumbar puncture and measured by sensitive assays. Thus, assays that recognize tau protein-derived fragments in CSF can be used to detect the presence of neurological diseases. Such tau assays require the ability to recognize tau species that are characteristic of neurodegenerative disorders. Hyperphosphorylated tau is a major example of AD-associated tau protein. Thus, an assay that detects hyperphosphorylated tau protein in CSF can be most effective in detecting the presence of AD.

[0007] Phosphorylation is not the only post-translational modification to consider when measuring tau. Recent studies have confirmed that in CSF, tau protein exists primarily as fragments rather than as full-length protein (Meredith et al., PLoS One. 8(10):e76523, 2013). In addition, the tau fragmentation pattern can be affected by disease, as proteolysis is often abnormal in pathological conditions. Thus, tau-based assays for neurodegeneration need to provide information not only about the phosphorylation status (e.g., phosphorylation sites) but also about the nature of the tau fragments being measured (e.g., the length, polarity of the tau fragments). However, the translation of this idea has been hindered by the low endogenous levels of phosphorylated tau, especially in samples from healthy subjects.

[0008] Generally speaking, there is still a need for sensitive, precise, and accurate methods for detecting hyperphosphorylated tau in biological fluids. Such methods would be useful for effectively detecting, diagnosing, staging, and tracking the disease progression of neurodegenerative diseases such as AD and other tauopathy diseases. These methods can also be used as pharmacodynamic markers to measure the levels of hyperphosphorylated tau bound by total antibodies, free antibodies, and therapeutic antibodies. The ability to detect and measure hyperphosphorylated tau fragments is even more important in this field because the transmissible tau species can be one or more tau fragments. SUMMARY OF THE INVENTION

[0009] The present invention meets the need to detect tau forms associated with neurodegenerative diseases in CSF. The present invention enables the detection of mono - phosphorylated or multi - phosphorylated tau as well as the detection of tau fragments.

[0010] According to an embodiment of the present invention, a highly sensitive enzyme - linked immunosorbent assay (ELISA) has been developed and is suitable for measuring p217+tau containing a phosphorylated tau epitope (“p217+tau epitope” or “pT3 epitope”), which phosphorylated tau epitope contains phosphorylated residues T212 and / or T217 having the sequence of (212)R(pT)PSLPTPPTR (SEQ ID NO:25), (217)RTPSLP(pT)PPTR (SEQ ID NO:26) or (212 and 217)R(pT)PSLP(pT)PPTR (SEQ ID NO:27).

[0011] The assays according to embodiments of the present invention are capable of measuring p217+tau species in a variety of fluid matrices, including but not limited to CSF, interstitial fluid (ISF), brain homogenate, serum, plasma and their denatured or enriched forms. The assays according to embodiments of the present invention use a first monoclonal antibody against the pT3 epitope of tau as a capture antibody and a second monoclonal antibody against a second epitope of tau as a detection antibody. These assays are highly sensitive, precise, accurate, inter - laboratory transferable, dilution - linear, and applicable to many sample types. In addition to measuring p217+tau species in the original biological fluid, these assays can be used to measure denatured or native samples, or for two complementary techniques after immunoprecipitation to quantify the amount of free p217+tau or p217+tau bound to endogenous or therapeutically administered antibodies. These assays can be coupled with reversed - phase high - performance liquid chromatography (rpHPLC) to measure fractionated CSF, allowing analysis of the fragment profile of p217+tau.

[0012] In a general aspect, the present invention relates to a method for measuring the amount of p217+tau peptide in a sample. The method comprises: (i) contacting the sample with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, and (ii) contacting the captured p217+tau peptide with a detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein, such as amino acid residues 116 to 127, or against an epitope containing amino acid residues 7 to 20 of the tau protein, thereby measuring the amount of p217+tau peptide and the amount of long p217+tau peptide, respectively, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1.

[0013] In one particular aspect, the present invention relates to a method for determining the relative amounts of long p217+tau peptides or short p217tau peptide fragments in a sample. The method comprises (i) contacting the sample with a capture antibody against a p217+tau epitope to capture p217+tau peptides in the sample, (ii) contacting the captured p217+tau peptides with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of tau protein to measure the amount of p217+tau peptides, (iii) contacting the captured p217+tau peptides with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of tau protein to measure the amount of long p217+tau peptides, and (iv) determining the relative amounts of long p217+tau peptides or short p217+tau peptides based on the amount of p217+tau peptides and the amount of long p217+tau peptides, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1.

[0014] In one embodiment of the present invention, the amount of short p217+tau peptides in the sample is calculated, for example, by subtracting the amount of long p217+tau peptides from the amount of p217+tau peptides, based on the amount of p217+tau peptides and the amount of long p217+tau peptides in the sample. In another embodiment, the ratio between the amount of short p217+tau peptides and the amount of p217+tau peptides, the ratio between the amount of long p217+tau peptides and the amount of p217+tau peptides, or the ratio between the amount of long p217+tau peptides and the amount of short p217+tau peptides is determined based on the amount of p217+tau peptides and the amount of long p217+tau peptides in the sample. According to an embodiment of the present invention, the amount of p217+tau peptides and / or the amount of long p217+tau peptides in the sample, and information based on the measured amounts, such as the calculated amount of short p217+tau peptides and one or more of the above ratios, can be used for one or more diagnostic purposes.

[0015] Accordingly, in one specific aspect, the present invention relates to a method for determining the ratio of p217+tau peptide to total tau peptide in a sample. The method comprises (i) contacting the sample with a capture antibody directed against a p217+tau epitope to capture the p217+tau peptide in the sample, and contacting the sample with a phosphorylation-independent capture antibody directed against an epitope between amino acids 150 and 250 of the tau protein, which epitope is preferably an epitope comprising amino acids 159 to 163 of the tau protein; (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody directed against an epitope comprising amino acids 119 to 126 of the tau protein, thereby measuring the amount of p217+tau peptide, and contacting the captured total tau peptide with the first detection antibody, thereby measuring the amount of total tau peptide; and (b) contacting the captured p217+tau peptide with a second detection antibody directed against an epitope comprising amino acids 7 to 20 of the tau protein, thereby measuring the amount of long p217+tau peptide, and contacting the captured total tau peptide with the second detection antibody, thereby measuring the amount of total long tau peptide; and (iii) determining the ratio of the amount of p217+tau peptide to the amount of total tau peptide or the ratio of the amount of long p217+tau peptide to the amount of total long tau peptide, wherein the amino acid numbering is referenced to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the amount of short p217+tau peptide is calculated by subtracting the amount of long p217+tau peptide from the amount of p217+tau peptide, the amount of total short tau peptide is calculated by subtracting the amount of total long tau peptide from the amount of total tau peptide, and the ratio of the amount of short p217+tau peptide to the amount of total short tau peptide is determined.

[0016] According to a specific aspect, the method of the present invention comprises (i) contacting a biological sample (preferably a CSF sample) from a subject with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein, thereby measuring the amount of the p217+tau peptide, and (b) contacting the captured p217+tau peptide with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein, thereby measuring the amount of the long p217+tau peptide, and (iii) determining whether the subject has a tauopathy or is at risk of developing a tauopathy based on at least one of the amount of the p217+tau peptide, the amount of the long p217+tau peptide, the amount of the short p217+tau peptide obtained by subtracting the amount of the long p217+tau peptide from the amount of the p217+tau peptide, and their ratios, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method further comprises administering to the subject a therapeutic agent for treating or preventing a tauopathy.

[0017] According to a specific aspect, the method of the present invention comprises (i) contacting a biological sample (preferably a CSF sample) from a subject with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, and contacting the sample with a phosphorylation-independent capture antibody against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 159 to 163 of the tau protein; (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of the p217+tau peptide, and contacting the captured total tau peptide with the first detection antibody to measure the amount of the total tau peptide; and (b) contacting the captured p217+tau peptide with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of the long p217+tau peptide, and contacting the captured total tau peptide with the second detection antibody to measure the amount of the total long tau peptide; and (iii) determining whether the subject has a tauopathy or is at risk of developing a tauopathy based on at least one of (a) the ratio of the amount of the p217+tau peptide to the amount of the total tau peptide, (b) the ratio of the amount of the long p217+tau peptide to the amount of the total long tau peptide, and (c) the ratio of the amount of the short p217+tau peptide to the amount of the total short tau peptide, wherein the amount of the short p217+tau peptide is obtained by subtracting the amount of the long p217+tau peptide from the amount of the p217+tau peptide, and the amount of the total short tau peptide is obtained by subtracting the amount of the total short tau peptide from the amount of the total tau peptide, and wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method further comprises administering to the subject a therapeutic agent for treating or preventing a tauopathy.

[0018] According to another specific aspect, the method of the present invention comprises (i) contacting a biological sample (preferably a CSF sample) from a subject being treated with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of the p217+tau peptide, and (b) contacting the captured p217+tau peptide with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of the long p217+tau peptide, and (iii) determining the effectiveness of the treatment in the subject based on at least one of the amount of the p217+tau peptide, the amount of the long p217+tau peptide, the amount of the short p217+tau peptide obtained by subtracting the amount of the long p217+tau peptide from the amount of the p217+tau peptide, and their ratios, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method further comprises administering to the subject a therapeutic agent for treating or preventing tau proteinopathies.

[0019] According to another specific aspect, the method of the present invention comprises (i) contacting a biological sample (preferably a CSF sample) from a subject being treated with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, and contacting the sample with a phosphorylation-independent capture antibody against an epitope between amino acids 150 and 250 of the tau protein, which epitope is preferably the epitope comprising amino acids 159 to 163 of the tau protein; (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of the p217+tau peptide, and contacting the captured total tau peptide with the first detection antibody to measure the amount of the total tau peptide; and (b) contacting the captured p217+tau peptide with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of the long p217+tau peptide, and contacting the captured total tau peptide with the second detection antibody to measure the amount of the total long tau peptide; and (iii) determining the effectiveness of the treatment in the subject based on at least one of (a) the ratio of the amount of the p217+tau peptide to the amount of the total tau peptide, (b) the ratio of the amount of the long p217+tau peptide to the amount of the total long tau peptide, and (c) the ratio of the amount of the short p217+tau peptide to the amount of the total short tau peptide, wherein the amount of the short p217+tau peptide is obtained by subtracting the amount of the long p217+tau peptide from the amount of the p217+tau peptide, and the amount of the total short tau peptide is obtained by subtracting the amount of the total short tau peptide from the amount of the total tau peptide, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method further comprises administering to the subject a therapeutic agent for treating or preventing tau proteinopathies.

[0020] According to another specific aspect, the method of the present invention includes (i) contacting a biological sample from a subject (preferably a CSF sample) with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample; (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein to measure the amount of the p217+tau peptide, and (b) contacting the captured p217+tau peptide with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein to measure the amount of the long p217+tau peptide; and (iii) determining whether the subject is suitable for an anti-p217+tau antibody based on at least one of the amount of the p217+tau peptide, the amount of the long p217+tau peptide, the amount of the short p217+tau peptide obtained by subtracting the amount of the long p217+tau peptide from the amount of the p217+tau peptide, and their ratios, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method further includes administering to the subject an anti-p217+tau antibody for treating or preventing tau proteinopathies.

[0021] According to another specific aspect, the method of the present invention comprises (i) contacting a biological sample from a subject (preferably a CSF sample) with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, and contacting the sample with a phosphorylation-independent capture antibody against an epitope between amino acids 150 and 250 of tau protein, preferably an epitope comprising amino acids 159 to 163 of tau protein; (ii) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of tau protein to measure the amount of p217+tau peptide, and contacting the captured total tau peptide with the first detection antibody to measure the amount of total tau peptide; and (b) contacting the captured p217+tau peptide with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of tau protein to measure the amount of long p217+tau peptide, and contacting the captured total tau peptide with the second detection antibody to measure the amount of total long tau peptide; and (iii) determining whether the subject is suitable for anti-p217+tau antibody therapy based on at least one of (a) the ratio of the amount of p217+tau peptide to the amount of total tau peptide, (b) the ratio of the amount of long p217+tau peptide to the amount of total long tau peptide, and (c) the ratio of the amount of short p217+tau peptide to the amount of total short tau peptide, wherein the amount of short p217+tau peptide is obtained by subtracting the amount of long p217+tau peptide from the amount of p217+tau peptide, and the amount of total short tau peptide is obtained by subtracting the amount of total short tau peptide from the amount of total tau peptide, and wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method further comprises administering to the subject an anti-p217+tau antibody for treating or preventing tau proteinopathies.

[0022] In another specific aspect, the present invention relates to a method for monitoring the treatment with an anti-p217+tau antibody in a subject, the method comprising: (i) obtaining a biological sample from the subject; (ii) separating the biological sample into a first sample containing p217+tau without the anti-p217+tau antibody (the first sample is preferably from IgG), and a second sample containing p217+tau peptides bound to the anti-p217+tau antibody; (iii) obtaining a third sample containing p217+tau without the anti-p217+tau antibody from the second sample, preferably by rpHPLC; (iv) contacting each of the first sample and the third sample with a capture antibody against a p217+tau epitope to capture the p217+tau peptides in each of the samples; (v) performing at least one of the following: (a) contacting the captured p217+tau peptides with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the tau protein, thereby measuring the amount of p217+tau peptides in each of the samples, and (b) contacting the captured p217+tau peptides with a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein, thereby measuring the amount of long p217+tau peptides in each of the samples; (vi) monitoring the treatment with the anti-p217+tau antibody based on at least one of the amount of p217+tau peptides and the amount of long p217+tau peptides in each of the samples, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. For example, the treatment with the anti-p217+tau antibody can be monitored based on the ratio of the amount of long p217+tau peptides in the first sample to the amount of long p217+tau peptides in the third sample, the ratio of the amount of p217+tau peptides in the first sample to the amount of p217+tau peptides in the third sample, or the ratio of the amount of short p217+tau peptides in the first sample (which can be calculated by subtracting the amount of long p217+tau peptides from the amount of p217+tau peptides) to the amount of short p217+tau peptides in the third sample. In one embodiment, the method further comprises administering to the subject an anti-p217+tau antibody for treating or preventing tau proteinopathies.

[0023] In another general aspect, the invention relates to a method of monitoring treatment with an anti-p217+tau antibody in a subject, the method comprising: (i) obtaining a biological sample from the subject; (ii) obtaining a semi-denatured sample from the biological sample containing total p217+tau, wherein the semi-denatured sample is heated to denature the antibodies in the sample, and obtaining a non-denatured sample from the biological sample containing p217+tau that does not contain anti-p217+tau antibody; (iii) contacting each of the semi-denatured sample and the non-denatured sample with a capture antibody directed against a p217+tau epitope to capture the p217+tau peptide in each of the samples; (v) performing at least one of the following: (a) contacting the captured p217+tau peptide with a first detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein, thereby measuring the amount of p217+tau peptide in each of the samples, and (b) contacting the captured p217+tau peptide with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein, thereby measuring the amount of long p217+tau peptide in each of the samples; and (vi) monitoring treatment with the anti-p217+tau antibody based on at least one of the amount of p217+tau peptide and the amount of long p217+tau peptide in each of the samples, wherein the amino acid numbering refers to the amino acid sequence shown in SEQ ID NO:1. For example, treatment with the anti-p217+tau antibody can be monitored based on the ratio of the amount of long p217+tau peptide in the semi-denatured sample to the amount of long p217+tau peptide in the non-denatured sample, the ratio of the amount of p217+tau peptide in the semi-denatured sample to the amount of p217+tau peptide in the non-denatured sample, or the ratio of the amount of short p217+tau peptide in the semi-denatured sample (which can be calculated by subtracting the amount of long p217+tau peptide from the amount of p217+tau peptide) to the amount of short p217+tau peptide in the non-denatured sample. In one embodiment, the method further comprises administering to the subject an anti-p217+tau antibody for treating or preventing tau proteinopathies.

[0024] According to one specific aspect, tau proteinopathies include, but are not limited to, one or more selected from the following: Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (punch-drunk disease).

[0025] Preferably, the tau proteinopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), FTDP-17, or progressive supranuclear palsy.

[0026] Most preferably, the tau proteinopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease).

[0027] According to one specific aspect, the lower limit of quantification of the method of the present invention is about 40 fg / ml of p217+tau peptide, and the lower limit of detection of the method of the present invention is about 2 fg / ml of p217+tau peptide.

[0028] According to one specific aspect, the sample is a biological sample from a subject in need thereof, such as a blood, brain homogenate, or cerebrospinal fluid (CSF) sample. Preferably, the biological sample is a CSF sample from a subject in need of diagnosing a tau proteinopathy, monitoring the effectiveness of treatment of a tau proteinopathy, or determining the suitability of anti-p217+tau antibody therapy.

[0029] According to a specific aspect, the capture antibody for use in the methods of the present invention is directed against the p217+tau epitope, preferably the p217+tau epitope comprising the amino acid sequence of SEQ ID NO:25, 26 or 27. In one embodiment, the capture antibody for use in the methods of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NO:32, 33 and 34, respectively, and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:35, 36 and 37, respectively. Preferably, the capture antibody has a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO:28 or 30 and a light chain variable region having the polypeptide sequence of SEQ ID NO:29 or 31.

[0030] According to a specific aspect, the detection antibody for use in the methods of the present invention is directed against an epitope comprising amino acid residues 119 to 126 of the tau protein, preferably an epitope comprising the amino acid sequence of SEQ ID NO:10, such as the amino acid sequence of SEQ ID NO:11. In one embodiment, the detection antibody for use in the methods of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NO:2, 3 and 4, respectively; and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:5, 6 and 7, respectively. Preferably, the detection antibody is the pT82 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO:8 and a light chain variable region having the polypeptide sequence of SEQ ID NO:9.

[0031] According to another specific aspect, the detection antibody for use in the methods of the present invention is directed against an epitope containing amino acid residues 7 to 20 of the tau protein, preferably an epitope having the amino acid sequence of SEQ ID NO:20. In one embodiment, the detection antibody for use in the methods of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 having the polypeptide sequences of SEQ ID NO:12, 13 and 14, respectively; and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having the polypeptide sequences of SEQ ID NO:15, 16 and 17, respectively. Preferably, the detection antibody is the hT43 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO:18 and a light chain variable region having the polypeptide sequence of SEQ ID NO:19.

[0032] According to another specific aspect, the phosphorylation-independent capture antibody useful in the present invention is directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acids 211 to 221 of the tau protein or an epitope comprising amino acids 159 to 163 of the tau protein, more preferably an epitope having the amino acid sequence of SEQ ID NO: 21. In one embodiment, the phosphorylation-independent capture antibody useful in the present invention is the hT7 antibody.

[0033] According to another specific aspect, after fractionating a biological sample using reverse-phase high performance liquid chromatography (rpHPLC), a sample for use in the method of the present invention is obtained.

[0034] In another general aspect, the present invention relates to an isolated detection antibody or an antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein, the isolated detection antibody or an antigen-binding fragment thereof comprising (a) immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NOs: 12, 13, and 14, respectively; and (b) immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NOs: 15, 16, and 17, respectively. According to a specific aspect, the isolated detection antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 19. Preferably, the isolated detection antibody or an antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 7 to 20 of the tau protein is the hT43 antibody.

[0035] In another general aspect, the present invention relates to a kit that comprises (a) a capture antibody directed against the p217+tau epitope, and (b) a detection antibody directed against a tau protein epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein. Optionally, the kit further comprises a phosphorylation-independent capture antibody directed against a tau epitope between amino acids 150 and 250 of the tau protein. The kit can be used, for example, to measure the amount of p217+tau peptide, the amount of long p217+tau peptide, the amount of short p217+tau peptide, the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide, the ratio of the amount of short p217+tau peptide to the total amount of short tau peptide, etc. in a sample. The kit can also be used for various diagnostic or monitoring purposes, such as determining whether a subject has a tauopathy or is at risk of developing a tauopathy, monitoring the efficacy of a treatment for a tauopathy, such as treatment with an anti-p217+tau antibody, determining whether a subject is suitable for an anti-p217+tau antibody, etc.

[0036] According to one specific aspect, the kit of the present invention comprises a capture antibody having immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 with polypeptide sequences of SEQ ID NO: 32, 33, and 34, respectively, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 with polypeptide sequences of SEQ ID NO: 35, 36, and 37, respectively. Preferably, the capture antibody has a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29.

[0037] According to another specific aspect, the kit of the present invention comprises a detection antibody comprising immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 with polypeptide sequences of SEQ ID NO: 2, 3, and 4, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 with polypeptide sequences of SEQ ID NO: 5, 6, and 7, respectively. Preferably, the detection antibody is the pT82 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 9.

[0038] According to another specific aspect, the kit of the present invention comprises a detection antibody comprising immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 with polypeptide sequences of SEQ ID NO: 12, 13, and 14, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 with polypeptide sequences of SEQ ID NO: 15, 16, and 17, respectively. Preferably, the detection antibody is the hT43 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 19.

[0039] Based on the following disclosure, including the detailed description of the present invention and its preferred embodiments as well as the appended claims, other aspects, features, and advantages of the present invention will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above - described invention content and the following detailed description of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the exact embodiments shown in the drawings.

[0041] Figure 1 Representative standard curves for the pT3xhT43 assay and the pT3xpT82 assay generated using calibration peptides are shown, where the mean + / - SD of duplicate measurements is shown at each point.

[0042] Figures 2A to 2EShows the dilution linearity of the pT3xhT43 assay and the pT3xpT82 assay in CSF samples, where (A, C, and E) the measured values are shown in pg / mL corrected for dilution or (B and D) the measured values are shown as the % corrected for dilution of the 1:4 measured values, where the dashed line indicates + / - 20% of the 1:4 measured values.

[0043] Figure 3 Shows the within - test precision and between - test precision of the (A) pT3xhT43 assay and (B) pT3xpT82 assay.

[0044] Figures 4A to 4B Shows the precision between test sites of the pT3xhT43 assay and the pT3xpT82 assay, where the data is plotted as signal - to - noise (S / N).

[0045] Figures 5A to 5B Shows the competition of soluble p217+tau - targeting antibody against the pT3 - based assay signals of the (A) pT3xhT43 assay and (B) pT3xpT82 assay.

[0046] Figure 6 Shows the phosphorylation dependence of the pT3xhT43 assay and the pT3xpT82 assay.

[0047] Figure 7 Shows the p217+tau fragment profile of AD CSF measured using the pT3xhT43 assay and the pT3xpT82 assay, where the data is plotted as signal minus noise.

[0048] Figures 8A to 8B Shows the temperature and freeze - thaw stability of the p217+tau signal in AD CSF samples using the (A) pT3xhT43 assay and (B) hT7xpT82 assay.

[0049] Figure 9 Shows the long - term stability of the p217+tau signal in CSF samples after storage at - 70°C. No change in the signal was detected.

[0050] Figures 10A to 10F Shows the correlation between p217+tau and the classical AD biomarkers Aβ42, tTau (total Tau), and pTau181 measured by the (A - C) pT3xhT43 assay and (D - F) pT3xpT82 assay.

[0051] Figures 11A to 11B Shows the correlation between brain biopsy IHC analysis measured by the (A) pT3xhT43 assay and (B) pT3xpT82 assay and p217+tau.

[0052] Figures 12A to 12D The following results are shown: (A) pT3xhT43, (B) pT3xpT82, (C) hT7xpT82, and (D) the ratio of pT3xpT82 analysis to hT7xpT82 analysis of crude CSF from AD patients and HV patients.

[0053] Figure 13 The predictive abilities of the pT3xhT43 (“343”) assay, the pT3xpT82 (“382”) assay, and the hT7xpT82 (“782”) assay in differentiating AD subjects from HV subjects are shown.

[0054] Figures 14A to 14F Signals from (A, B) pT3xhT43 (“343”) assays, (C, D) pT3xpT82 (“382”) assays, and (E, F) hT7xpT82 (“782”) assays of rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects are shown.

[0055] Figures 15A to 15O Signals from (A to E) pT3xhT43 assays, (F to J) pT3xpT82 assays, and (K to O) hT7xpT82 assays of rp-HPLC fractions of CSF from CDR 0 subjects and CDR 0.5 subjects are shown.

[0056] Figures 16A to 16B The following results are shown: (A) the ratio of pT3xpT82 analysis to hT7xpT82 analysis (short pTau) or the ratio of pT3xhT43 analysis to hT7xpT82 analysis (long pTau) of crude CSF, and (B) the ratio of pT3xpT82 analysis to hT7xpT82 analysis of rp-HPLC fractions of CSF; all from a blinded cohort of CDR 0 subjects and CDR 1 subjects, compared to the MMSE score.

[0057] Figures 17A to 17TThe following results are shown: (A) pT3xhT43 analysis of crude CSF, (B) pT3xpT82 analysis of crude CSF, and (C) hT7xpT82 analysis of crude CSF, (D) correlation of two pT3 measurements of crude CSF, (E) correlation of pT3xpT82 and hT7xpT82 of crude CSF, (F) correlation of pT3xhT43 of crude CSF with Innotest tTau, (G) correlation of pT3xhT43 of crude CSF with Innotest pTau181, (H) correlation of pT3xhT43 of crude CSF with Innotest AB42, (I) correlation of pT3xhT43 of crude CSF with Innotest AB42 / 40 ratio; (J, M, Q) pT3xhT43 signal, (M to P) pT3xpT82 signal, or (Q to T) hT7xpT82 signal in (J to I) all rp-HPLC fractions and (K, N, R) the sum of all fractions, (Q, S) the sum of early peak fractions (short tau fragments) or (L, P, T) the sum of late peak fractions (longer tau fragments); all from a cohort of HV, MCI, and AD subjects.

[0058] Figures 18A to 18P The following results are shown: (A) correlation of pT3xpT82 (p217+ short) with pT3xhT43 (p217+ long), (B) correlation of pT3xpT82 with hT7xpT82 (tTau short), (C) correlation of pT3xpT82 with NFL, and (D) correlation of pT3xhT43 or (E) pT3xpT82 with amyloid status, and (F to I, N to P) correlation of pT3xhT43 or (J to M) pT3xpT82 with (F to M) various cognitive scores or (N to P) changes in these scores over 78 weeks; all from a cohort of 235 subjects (90 of whom were followed for 78 weeks) from the Janssen study ELN115727301 / 302 for mild to moderate AD subjects. Subjects were initially enrolled based on cognition (and classified as AD), however, 27 of the subjects were determined to be amyloid negative at biochemical assessment (AB40 and AB42) and may therefore represent dementia of non-AD origin. These subjects were analyzed as a separate cohort in the figures above and were designated amyloid negative = 0, while amyloid positive subjects = 1.

[0059] Figure 19 The signal from pT3xhT43 measurements of rp-HPLC fractions of AD CSF samples spiked with IgG, pT3 mAb, humanized pT3 mAb, or mock control, followed by immunoprecipitation for collection of antibody-bound p217+ tau, is shown.

[0060] Figures 20A to 20B Shows the antibody dose-dependence of an immunocapture / rpHPLC method for quantifying (A) antibody-free p217+tau and (B) antibody-bound p217+tau, where the data are plotted as the sum of the signals in rpHPLC fractions 12 to 16.

[0061] Figures 21A to 21C Shows the kinetic differences in antibody-induced p217+tau protein damage in the presence (A, C) or absence (B) of heat-mediated denaturation. (A) Humanized PT3 mAb / CSF mixture; (B) untreated CSF; (C) humanized PT3 mAb.

[0062] Figures 22A to 22C Shows (A) heat-mediated denaturation and (B) an immunocapture / rpHPLC method for quantifying antibody-free p217+tau and antibody-bound p217+tau; (C) shows a comparison of these methods.

[0063] Figure 23 Shows the lack of pT3-based assay recognition of p217+tau in cynomolgus macaque CSF.

[0064] Figures 24A to 24C Shows the measurement of p217+tau in marmoset CSF determined using (A) the pT3xhT43 assay, (B) the pT3xpT82 assay, and (C) the hT7xpT82 assay.

[0065] Figures 25A to 25D Shows the measurement of (A, B) hT7xpT82 (tTau) or (C, D) pT3xpT82 (short p217+tau) in crude sera from 4 AD subjects and 4 HV subjects. Measurements were made at (A, C) a 1:4 or (B, D) a 1:16 dilution, noting the lack of dilution linearity and sensitivity.

[0066] Figures 26A to 26B Shows in sera from the same 4 AD subjects and 4 HV subjects evaluated in Figures 25A to 25D the measurement of (A) hT7xpT82 (tTau) or (B) pT3xpT82 (short p217+tau) in sera pretreated with NaOAc and heat denaturation.

[0067] Figure 27 Shows in sera from the same 4 AD subjects and 4 HV subjects evaluated in Figures 25A to 25D as well as Figures 26A to 26B the measurement of pT3xpT82 (short p217+tau) in pT3-immunoprecipitated (IP) sera. Detailed implementation manners

[0068] The background and various publications, articles, and patents are cited or described throughout the specification; each of these references is hereby incorporated by reference in its entirety. The discussions of documents, acts, materials, devices, articles, etc. included in this specification are intended to provide context for the present invention. Such discussions do not admit that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0069] Definition

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings set forth in this specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein. It should be noted that, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" as used herein and in the appended claims include plural referents.

[0071] Unless otherwise indicated, any numerical values, such as the concentrations or concentration ranges described herein, are understood to be modified in all cases by the term "about". Thus, the numerical values generally include the stated value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, the numerical ranges used expressly include all possible sub-ranges, all individual numerical values within the range, including integers within such range and fractions within such range.

[0072] As used herein, the term "antibody" or "immunoglobulin" is used broadly and includes immunoglobulin or antibody molecules (including polyclonal antibodies), monoclonal antibodies (including murine, human, human - adapted, humanized, and chimeric monoclonal antibodies and antibody fragments).

[0073] Generally speaking, an antibody is a protein or peptide chain that exhibits binding specificity for a specific antigen. The antibody structure is well-known. Immunoglobulins can be designated into five main classes, namely IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, the antibody of the present invention can be any one of the five main classes or the corresponding subclasses. Preferably, the antibody of the present invention is IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of its constant domain, the light chain of an antibody from any vertebrate species can be designated as one of two completely different types, namely κ and λ. Thus, the antibody of the present invention can contain a κ or λ light chain constant domain. According to a particular embodiment, the antibody of the present invention comprises the heavy and / or light chain constant regions from a murine antibody or a human antibody.

[0074] In addition to the heavy and light chain constant domains, an antibody also contains light and heavy chain variable regions. The variable region of an immunoglobulin light or heavy chain consists of "framework" regions interrupted by "antigen-binding sites". The antigen-binding sites are defined by the following various terms and numbering schemes:

[0075] (i) Kabat: "Complementary determining regions" or "CDRs" are based on sequence variability (Wu and Kabat, J Exp Med. 132:211-50, 1970). Generally speaking, an antigen-binding site has three CDRs in each variable region (e.g., HCDR1, HCDR2, and HCDR3 in the heavy chain variable region (VH), and LCDR1, LCDR2, and LCDR3 in the light chain variable region (VL));

[0076] (ii) Chothia: The term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk, J Mol Biol. 196:901-17, 1987). Generally, an antigen-binding site has three hypervariable regions in each VH (H1, H2, H3) and VL (L1, L2, L3). The numbering systems and annotations for CDRs and HVRs have been revised by Abhinandan and Martin (Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008);

[0077] (iii) IMGT: Another definition of the regions forming the antigen-binding site has been proposed by Lefranc (Lefranc et al., Dev Comp Immunol. 27:55-77, 2003) based on a comparison of the V domains of immunoglobulins and T cell receptors. The International ImMunoGeneTics (IMGT) database (http: / / www.imgt.org) provides standardized numbering and definitions of these regions. The correspondence between CDR, HVR, and IMGT delineations is described in Lefranc et al., 2003 (ibid);

[0078] (iv) The antigen-binding site can also be described based on "Specificity Determining Residue Usage" (SDRU) (Almagro, Mol Recognit. 17:132-43, 2004), where SDR refers to the amino acid residues of the immunoglobulin that are directly involved in antigen contact.

[0079] A "framework" or "framework sequence" is the remaining sequence within the variable region of an antibody other than those defined as antigen-binding site sequences. Since the exact definition of the antigen-binding site can be determined by multiple partitions as described above, the exact framework sequence depends on the definition of the antigen-binding site. The framework region (FR) is the more highly conserved part of the variable domain. The variable domains of native heavy and light chains each include four FRs (FR1, FR2, FR3, and FR4, respectively), which generally adopt a β-sheet conformation and are linked by three hypervariable loops. The hypervariable loops in each chain pass through the FRs and bind tightly to the hypervariable loops of the other chain, and contribute to the formation of the antigen-binding site of the antibody. Structural analysis of antibodies has shown the relationship between the sequence and shape of the binding site formed by the complementarity-determining regions (Chothia et al., J. Mol. Biol. 227:799-817, 1992; Tramontano et al., J. Mol. Biol. 215:175-182, 1990). Although their sequence variability is high, five of the six loops adopt only small-scale backbone conformations, called "canonical structures". These conformations are determined first by the length of the loop and second by the presence of key residues at specific positions in the loop and framework regions that determine the conformation through their ability to pack, hydrogen bond, or present a unique backbone conformation.

[0080] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or parent antibody fragment binds. According to specific embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region. According to other specific embodiments, the antigen-binding fragment comprises Fab and F(ab’).

[0081] As used herein, the term "epitope" refers to the site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof specifically binds. Epitopes can be formed both by contiguous amino acids or by non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by contiguous amino acids generally remain upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding generally are lost upon treatment with denaturing solvents. Epitopes generally include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, edited by G.E. Morris (1996).

[0082] As used herein, the term "tau" or "tau protein" refers to an abundant protein of the central and peripheral nervous systems that has multiple isoforms. In the human central nervous system (CNS), due to alternative splicing, there are six major tau isoforms with lengths ranging from 352 to 441 amino acids (Hanger et al., Trends Mol Med. 15:112-9, 2009). The isoforms differ from each other by the regulated inclusion of 0 to 2 N-terminal insert sequences and 3 or 4 tandemly arrayed microtubule-binding repeats, and are designated 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R. As used herein, the term "control tau" refers to the tau isoform of SEQ ID NO:1, which lacks phosphorylation and other post-translational modifications. As used herein, the term "tau" includes proteins containing mutations of full-length wild-type tau, such as point mutations, fragments, insertions, deletions, and splice variants. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0083] Unless otherwise specified, as used herein, the amino acid numbering in tau protein or its fragments refers to the amino acid sequence shown in SEQ ID NO:1.

[0084] As used herein, the terms "p217+tau peptide", "p217+tau", or "p217+tau protein" refer to human tau protein or tau fragments phosphorylated at one or both of residues 217 (pT217) and 212 (pT212) of the tau protein, where the numbering of these positions is based on the numbering in SEQ ID NO:1.

[0085] As used herein, the term "p217+tau epitope" refers to a tau epitope containing at least one of phosphorylated T217 and phosphorylated T212, where the numbering of these positions is based on the numbering in SEQ ID NO:1. Examples of p217+tau epitopes include, for example, the pT3 epitope. As used herein, the term "pT3 epitope" refers to an epitope containing amino acids 210 to 220 of human tau protein, which is phosphorylated at at least one residue of T217 and T212 of human tau, where the numbering of these positions is based on the numbering in SEQ ID NO:1. Examples of the pT3 epitope include, for example, SEQ ID NO:25, 26, and 27.

[0086] As used herein, each of the terms "long p217+tau peptide", "long p217+tau", "long form of the p217+tau peptide", or "long p217+tau peptide fragment" has the same meaning and refers to a p217+tau peptide that contains a p217+tau epitope and an epitope that includes amino acid residues 7 to 20 of the tau protein. The "long p217+tau peptide" according to an embodiment of the present invention may have different lengths. For example, the amino terminus of the "long p217+tau peptide fragment" may be amino acid residue 1, 2, 4, 5, 6, or 7 of the tau protein.

[0087] As used herein, each of the terms "short p217+tau peptide", "short p217+tau", "short form of the p217+tau peptide", or "short p217+tau peptide fragment" has the same meaning and refers to a p217+tau peptide that contains a p217+tau epitope and an epitope that includes amino acid residues 119 to 126 of the tau protein, but does not contain an epitope that includes amino acid residues 7 to 20 of the tau protein. The "short p217+tau peptide" according to an embodiment of the present invention may have different lengths. For example, the amino terminus of the "short p217+tau peptide" may be any one of the amino acid residues between the epitope that includes amino acid residues 7 to 20 of the tau protein and the epitope that includes amino acid residues 119 to 126 of the tau protein.

[0088] As used herein, each of the terms "long tau peptide", "long tau", "long form of the tau peptide", or "long tau peptide fragment" has the same meaning and refers to a tau peptide that contains a tau epitope recognized by a phosphorylation-independent capture antibody and an epitope that includes amino acid residues 7 to 20 of the tau protein. The "long tau peptide fragment" according to an embodiment of the present invention may have different lengths. For example, the amino terminus of the "long tau peptide fragment" may be amino acid residue 1, 2, 4, 5, 6, or 7 of the tau protein.

[0089] As used herein, each of the terms "short tau peptide", "short tau", "short form of the tau peptide", or "short tau peptide fragment" has the same meaning and refers to a tau peptide that contains a tau epitope recognized by a phosphorylation-independent capture antibody and an epitope that includes amino acid residues 119 to 126 of the tau protein, but does not contain an epitope that includes amino acid residues 7 to 20 of the tau protein. The "short tau peptide fragment" according to an embodiment of the present invention may have different lengths. For example, the amino terminus of the "short tau peptide" may be any one of the amino acid residues between the epitope that includes amino acid residues 7 to 20 of the tau protein and the epitope that includes amino acid residues 119 to 126 of the tau protein.

[0090] As used herein, the term "capture antibody" refers to an antibody that binds to an antigen of interest and is directly or indirectly linked to a solid-phase support. Examples of solid-phase supports include, but are not limited to, microparticles or beads, such as magnetic beads. Examples of capture antibodies include, but are not limited to, monoclonal antibodies that bind to the p217+tau epitope. According to an embodiment of the invention, the capture antibody can be a monoclonal antibody that comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NOs: 32, 33, and 34, respectively, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NOs: 35, 36, and 37. In a specific embodiment, the capture antibody is pT3. As used herein, the term "pT3" refers to an antibody that binds to the p217+tau peptide and has a heavy chain variable region amino acid sequence of SEQ ID NO: 28 and a light chain variable region amino acid sequence of SEQ ID NO: 29. In one embodiment, the pT3 monoclonal antibody is expressed by a murine hybridoma. In another embodiment, the capture antibody is a humanized antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 30 and a light chain variable region amino acid sequence of SEQ ID NO: 31.

[0091] According to other embodiments of the invention, the capture antibody can be a monoclonal antibody that binds to an epitope between amino acids 150 and 250 of the tau protein, preferably amino acids 211 to 221 or amino acids 159 to 163 of human tau protein, in a phosphorylation-independent manner, and the numbering of these positions is according to the numbering in SEQ ID NO: 1. In a specific embodiment, the capture antibody is hT7. As used herein, the term "hT7" refers to a commercially available monoclonal antibody that binds to an epitope comprising amino acids 159 to 163 of human tau protein, wherein the numbering of these positions is according to the numbering in SEQ ID NO: 1. The hT7 monoclonal antibody can be purchased, for example, from Thermo Fisher (e.g., catalog number: MN1000).

[0092] As used herein, the term "detection antibody" refers to an antibody that binds to an antigen of interest and has a detectable label or is linked to an auxiliary detection system. Examples of detectable labels include, but are not limited to, various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of detection antibodies include, but are not limited to, monoclonal antibodies that bind to tau protein, preferably epitopes comprising amino acids 7 to 20 or 116 to 127 of human tau protein, wherein the numbering of these positions is according to the numbering in SEQ ID NO:1. When a monoclonal antibody that binds to tau protein at the epitope comprising amino acids 7 to 20 is used as the detection antibody for the captured p217+tau peptide, long tau fragments are detected. When a monoclonal antibody that binds to tau protein at the epitope comprising amino acids 116 to 127 is used as the detection antibody for the captured p217+tau peptide, both short and long tau fragments are detected.

[0093] In one specific embodiment, the detection antibody is hT43. As used herein, the term "hT43" refers to a monoclonal antibody that binds to an epitope comprising amino acids 7 to 20 of human tau protein, wherein the numbering of these positions is according to the numbering in SEQ ID NO:1, and the antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO:8 and the amino acid sequence of the light chain variable region of SEQ ID NO:9. In another specific embodiment, the detection antibody is pT82. As used herein, the term "pT82" refers to a monoclonal antibody that binds to an epitope comprising amino acids 119 to 126, preferably 116 to 127, of human tau protein, wherein the numbering of these positions is according to the numbering in SEQ ID NO:1, and the antibody has the amino acid sequence of the heavy chain variable region of SEQ ID NO:18 and the amino acid sequence of the light chain variable region of SEQ ID NO:19.

[0094] As used herein, the term "assay based on pT3" refers to an assay according to an embodiment of the present invention, wherein the pT3 antibody is used as the capture antibody. As used herein, the term "pT3xhT43" refers to an assay according to an embodiment of the present invention, wherein the pT3 antibody is used as the capture antibody and the hT43 antibody is used as the detection antibody. As used herein, the term "pT3xpT82" refers to an assay according to an embodiment of the present invention, wherein the pT3 antibody is used as the capture antibody and the pT82 antibody is used as the detection antibody.

[0095] As used herein, the term "assay based on hT7" refers to an assay according to an embodiment of the present invention, wherein the hT7 antibody is used as the capture antibody. As used herein, the term "hT7xpT82" refers to an assay according to an embodiment of the present invention, wherein the hT7 antibody is used as the capture antibody and the pT82 antibody is used as the detection antibody.

[0096] As used herein, the term "subject" refers to an animal, and preferably a mammal. According to a particular embodiment, the subject is a mammal, including non - primates (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, marmoset or mouse) or primates (e.g., monkey, chimpanzee or human). In a specific embodiment, the subject is a human.

[0097] As used herein, "tauopathy" encompasses any neurodegenerative disease involving pathological aggregation of tau within the brain. In addition to familial and sporadic AD, other exemplary tauopathies are chromosome 17 - related frontotemporal dementia with parkinsonism (FTDP - 17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle - only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis - parkinsonism - dementia complex, Down syndrome, Gerstmann - Straussler - Scheinker disease, Hallervorden - Spatz disease, inclusion body myositis, Creutzfeldt - Jakob disease, multiple system atrophy, Niemann - Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non - Guamanian motor neuron disease with neurofibrillary tangles, post - encephalitic parkinsonism, and chronic traumatic encephalopathy such as pugilistic dementia (punch drunk syndrome) (Morris et al., Neuron, 70:410 - 26, 2011).

[0098] As used herein, the terms "determine", "measure", "assess" and "assay" are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement and include determining whether a property, trait or characteristic is present. An assessment can be relative or absolute. "Assessing the presence" includes determining the amount of something that is present and determining whether it is present.

[0099] As used herein, the term "diagnosis" means detecting a disease or disorder or determining the stage or extent of a disease or disorder such as a tauopathy. Generally, the diagnosis of a disease or disorder is based on the assessment of one or more factors and / or symptoms indicative of the disease. A diagnosis can be made based on the presence, absence, or amount of a factor (e.g., p217+tau) indicative of the presence or absence of a disease or condition. Each factor or symptom considered indicative of a diagnosis of a particular disease need not be associated solely with that particular disease, i.e., there can be differential diagnoses that can be inferred from the diagnostic factor or symptom. Similarly, there can be cases where a factor or symptom indicative of a particular disease is present in an individual who does not have that particular disease. The term "diagnosis" also encompasses determining the therapeutic efficacy of a drug therapy (e.g., anti-p217+tau antibody therapy) or predicting the response pattern to a drug therapy (e.g., anti-p217+tau antibody therapy). Diagnostic methods can be used independently or in combination with other diagnostic and / or staging methods known in the medical field for a particular disease or disorder (e.g., Alzheimer's disease).

[0100] As used herein, the terms "increase" and "decrease" refer to the difference in the amount of a particular biomarker in a sample compared to a control level or reference level. For example, the amount of a particular peptide can be present in a sample from a patient with a disease in an increased amount or a decreased amount compared to a reference level. In one embodiment, an "increase in level" or "decrease in level" can be a difference in the level of a biomarker present in a sample compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more. In one embodiment, an "increase in level" or "decrease in level" can be a statistically significant difference in the level of a biomarker present in a sample compared to a control. For example, if the measured level of a biomarker falls outside of about 1.0 standard deviation, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 standard deviations of the mean of any control group or reference group, the difference can be statistically significant. The reference or control can be, for example, a sample from a healthy individual or a sample taken from the same individual at an earlier time point (such as a time point before administration of a therapeutic agent or an earlier time point during a treatment regimen).

[0101] As used herein, the term "isolated" means that a biological component, such as a nucleic acid, peptide, or protein, has been substantially separated from, obtained separately from, or purified from other biological components of the organism in which the component naturally occurs (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins). Thus, nucleic acids, peptides, and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. An "isolated" nucleic acid, peptide, and protein can be part of a composition, and is still isolated if such composition is not part of the nucleic acid, peptide, or protein's natural environment. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0102] As used herein, an "isolated antibody that binds to the tau protein" or an "isolated anti-tau antibody" is intended to mean an antibody that specifically binds to the tau protein and is substantially free of other antibodies having different antigen specificities (e.g., an isolated anti-tau detection antibody is substantially free of antibodies that specifically bind antigens other than tau). However, an isolated anti-tau detection antibody may be cross-reactive with other related antigens, such as tau species homologs, from other species.

[0103] As used herein, the terms "specifically binds" or "binds specifically" mean that an anti-tau antibody of the invention binds to a predetermined target with a dissociation constant (K -6 d) of about 1×10 -7 M or lower (e.g., about 1×10 -8 M or lower, about 1×10 -9 M or lower, about 1×10 -10 M or lower, about 1×10 -11 M or lower, about 1×10 -12 M or lower, or about 1×10 -13 M or lower). The KD is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). According to the present disclosure, the KD value of an antibody can be determined using methods in the art. For example, the KD value of an anti-tau antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., D system, Proteon instrument (BioRad), KinExA instrument (Sapidyne), ELISA, or competitive binding assays known to those skilled in the art. Generally, the K d of an anti-tau antibody binding to a predetermined target (i.e., tau) is D for its K Dless than 1 / 10 of that measured, e.g., by surface plasmon resonance using, for example, a Proteon instrument (BioRad). However, anti-tau antibodies that specifically bind to tau can be cross-reactive with other related targets, e.g., with the same predetermined target from other species (homologs), such as from mouse, rat, marmoset, dog, or pig.

[0104] As used herein, the term "polynucleotide", which is used synonymously with "nucleic acid molecule", "nucleotide", or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that contain DNA and RNA, which may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases, as well as DNA or RNA having a backbone that has been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides that are typically found in nature, as well as the chemical forms of DNA and RNA that are unique to viruses and cells. "Polynucleotide" also includes relatively short nucleic acid chains, commonly referred to as oligonucleotides.

[0105] As used herein, the term "vector" is a replicon into which another nucleic acid segment can be operably inserted to cause the replication or expression of that segment.

[0106] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the present invention. A "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell transfected with a nucleic acid molecule of the present invention. In another embodiment, a "host cell" is a progeny or potential progeny of such a transfected cell. The progeny of a cell may or may not be the same as the parental cell, e.g., due to mutations that may occur in the progeny or environmental influences or due to integration of the nucleic acid molecule into the host cell genome.

[0107] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene to RNA. The term also encompasses the translation of RNA to one or more polypeptides and also encompasses all naturally occurring post-transcriptional and post-translational modifications. A detection antibody that binds tau in its expressed form or an antigen-binding fragment thereof can be within the cytoplasm of a host cell, within an extracellular environment such as the growth medium of a cell culture, or anchored to the cell membrane.

[0108] Anti-tau antibody

[0109] In one general aspect, the invention relates to an isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein immobilized by a capture antibody. Such anti-tau antibodies can have the property of binding to a phosphorylated epitope on tau or binding to a non-phosphorylated epitope on tau. The anti-tau detection antibody can be used as a research or diagnostic reagent to detect tau in a biological sample.

[0110] According to a specific aspect, the invention relates to an isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 119 to 126 of tau protein, preferably amino acid residues 116 to 127.

[0111] According to a specific aspect, an isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 116 to 127 of tau protein comprises (a) immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO:2, 3, and 4, respectively; and (b) immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO:5, 6, and 7, respectively.

[0112] According to a specific aspect, an isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 116 to 127 of tau protein comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 80%, preferably at least 85% or 90%, more preferably at least 95% and most preferably 100% identical to SEQ ID NO:8, and the light chain variable region having a polypeptide sequence that is at least 80%, preferably at least 85% or 90%, more preferably at least 95% and most preferably 100% identical to SEQ ID NO:9.

[0113] Preferably, the isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 116 to 127 of tau protein is the pT82 antibody.

[0114] According to a specific aspect, the present invention relates to an isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 7 to 20 of tau protein.

[0115] According to a specific aspect, the isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 7 to 20 of tau protein comprises (a) immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 12, 13, and 14, respectively; and (b) immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 15, 16, and 17, respectively.

[0116] According to a specific aspect, the isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 7 to 20 of tau protein comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 80%, preferably at least 85% or 90%, more preferably at least 95% and most preferably 100% identical to SEQ ID NO: 18, and the light chain variable region having a polypeptide sequence that is at least 80%, preferably at least 85% or 90%, more preferably at least 95% and most preferably 100% identical to SEQ ID NO: 19.

[0117] Preferably, the isolated detection antibody or an antigen-binding fragment thereof that binds to tau protein at an epitope comprising amino acid residues 7 to 20 of tau protein is the hT43 antibody.

[0118] The antibodies of the present invention can be generated by a variety of techniques, such as the hybridoma method (Kohler and Milstein, Nature. 256:495-7, 1975). The chimeric mAbs can be prepared by the method disclosed in US4816567, which contains the light and heavy chain variable regions derived from a donor antibody (usually murine) and the light and heavy chain constant regions derived from a receptor antibody (usually another mammalian species, such as human) linked thereto. The CDR-grafted mAbs can be prepared by techniques known to those skilled in the art (such as the techniques disclosed in US5225539), which have the CDRs derived from a non-human donor immunoglobulin (usually murine) and the remaining portions of the molecule derived from one or more human immunoglobulins. The fully human mAbs lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice by the techniques mentioned in the following references (Lonberg et al., Nature. 368:856-9, 1994; Fishwild et al., Nat Biotechnol. 14:845-51, 1996; Mendez et al., Nat Genet. 15:146-56, 1997). Human mAbs can also be prepared and optimized from phage display libraries (Knappik et al., J Mol Biol. 296:57-86, 2000; Krebs et al., J Immunol Methods. 254:67-84, 2001; Shi et al., J Mol Biol. 397:385-96, 2010).

[0119] The functional activities of the tau-binding detection antibodies and their antigen-binding fragments can be characterized by methods known in the art. The methods for characterizing the antibodies and their antigen-binding fragments that bind to tau include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and FACS analysis, immunohistochemical analysis, etc.

[0120] Several well-known methods can be employed to determine the binding epitopes of the antibodies of the present invention. For example, when the structures of two individual components are known, in silico protein-protein docking can be performed to identify compatible interaction sites. Hydrogen-deuterium (H / D) exchange can be carried out with the antigen-antibody complex to localize the regions on the antigen that are bound by the antibody. Segment mutagenesis and site-directed mutagenesis of the antigen can be used to localize the amino acids important for antibody binding. The co-crystal structure of the antibody-antigen complex can be used to identify the residues contributing to the epitope and paratope.

[0121] In another general aspect, the invention relates to isolated polynucleotides encoding the detection antibodies of the invention or antigen-binding fragments thereof. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the detection antibodies of the invention or antigen-binding fragments thereof can be varied without changing the amino acid sequence of the protein. Exemplary isolated polynucleotides are polynucleotides encoding polypeptides comprising immunoglobulin heavy chain CDRs HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 2, 3, and 4, respectively, or polypeptides comprising immunoglobulin light chain CDRs LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 5, 6, and 7, respectively. Other exemplary isolated polynucleotides are polynucleotides encoding polypeptides comprising immunoglobulin heavy chain CDRs HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 12, 13, and 14, respectively, or polypeptides comprising immunoglobulin light chain CDRs LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 15, 16, and 17, respectively. Other exemplary isolated polynucleotides are polynucleotides encoding the variable regions of the antibodies of the invention. Other polynucleotides encoding the antibodies of the invention are also within the scope of the invention in view of the degeneracy of the genetic code or codon preference in a given expression system. The isolated nucleic acids of the invention can be prepared using well-known recombinant or synthetic techniques. Using methods known in the art, the DNA encoding a monoclonal antibody can be readily isolated and sequenced. In the case of hybridoma generation, such cells can be used as a source of such DNA. Alternatively, display techniques in which the coding sequence and the translation product are related, such as phage or ribosome display libraries, can be used.

[0122] In another general aspect, the invention relates to vectors comprising isolated polynucleotides encoding the detection antibodies of the invention or antigen-binding fragments thereof. According to the disclosure, any vector known to those skilled in the art can be used, such as plasmids, cosmid plasmids, phage vectors, or viral vectors. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element that performs the conventional functions of an expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to the embodiments of the invention.

[0123] In another general aspect, the invention relates to a host cell comprising an isolated polynucleotide encoding a detection antibody of the invention or an antigen-binding fragment thereof. Given the present disclosure, any host cell known to the person skilled in the art can be used for the recombinant expression of the antibody of the invention or an antigen-binding fragment thereof. Such host cells can be eukaryotic cells, bacterial cells, plant cells or archaeal cells. Exemplary eukaryotic cells can be of mammalian, insect, avian or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other available cell lines include cell lines derived from Chinese hamster ovary (CHO) cells such as CHO-K1 SV (Lonza Biologics), CHO-K1 (ATCC CRL-61, Invitrogen) or DG44.

[0124] In another general aspect, the invention relates to a method for producing a detection antibody of the invention or an antigen-binding fragment thereof, the method comprising culturing a cell comprising a polynucleotide encoding the detection antibody or an antigen-binding fragment thereof under conditions for producing the detection antibody of the invention or an antigen-binding fragment thereof, and recovering the antibody or an antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). The expressed antibody or an antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art.

[0125] Diagnostic method

[0126] The invention relates to measuring p217+ tau species enriched in AD, for example by using a capture antibody such as pT3 in combination with an anti-tau detection antibody, which selectively immobilizes the p217+ tau species, and the anti-tau detection antibody is labeled with a reporter element that allows detection of the captured p217+ tau species. The methods of the invention can be used for a variety of diagnostic purposes, such as for diagnosing AD or other tauopathies in a subject, monitoring the effectiveness of a treatment, identifying a subject suitable for anti-p217+ tau treatment, etc.

[0127] According to one embodiment of the present invention, a capture antibody directed against a p217+tau epitope, such as an epitope having the amino acid sequence of SEQ ID NO: 25, 26 or 27, is used to capture the p217+tau peptide in a sample of interest. The captured p217+tau peptides, although all containing the p217+tau epitope, may have different lengths, which can be detected by a detection antibody that binds to different epitopes. For example, a detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein can only detect the captured p217+tau peptide or a fragment thereof ("long p217+tau peptide") that still contains amino acid residues 7 to 20 of the tau protein, while a detection antibody directed against an epitope comprising amino acid residues 119 to 126 of the tau protein can detect not only the long p217+tau peptide but also short p217+tau peptides. The captured p217+tau peptides can be contacted with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein to detect and measure the amount of long p217+tau peptide or p217+tau peptides (long and short p217+tau peptides) in the sample. The amount of short p217+tau peptide in the sample is calculated by subtracting the amount of long p217+tau peptide from the amount of p217+tau peptide.

[0128] According to another embodiment of the present invention, in addition to capturing and measuring the amount of p217+tau peptide in a sample, a phosphorylation-independent capture antibody, such as an antibody directed against an epitope between amino acids 150 and 250 of the tau protein, preferably an epitope comprising amino acid residues 159 to 163 of the tau protein, is used to capture total tau peptide in the sample. The captured total tau peptide can be contacted with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein to detect and measure the amount of total long tau peptide or total tau peptide (long tau peptide fragments and short tau peptide fragments) in the sample. The amount of short total tau peptide in the sample is calculated by subtracting the amount of total long tau peptide from the amount of total tau peptide.

[0129] According to an embodiment of the present invention, values related to the p217+tau peptide in a sample, such as the amount of p217+tau peptide and the amount of long p217+tau peptide, optionally the amount of total tau peptide and the amount of total long tau fragments in the sample, and information based on the measured amounts, such as the calculated short p217+tau peptide and short total tau peptide, or ratios related to the p217+tau peptide, such as the ratio of the amount of short tau peptide fragments to the amount of long tau peptide fragments, the ratio of the amount of short p217+tau peptide to the total amount of short tau fragments, the ratio of the amount of long p217+tau peptide to the total amount of long tau fragments, etc., can be used for one or more diagnostic purposes.

[0130] Diagnosis is performed by comparing the value associated with the p217+tau peptide in a sample from a subject with a corresponding baseline value. The baseline value can represent the average level in a population of healthy individuals. The baseline value can also represent a previous level determined in the same subject. In one embodiment, if the value associated with the p217+tau peptide in a biological sample from a subject, such as the amount of long or short p217 tau peptide or the ratio associated with the p217+tau peptide, e.g., the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide, is significantly higher than the corresponding baseline value, then the subject is determined to have a tauopathy. As used herein, "significantly higher" means having statistical significance rather than being a higher value due only to chance, and the value has a p-value of 0.05 or less. At a p-value of less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc., "significantly higher" can be at least about 1%, 2%, 5%, or 10% higher than that found in healthy volunteers.

[0131] In one embodiment, the method of the invention comprises (i) contacting a biological sample (preferably a CSF sample) with a capture antibody directed against an epitope comprising phosphorylated p217+tau to capture the p217+tau peptide in the sample, (ii) contacting the captured p217+tau peptide with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 to measure the amount of long p217+tau peptide in the sample, and / or contacting the captured p217+tau peptide with a detection antibody directed against an epitope comprising amino acid residues 119 to 126 of tau protein to measure the amount of long and short p217+tau peptides in the sample, and (iii) determining whether the subject has a tauopathy or is at risk of developing a tauopathy based on the amount of p217+tau peptide or the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide. Diagnosis can be performed by comparing the amount or concentration of p217+tau peptide in a sample from a subject with a corresponding baseline value. Diagnosis can also be performed by comparing the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide in a sample from a subject with a corresponding baseline value.

[0132] In another embodiment, the method of the invention comprises (i) contacting a biological sample (preferably a CSF sample) with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, or with a phosphorylation-independent capture antibody against the tau epitope between amino acids 150 and 250 of the tau protein to capture the total tau peptide in the sample, (ii) contacting the captured p217+tau peptide or the captured total tau peptide with a detection antibody against the epitope comprising amino acid residues 116 to 127 of the tau protein, thereby measuring the amount of long and short p217+tau peptides or the amount of total short tau peptides in the sample, and (iii) determining whether a subject has a tauopathy or is at risk of developing a tauopathy based on the amount of the ratio of the amount of short p217+tau peptide to the amount of total short tau peptide in the biological sample. Diagnosis can be performed by comparing the ratio of the amount of short p217+tau peptide in a sample from a subject to the amount of total short tau peptide comprising the same region of the tau protein recognized by the pT3 antibody (i.e., amino acids 211 to 221 of tau) to a corresponding baseline value.

[0133] In another embodiment, the method of the invention comprises (i) contacting a biological sample (preferably a CSF sample) with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, (ii) contacting the captured p217+tau peptide with a detection antibody against the epitope comprising amino acid residues 7 to 20, thereby measuring the amount of long p217+tau peptide, and / or with a detection antibody against the epitope comprising amino acid residues 116 to 127 of the tau protein, thereby measuring the amount of long and short p217+tau peptides in the sample, and (iii) determining the efficacy of treatment in a subject based on the amount of p217+tau peptide or the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide.

[0134] In yet another embodiment, the method of the invention comprises (i) contacting a biological sample (preferably a CSF sample) with a capture antibody against the p217+tau epitope to capture the p217+tau peptide in the sample, or with a phosphorylation-independent capture antibody against the tau epitope between amino acids 150 and 250 of the tau protein to capture the total tau peptide in the sample, (ii) contacting the captured p217+tau peptide or the captured total tau peptide with a detection antibody comprising the epitope of amino acid residues 116 to 127 of the tau protein, thereby measuring the amount of long and short p217+tau peptides or the amount of total short tau peptides in the sample, and (iii) determining the efficacy of treatment in a subject based on the amount of the ratio of the amount of short p217+tau peptide to the amount of total short tau peptide in the biological sample.

[0135] In yet another embodiment, the effectiveness of treatment in a subject is determined by monitoring the amount of p217+ tau peptide, the ratio of the amount of short p217+ tau peptide to the amount of long p217+ tau peptide, or the ratio of the amount of short p217+ tau peptide to the total amount of short tau peptide before, during, or after treatment. A decrease relative to a baseline value signals a positive response to the treatment. The value may also transiently increase in a biological fluid as pathological tau is cleared from the brain.

[0136] According to one particular aspect, tauopathies include, but are not limited to, one or more selected from the following: Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), chromosome 17-related frontotemporal dementia with parkinsonism (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam type motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (punch-drunk disease).

[0137] Preferably, the tauopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), FTDP-17, or progressive supranuclear palsy.

[0138] Most preferably, the tauopathy is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease).

[0139] According to one embodiment, the method of the invention comprises (i) contacting a biological sample (preferably a CSF sample) with a capture antibody against a p217+ tau epitope to capture p217+ tau peptide in the sample, (ii) contacting the captured p217+ tau peptide with a detection antibody against an epitope comprising amino acid residues 7 to 20 to measure the amount of long p217+ tau peptide, and / or with a detection antibody against an epitope comprising amino acid residues 116 to 127 of tau protein to measure the amount of long and short p217+ tau peptides in the sample, and (iii) determining whether a subject is suitable for anti-p217+ tau antibody therapy based on the amount of p217+ tau peptide or the ratio of the amount of short p217+ tau peptide to the amount of long p217+ tau peptide.

[0140] According to one specific aspect, if the amount of p217+tau peptide in a biological sample or the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide in the biological sample is significantly higher than the corresponding baseline value, it is determined that the subject is suitable for anti-p217+tau antibody therapy.

[0141] According to another specific aspect, the method of the present invention comprises (i) contacting a biological sample (preferably a CSF sample) with a capture antibody against a p217+tau epitope to capture the p217+tau peptide in the sample, or contacting with a phosphorylation-independent capture antibody against a tau epitope between amino acids 150 and 250 of tau protein to capture the total tau peptide in the sample, (ii) contacting the captured p217+tau peptide or the captured total tau peptide with a detection antibody against an epitope comprising amino acid residues 116 to 127 of tau protein, thereby measuring the amount of long and short p217+tau peptides or the amount of total short tau peptides in the sample, and (iii) determining whether the subject is suitable for anti-p217+tau antibody therapy based on the amount of the ratio of the amount of short p217+tau peptide to the amount of total short tau peptides in the biological sample.

[0142] According to one embodiment, if the ratio of the amount of short p217+tau peptide to the amount of total short tau peptides is significantly higher than the corresponding baseline value, it is determined that the subject is suitable for anti-p217+tau antibody therapy.

[0143] The present invention also relates to measuring p217+tau complexed with an antibody in a biological sample and free p217+tau that is non-antibody bound in the sample. In one embodiment, total antibody is captured using an affinity technique, followed by denaturing conditions including chaotropic agents, heat inactivation, or other protein-disrupting techniques. p217+tau is separated from the antibody using rpHPLC and measured using the method of the present invention, thereby allowing quantification of antibody-bound p217+tau.

[0144] According to one general aspect, the present invention relates to a method for monitoring treatment with an anti-p217+tau antibody in a subject, the method comprising: (i) obtaining a biological sample from the subject, (ii) separating the biological sample into an IgG-enriched sample containing antibody-bound p217+tau and an IgG-depleted sample containing antibody-free p217+tau, (iii) purifying p217+tau from IgG by rpHPLC to obtain an antibody-free p217+tau sample, (iv) contacting each of the IgG-enriched sample and the antibody-free p217+tau sample with a capture antibody directed against a p217+tau epitope to capture the p217+tau peptide in each of the samples, (v) contacting the captured p217+tau peptide in each of the samples with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 to measure the amount of long p217+tau peptide in each of the samples, or contacting the captured p217+tau peptide in each of the samples with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein to measure the amount of long and short p217+tau peptides in each of the samples, (vi) calculating the ratio of the amount of antibody-bound p217+tau to the amount of antibody-free p217+tau, and (vii) monitoring treatment with the anti-p217+tau antibody in the subject based on the calculated ratio.

[0145] According to another general aspect, the present invention relates to a method for monitoring treatment with an anti-p217+tau antibody in a subject, the method comprising: (i) obtaining a biological sample from the subject, (ii) obtaining a semi-denatured sample from the biological sample containing total p217+tau and a non-denatured sample from the biological sample containing antibody-free p217+tau, wherein the semi-denatured sample is heated to denature the antibody in the sample, (iii) contacting each of the semi-denatured sample and the non-denatured sample with a capture antibody directed against a p217+tau epitope to capture the p217+tau peptide in each of the samples, (iv) contacting the captured p217+tau peptide in each of the samples with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 to measure the amount of long p217+tau peptide in each of the samples, or contacting the captured p217+tau peptide in each of the samples with a detection antibody directed against an epitope comprising amino acid residues 116 to 127 of the tau protein to measure the amount of long and short p217+tau peptides in each of the samples, (v) calculating the amount of antibody-bound p217+tau in the sample by subtracting the amount of antibody-free p217+tau from the amount of total p217+tau, (vi) calculating the ratio of antibody-bound p217+tau to antibody-free p217+tau, and (vii) monitoring treatment with the anti-p217+tau antibody in the subject based on the calculated ratio.

[0146] According to one specific aspect, the effectiveness of treatment in a subject is determined by monitoring the amounts of antibody-bound and antibody-free p217+tau peptides before, during, or after treatment. A decrease in the value of antibody-free p217+tau relative to baseline or an increase in the value of antibody-bound p217+tau relative to baseline, and thus an increase in the ratio of antibody-bound p217+tau to antibody-free p217+tau relative to baseline, signals a positive response to the treatment. As pathological tau is cleared from the brain, the value of antibody-free p217+tau may also transiently increase in a biological fluid.

[0147] According to a specific aspect, the capture antibody of the method of the invention is conjugated to beads, such as magnetic beads. According to other specific aspects, the detection antibody is biotinylated.

[0148] According to a specific aspect, the amount of p217+tau peptide measured in the method of the invention can be determined using any suitable technique known in the art, including ELISA and single molecule array platforms. According to a specific aspect, the method of the invention uses a high-sensitivity array platform such as Quanterix Simoa or MSD S-plex to measure the amount of p217+tau peptide in a sample. According to one specific aspect, the lower limit of quantification of the method of the invention is about 40 fg / ml, and the lower limit of detection of the method is about 2 fg / ml.

[0149] According to a specific aspect, the sample used in the method of the invention is a biological sample, such as a blood, brain homogenate, or cerebrospinal fluid (CSF) sample. Preferably, the sample is a CSF sample. According to a specific aspect, the sample is a crude CSF sample. According to another specific aspect, the sample is obtained after fractionating a biological sample such as CSF using reverse phase high performance liquid chromatography (rpHPLC) that separates full-length tau protein and tau fragments of different sizes.

[0150] According to a specific aspect, the capture antibody of the method of the invention comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO:32, 33, and 34, respectively, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO:35, 36, and 37, respectively. Preferably, the capture antibody is the pT3 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the polypeptide sequence of SEQ ID NO:28 and the light chain variable region having the polypeptide sequence of SEQ ID NO:29.

[0151] According to one specific aspect, the detection antibody of the method of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 2, 3, and 4, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 5, 6, and 7, respectively. Preferably, the detection antibody is the pT82 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 9.

[0152] According to another specific aspect, the detection antibody of the method of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 12, 13, and 14, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 15, 16, and 17, respectively. Preferably, the detection antibody is the hT43 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 19.

[0153] Kit

[0154] In another general aspect, the present invention relates to a kit comprising (a) a capture antibody against the p217+tau epitope, optionally a phosphorylation-independent capture antibody against a tau epitope between amino acids 150 and 250 of the tau protein, and (b) at least one detection antibody against a tau protein epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein. The kit is used for measuring the amount of the p217+tau peptide, which is used for the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide and / or the ratio of the amount of the short p217+tau peptide to the amount of the total short tau peptide in a sample.

[0155] The detection antibody may contain any detectable label (e.g., a fluorescent molecule, biotin, etc.), which can be detected directly or can be detected through a secondary reaction (e.g., reaction with streptavidin). Alternatively, a second reagent containing a detectable label can be used, wherein the second reagent has binding specificity for the primary antibody. In a diagnostic kit suitable for measuring p217+tau in a biological sample, the antibodies of the kit can be pre-bound to a solid phase such as the wells of a microtiter plate or to beads.

[0156] According to a specific aspect, the capture antibody of the kit of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 32, 33, and 34, respectively, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 35, 36, and 37, respectively. Preferably, the capture antibody is a pT3 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28, and the light chain variable region having the polypeptide sequence of SEQ ID NO: 29.

[0157] According to a specific aspect, the detection antibody of the kit of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 2, 3, and 4, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 5, 6, and 7, respectively. Preferably, the detection antibody is a pT82 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 9.

[0158] According to another specific aspect, the detection antibody of the kit of the present invention comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 12, 13, and 14, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 15, 16, and 17, respectively. Preferably, the detection antibody is an hT43 antibody comprising a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 19.

[0159] According to another specific aspect, the kit of the present invention is used to measure the amount of p217+ tau peptide, the ratio of the amount of short p217+ tau peptide to the amount of long p217+ tau peptide, and / or the ratio of the amount of short p217+ tau peptide to the amount of total short tau peptide in a sample using the method of the present invention.

[0160] The content of all references cited in this patent application (including references, published patents, published patent applications, and co-pending patent applications) is hereby expressly incorporated by reference.

[0161] Embodiment

[0162] The present invention also provides the following non-limiting embodiments.

[0163] Embodiment 1 is a method for measuring the amount of p217+tau peptide in a sample, comprising:

[0164] (i) contacting the sample with a capture antibody against a p217+tau epitope to capture the p217+tau peptide in the sample, and

[0165] (ii) contacting the captured p217+tau peptide with a detection antibody against an epitope comprising amino acid residues 119 to 126 of tau protein, such as an epitope of amino acid residues 116 to 127 or an epitope containing amino acid residues 7 to 20 of tau protein, so as to measure the amount of p217+tau peptide or the amount of long p217+tau peptide, respectively.

[0166] Embodiment 2 is a method for determining the relative amount of long p217+tau peptide or short p217 tau peptide fragment in a sample, comprising

[0167] (i) contacting the sample with a capture antibody against a p217+tau epitope to capture the p217+tau peptide in the sample,

[0168] (ii) contacting the captured p217+tau peptide with a first detection antibody against an epitope comprising amino acid residues 119 to 126 of tau protein, so as to measure the amount of p217+tau peptide,

[0169] (iii) contacting the captured p217+tau peptide with a second detection antibody against an epitope containing amino acid residues 7 to 20 of tau protein, so as to measure the amount of long p217+tau peptide, and (iv) determining the relative amount of long p217+tau peptide or short p217+tau peptide based on the amount of the p217+tau peptide and the amount of the long p217+tau peptide.

[0170] Embodiment 3 is the method according to Embodiment 1 or 2, wherein the capture antibody is conjugated to beads, and wherein the detection antibody is biotinylated.

[0171] Embodiment 4 is the method according to any one of Embodiments 1 to 3, wherein the amount of the p217+tau peptide in the sample is measured using a high-sensitivity platform.

[0172] Embodiment 5 is the method according to any one of Embodiments 1 to 4, wherein the lower limit of quantification of the method is about 40 fg / ml of the p217+tau peptide, and the lower limit of detection of the method is about 2 fg / ml of the p217+tau peptide.

[0173] Embodiment 6 is the method according to any one of Embodiments 1 to 5, wherein the sample is a biological sample from a subject (preferably a CSF sample), and the method further comprises determining whether the subject has a tauopathy or is at risk of developing a tauopathy based on the amount of the p217+tau peptide, the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide, or the ratio of the amount of the short p217+tau peptide to the total amount of short tau peptides in the biological sample.

[0174] Embodiment 7 is the method according to Embodiment 6, wherein if the amount of the p217+tau peptide, the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide, or the ratio of the amount of the short p217+tau peptide to the total amount of short tau peptides in the biological sample is significantly higher than the corresponding baseline value, such as the average corresponding value of healthy volunteers, it is determined that the subject has a tauopathy or is at risk of developing a tauopathy.

[0175] Embodiment 8 is the method according to any one of Embodiments 1 to 5, wherein the sample is a biological sample from a subject receiving treatment for a tauopathy (preferably a CSF sample), and the method further comprises determining the effectiveness of the treatment in the subject based on the amount of the p217+tau peptide, the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide, or the ratio of the amount of the short p217+tau peptide to the total amount of short tau peptides in the biological sample.

[0176] Embodiment 9 is the method according to Embodiment 8, wherein if the amount of the p217+tau peptide in the biological sample decreases during the course of the treatment, it is determined that the treatment is effective.

[0177] Embodiment 10 is the method according to any one of Embodiments 6 to 9, wherein the tau proteinopathy is selected from Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam type motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (punch disease).

[0178] Embodiment 11 is the method according to Embodiment 10, wherein the tau proteinopathy is Alzheimer's disease.

[0179] Embodiment 12 is the method according to any one of Embodiments 1 to 5, wherein the sample is a biological sample from a human subject (preferably a CSF sample), and the method further comprises determining whether the subject is suitable for anti-p217+tau antibody therapy based on the amount of p217+tau peptide, the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide, or the ratio of the amount of short p217+tau peptide to the total amount of short tau peptide in the biological sample.

[0180] Embodiment 13 is the method according to Embodiment 12, wherein if the amount of p217+tau peptide, the ratio of the amount of short p217+tau peptide to the amount of long p217+tau peptide, or the ratio of the amount of short p217+tau peptide to the total amount of short tau peptide in the biological sample is significantly higher than the corresponding baseline value, such as the average corresponding value of healthy volunteers, then it is determined that the subject is suitable for anti-p217+tau antibody therapy.

[0181] Embodiment 14 is a method for monitoring the treatment using anti-p217+tau antibody in a subject, the method comprising:

[0182] i. obtaining a biological sample from the subject,

[0183] ii. separating the biological sample into an IgG-enriched sample containing antibody-bound p217+tau and an IgG-depleted sample containing antibody-free p217+tau,

[0184] iii. Contact each of the IgG-enriched sample and the IgG-depleted sample with a capture antibody directed against an epitope comprising phosphorylated T212 and / or phosphorylated T217 of the tau protein to capture the p217+tau peptide in each of the samples,

[0185] iv. Contact the captured p217+tau peptide with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein, thereby measuring the amount of antibody-bound p217+tau and the amount of antibody-free p217+tau in the biological sample,

[0186] v. Calculate the ratio of the antibody-bound p217+tau to the antibody-free p217+tau, and

[0187] vi. Monitor the treatment with the anti-p217+tau antibody in the subject based on the calculated ratio.

[0188] Embodiment 15 is a method for monitoring the treatment with an anti-p217+tau antibody in a subject, the method comprising:

[0189] i. Obtain a biological sample from the subject,

[0190] ii. Obtain a semi-denatured sample from the biological sample containing total p217+tau and a non-denatured sample from the biological sample containing antibody-free p217+tau, wherein the semi-denatured sample is heated to denature the antibody in the sample,

[0191] iii. Contact each of the semi-denatured sample and the non-denatured sample with a capture antibody directed against an epitope comprising phosphorylated T212 and / or phosphorylated T217 of the tau protein to capture the p217+tau peptide in each of the samples,

[0192] iv. Contact the captured p217+tau peptide with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 or 116 to 127 of the tau protein, thereby measuring the amount of total p217+tau and the amount of antibody-free p217+tau in the biological sample,

[0193] v. Calculate the amount of antibody-bound p217+tau in the sample by subtracting the amount of antibody-free p217+tau from the amount of total p217+tau,

[0194] vi. Calculate the ratio of the antibody-bound p217+tau to the antibody-free p217+tau, and

[0195] vii. Monitoring the treatment with the anti-p217+tau antibody in the subject based on the calculated ratio.

[0196] Embodiment 16 is the method according to any one of Embodiments 1 to 15, wherein the capture antibody comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 32, 33, and 34, respectively, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 35, 36, and 37, respectively; preferably, the capture antibody has a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29.

[0197] Embodiment 17 is the method according to any one of Embodiments 1 to 16, wherein the detection antibody comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 2, 3, and 4, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 5, 6, and 7, respectively; preferably, the detection antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 9.

[0198] Embodiment 18 is the method according to any one of Embodiments 1 to 16, wherein the detection antibody comprises immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO: 12, 13, and 14, respectively; and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 15, 16, and 17, respectively; preferably, the detection antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 19.

[0199] Embodiment 19 is the method according to any one of Embodiments 1 to 18, wherein the sample is a blood, brain homogenate, or cerebrospinal fluid (CSF) sample.

[0200] Embodiment 20 is the method according to any one of Embodiments 1 to 19, wherein the sample is obtained after fractionating a biological sample using reverse-phase high performance liquid chromatography (rpHPLC).

[0201] Embodiment 21 is an isolated detection antibody or an antigen-binding fragment thereof that binds to the tau protein at an epitope comprising amino acid residues 116 to 127 of the tau protein, comprising:

[0202] a. Immunoglobulin heavy chains having polypeptide sequences of SEQ ID NO: 2, 3, and 4 respectively

[0203] HCDR1, HCDR2, and HCDR3; and

[0204] b. Immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 5, 6, and 7 respectively.

[0205] Embodiment 22 is the isolated detection antibody or antigen-binding fragment according to Embodiment 21, and the isolated detection antibody or antigen-binding fragment preferably comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 8 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 9.

[0206] Embodiment 23 is an isolated detection antibody or its antigen-binding fragment that binds to tau protein at the epitope comprising amino acid residues 7 to 20 of tau protein, comprising:

[0207] a. Immunoglobulin heavy chains having polypeptide sequences of SEQ ID NO: 12, 13, and 14 respectively

[0208] HCDR1, HCDR2, and HCDR3; and

[0209] b. Immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO: 15, 16, and 17 respectively.

[0210] Embodiment 24 is the isolated detection antibody or antigen-binding fragment according to Embodiment 23, and the isolated detection antibody or antigen-binding fragment preferably comprises a heavy chain variable region having a polypeptide sequence of SEQ ID NO: 18 and a light chain variable region having a polypeptide sequence of SEQ ID NO: 19.

[0211] Embodiment 25 is an isolated nucleic acid encoding the detection antibody or its antigen-binding fragment according to any one of Embodiments 21 to 24.

[0212] Embodiment 26 is a vector comprising the nucleic acid according to Embodiment 25.

[0213] Embodiment 27 is a host cell comprising the nucleic acid according to Embodiment 25.

[0214] Embodiment 28 is a method of generating a detection antibody or an antigen-binding fragment thereof according to any one of Embodiments 21 to 24, the method comprising culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment under conditions for generating the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the cell or cell culture.

[0215] Embodiment 29 is a kit comprising:

[0216] a. a capture antibody against a mono- or multi-phosphorylated tau protein epitope comprising phosphorylated T212 and / or phosphorylated T217 of tau protein, and

[0217] b. a detection antibody against a tau protein epitope comprising amino acid residues 7 to 20 or 116 to 127 of tau protein;

[0218] wherein the kit is for measuring the amount of p217+tau peptide in a sample.

[0219] Embodiment 30 is the kit according to Embodiment 29, wherein the capture antibody comprises immunoglobulin heavy chain HCDR1, HCDR2 and HCDR3 having polypeptide sequences of SEQ ID NO: 32, 33 and 34 respectively, and immunoglobulin light chain LCDR1, LCDR2 and LCDR3 having polypeptide sequences of SEQ ID NO: 35, 36 and 37 respectively; preferably, the capture antibody has a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 29.

[0220] Embodiment 31 is the kit according to Embodiment 29 or 30, wherein the detection antibody is an isolated detection antibody according to any one of Embodiments 20 to 23.

[0221] Example

[0222] The following examples of the present invention are intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is determined by the appended claims.

[0223] Example 1: Highly sensitive assay for detecting p217+tau

[0224] The reagents for the assay are as follows: Simoa Homebrew kit (Quanterix, catalog number 101351), auxiliary beads (Quanterix, catalog number 101732), pT3 mouse monoclonal antibody (mAb), hT43 mAb, pT82 mAb, and hT7 mAb. pT3 is a parental antibody developed by Janssen that recognizes p217+tau, and its humanized form is referred to herein as the humanized pT3 mAb.

[0225] The samples are diluted in 50 mM Tris, 50 mM NaCl, 5 mM EDTA, 2% bovine serum albumin, 0.1% Tween 20, and 0.05% ProClin 300 (pH 7.8).

[0226] Three custom peptides produced by New England Peptide are used to calibrate the assay (calibration peptides).

[0227] Peptide pT3xhT43 contains hT43, PT51, and pT3 epitopes linked by a PEG4 linker and has a molecular weight of 6893 g / mol. The amino acid sequence of peptide pT3xhT43 is PRQEFEVMEDHAGTYGLGDR(dPEG4)GKTKIATPRGAAPPGQKG(dPEG4)GSRSR(pT)PSLP(pT)PPTREPKKV-amide (SEQ ID NO:22).

[0228] Peptide pT3xpT82 contains pT82 and pT3 epitopes linked by a PEG4 linker and has a molecular weight of 4551 g / mol. The amino acid sequence of peptide pT3xpT82 is acetyl-SLEDEAAGHVTQARMVSK)(dPEG4)GSRSR(pT)PSLP(pT)PPTREPKKV-amide (SEQ ID NO:23).

[0229] Peptide hT7xpT82 contains pT82 and hT7 epitopes linked by a PEG4 linker and has a molecular weight of 3619 g / mol. The amino acid sequence of peptide hT7xpT82 is acetyl-SLEDEAAGHVTQARMVSK(dPEG4)PRGAAPPGQKGQANA-amide (SEQ ID NO:24).

[0230] Reagent preparation

[0231] Coat the capture beads with 0.3 mg / ml capture Ab according to the protocol provided in the Quanterix manual. Dilute the coated capture beads to 200,000 beads / ml in bead dilution buffer, and then add 200,000 beads / ml of auxiliary beads so that the total bead concentration is 400,000 beads / ml.

[0232] Biotinylate the detection antibody 60-fold according to the protocol provided in the Quanterix manual and dilute it to 1.8 μg / ml in Homebrew detector / sample diluent.

[0233] Reconstitute the calibration peptide to 5 mg / ml in 0.1% phosphoric acid / water, aliquot into 20 μl and freeze. When ready to use, thaw the calibration peptide aliquots and dilute 1:1000 (e.g., dilute 1.5 μl into 1498.5 μl), and then dilute the dilution 1:1000 so that the final concentration of the peptide is 5000 pg / ml. Make a standard curve with 3 jumps starting from 30 pg / ml.

[0234] Dilute the CSF samples at least 1:4 in sample diluent. Dilute the healthy volunteer (HV) samples 1:5 or 1:10, and dilute the AD samples at least 1:20.

[0235] Simoa assay

[0236] Created a custom Simoa assay consisting of a two-step protocol that includes incubating with capture Ab, sample, and detection Ab for 35 minutes and washing, followed by incubating with streptavidin β-galactosidase (SBG) for 5 minutes. Each reaction consists of 25 μl of bead solution, 100 μl of sample or calibrator, 20 μl of detection solution, and 100 μl of SBG. Specify the names of the antibodies, and up to five capture antibodies and five detection antibodies can be loaded each time. React in a Simoa cuvette by the instrument, load the measurement plate with the β-galactosidase substrate (RGP) after the last wash, and then measure with the instrument.

[0237] Example 2: Isolation of native tau fragments on rpHPLC

[0238] The reagents are as follows: trifluoroacetic acid (HPLC grade), water (HPLC grade), acetonitrile (HPLC grade), phosphoric acid (analytical grade), HPLC binary gradient system, immunoassay buffer (100 mM TrisHCl, 100 mM NaCl, 0.05% Tween, and BSA, pH 7.8).

[0239] The protocol is as follows: Thaw 500 μl of frozen CSF for 30 minutes on ice. Add the thawed CSF to 1.5 ml of 100 mM sodium phosphate (pH 2.5) containing 100 mM sodium chloride and mix. Apply 1.8 ml of the resulting mixture to a C18 or similar reversed-phase chromatography column equilibrated in 0.1% aqueous trifluoroacetic acid. Then develop the HPLC column in an increasing acetonitrile gradient. Collect fractions during elution. Adjust the fractions to 10 mM with guanidine hydrochloride and then dry in a vacuum concentrator. Resuspend the dried fractions in immunoassay buffer and measure the tau peptides in the fractions based on the anti-tau capture and detection antibody pairs of the present invention.

[0240] Example 3: Quantification of free p217+tau or p217+tau bound by an antibody

[0241] Through additional upstream sample manipulation, the high-sensitivity pT3 assay can be used to measure the binding of p217+tau by antibodies produced in vivo or exogenously administered to the patient (e.g., humanized pT3 mAb). This technique can be used as a pharmacodynamic assay to study therapeutic anti-p217+tau antibodies, such as humanized pT3 mAb. For example, the following method can be used to measure antibody-free p217+tau and antibody-bound p217+tau.

[0242] Assay 1: Quantification of free p217+tau and bound p217+tau in biological fluids using immunocapture / depletion followed by rpHPLC and binding p217+tau

[0243] Incubate a biological fluid (e.g., CSF) with protein A / G-coated magnetic beads (15 μl bead slurry per 0.5 mL CSF) with shaking at room temperature for 2 hours to capture immunoglobulins in the sample. Precipitate the beads by magnet and transfer the supernatant to a second tube (sample = "IgG-depleted supernatant"). Wash the beads 4 times with 1 mL cold phosphate-buffered saline (PBS). Then add 0.5 mL of 6M GuHCl to the tube containing (a) the washed beads and (b) the IgG-depleted supernatant, and incubate the tube with shaking at room temperature for 20 minutes. Then precipitate the beads by magnet and transfer the resulting supernatant to a third tube (sample = "IgG-concentrated supernatant"). Finally, add 0.1M phosphoric acid (pH 2) to both solutions (add 1.0 mL phosphoric acid to the denatured IgG-depleted supernatant and 1.5 mL phosphoric acid to the IgG-concentrated supernatant to obtain a final volume of 2.0 mL of the sample), and then separate by rpHPLC as performed in Example 2. Reconstitute the resulting rpHPLC fractions as described in Example 2 and measure using the Simoap 217+tau assay of Example 1. The signal from the IgG-depleted supernatant represents free p217+tau (i.e., p217+tau not bound by antibodies), while the signal from the IgG-concentrated supernatant represents bound p217+tau (i.e., p217+tau bound by antibodies such as the humanized pT3 mAb). Analyze the rpHPLC separation and Simoa p217+ measurement of the same parental biological fluid (e.g., CSF) that has not undergone the immunocapture / depletion process simultaneously to evaluate the total p217+tau signal as a control or normalizer for the free measurement and the bound measurement.

[0244] Assay 2: Quantification of free p217+tau and bound p217+tau in biological fluids using heat denaturation of the antibody and quantification

[0245] Heat an aliquot of the biological fluid of interest (e.g., CSF) at 95 °C for 4 minutes and then cool on wet ice for 4 minutes (sample = "semi-denatured fluid"). Simultaneously, cool a second aliquot of the same fluid on wet ice for 8 minutes (sample = "non-denatured fluid"). Then measure both samples using the Simoa p217+tau assay of Example 1. The semi-denatured fluid signal represents total p217+tau, while the non-denatured fluid represents free p217+tau. Subtract the latter from the former to obtain a measurement of bound tau. Determine the precise heating time and temperature to irreversibly modify any antibodies in the fluid such that they can no longer interfere with the Simoap 217+tau assay while the p217+tau signal itself is not affected in any way. This assay is not a direct measure of whether an antibody binds to p217+tau, but it confirms the presence of antibodies competing in the assay. However, this assay gives results similar to the more laborious Assay 1.

[0246] Example 4: Biological samples

[0247] Samples for assay development and technology validation

[0248] The assays of Examples 1 to 3 were developed using CSF collected from human subjects with high tau levels. Some experiments were also conducted using CSF collected from human subjects with low tau levels to ensure the assay sensitivity required to test healthy volunteers in the Phase 1 trial. The CSF from cynomolgus monkeys (Macaca fascicularis) and common marmosets (Callithrix jacchus) was also measured using the assays of Examples 1 to 3. The CSF was obtained from Neu Encepharms GmbH (animal experiment CRO). Additionally, rapidly frozen brain samples from cognitively normal human subjects and common marmosets were homogenized and measured using the assays of Examples 1 and 3. Some experiments were conducted using the individual sera from clinically defined HV subjects and AD subjects.

[0249] Samples for preliminary clinical validation

[0250] Cohort 1 ("Inter-assay correlation cohort") : Ventricular fluid (VF) and lumbar fluid (LF) CSF samples were obtained from subjects with normal pressure hydrocephalus (NPH) (n = 11) (Professor Ville Lenoinen, University of Kuopio). These samples were separated by CSF Aβ42, total tau (tTau), and pTau181 measurements determined by the Innotest assay conducted at the University of Gothenburg (Professor Kaj Blennow), as well as by amyloid and tau immunohistochemistry (IHC) measurements from brain biopsies. RpHPLC and Simoa p217+tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla.

[0251] Cohort 2 ("Definite HV vs. definite AD cohort"): LF CSF samples from biochemically defined Alzheimer's disease (AD) subjects and healthy volunteer (HV) subjects (n = 20 per group) were obtained from the Sahlgrenska University (Professor Kaj Blennow). CSF Aβ42, tTau, and pTau181 measurements by Innotest were performed at the Sahlgrenska University. Samples were selected from a large batch of samples based on separation into predefined AD and HV cut-off metrics (AD = CSF Aβ42 < 400 pg / ml and CSF tTau > 600 pg / ml, HV = CSF Aβ42 > 400 pg / ml and CSF tTau < 600 pg / ml). rpHPLC and Simoa p217+tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla.

[0252] Cohort 3 ("HV vs. ARAD vs. early AD cohort") : LF CSF samples from clinically defined normal (Clinical Dementia Rating scale 0; CDR 0) subjects and mild memory impairment (CDR 0.5) subjects (n = 20 per group) were obtained from the Janssen study ALZ1005 / 1002. CSF Aβ42, tTau, and pTau181 measurements by Innotest were performed at the Sahlgrenska University. Subjects were classified into (a) HV = CDR 0 and Aβ42 > 600 pg / ml, (b) at risk of AD (ARAD) = CDR 0 and Aβ42 < 600 pg / ml, (c) potential non-AD dementia = CDR 0.5 and Aβ42 > 600 pg / ml, and (d) early AD = CDR 0.5 and Aβ42 < 600 pg / ml based on CDR and CSF Aβ42 scores. rpHPLC and Simoa p217+tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla.

[0253] Cohort 4 ("CDR 0 vs. CDR 1 cohort"): LF CSF samples from clinically defined normal (Clinical Dementia Rating scale 0; CDR 0) subjects and subjects with mild memory impairment (CDR 1) (n = 5 per group) were obtained from the University of Washington. CDR and MMSE determined by Innotest, as well as CSF Aβ42, tTau, and pTau181 measurements, were obtained at the University of Washington. Samples were coded prior to shipment such that Janssen was blinded to sample identity or characteristics. rpHPLC and Simoa tTau&p217+tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla and the measurement results were sent to the University of Washington for analysis.

[0254] Cohort 5 ("HV vs. MCI vs. AD cohort") : LF CSF samples from HV (n = 7) defined clinically and biochemically (Innotest AB42 > 600 pg / ml) were obtained from Precision Medicine in San Diego. LF CSF samples from MCI (n = 28) and AD (n = 12) defined clinically and biochemically (Innotest AB42 < 600 pg / ml) were obtained from the University of Antwerp. rpHPLC and Simoa p217+tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla.

[0255] Cohort 6 ("Disease severity and progression cohort"): LF CSF samples from clinically defined AD (Clinical Dementia Rating grade 1+) subjects (n = 235) were obtained from the Janssen study ELN115727301 / 302. These samples were baseline (pre-dose) samples from all subjects in the trial. In addition, CSF samples from placebo subjects (n = 90) at 78-week follow-up were included to evaluate biomarkers of disease progression. Cognitive assessments (ADAS-COG, MMSE, NTB, CDR.SOB), ApoE genotype, gender, and age were obtained from the trial. Innotest AB42, Innotest AB40, Simoa NFL, pT3xpT82, pT3xhT43, and hT7xpT82 assays were performed at Janssen Neuroscience Biomarkers in La Jolla. Subjects were confirmed as amyloid positive or negative based on an AB42 / 40 ratio cut-off of 0.09 (e.g., subjects with a ratio < 0.09 = amyloid positive = AD, while those with a ratio > 0.09 = amyloid negative = dementia of non-AD origin). Twenty-seven of the 235 subjects were determined to be amyloid negative, and the two groups were analyzed separately.

[0256] Samples for evaluating target engagement after treatment with an anti-p217+ agent

[0257] LF CSF from HV subjects (n = 40) treated with placebo or JNJ63733657 (single intravenous injection) was obtained from the Janssen trial JNJ63733657EDI1001. pT3xpT82 assays were performed at Janssen Neuroscience Biomarkers in La Jolla. pT3xhT43 assays were performed at Quanterix Corporation in Lexington MA.

[0258] Example 5: Screening of capture and detection antibody pairs using the Simoa platform

[0259] Previous reports from Janssen Neuroscience Discovery and the literature (e.g., Meredith et al., PLoS One. 8(10):e76523, 2013; Barthelemy et al., J Amheimers Dis. 51(4):1033-43, 2016; Russell et al., J Alzheimers Dis. 55(1):303-313, 2017; Hanger et al., J Biol Chem. 282(32):23645-54, 2007) have shown that tau fragments containing amino acids 200 to 220, particularly some combinations phosphorylated at amino acids 212, 214, 217, are enriched in AD. Development of an assay to measure this specific tau species (“p217+tau”) could thus yield improved biomarkers for AD diagnosis and / or staging, as well as potential predictive and / or pharmacodynamic assays for new drugs targeting this tau moiety. However, tau can be present at low levels (<200 pg / ml) in healthy volunteers, and p217+tau is a minor component of total tau, so p217+tau assays require optimal antibody pairs and high sensitivity.

[0260] To achieve this goal, the ability of a panel of anti-tau mAbs discovered at Janssen and some high-affinity commercial anti-tau mAbs to generate signals in a sandwich ELISA (sELISA) format when paired with pT3 was evaluated. A series of dilutions of CSF pools from AD subjects were used to screen the antibody pairs on the Simoa HD-1 analyzer platform (Quanterix Corporation) to provide the required sensitivity. Assay performance was based on signal / noise = average enzyme per bead (AEB) of the sample diluted in sample diluent / AEB of the assay diluent alone. The best-detecting antibodies paired with pT3 were hT43, pT82, Quanterix tau 2.0 detection reagent, and BT2, in order of sensitivity (Table 1). hT43 and Quanterix tau 2.0 detection reagent recognize the N-terminal region of tau, while pT82 and BT2 recognize sequences closer to the middle region of tau. The best N-terminal (hT43) and middle region (pT82) mAbs were selected for further optimization. Screening was performed simultaneously at Janssen Neuroscience Biomarkers and Quanterix Corporation, yielding similar results.

[0261] Table 1: Screening of the specificity of tau detection antibodies paired with pT3 in AD CSF

[0262]

[0263] Shows antibody epitopes on tau and signal / noise (S / N) ratios when measuring pooled CSF from AD subjects; NT = not tested.

[0264] Example 6: Optimization of the pT3xhT43 assay and the pT3xpT82 assay

[0265] A series of optimization experiments were conducted based on the general Quanterix experience of optimizing assays on the Simoa platform. 10% mouse serum or 500 μg / ml mouse IgG was added to the detector diluent, but assay sensitivity was not improved. Titrations of detector mAb concentration (0.15 μg / ml, 0.3 μg / ml, 0.6 μg / ml, 1.2 μg / ml, and 1.8 μg / ml), SβG concentration (100 pM, 200 pM, or 300 pM), and capture mAb bead concentration (300K / well, 150K + 200K auxiliary beads) were evaluated. Incubation time of the protocol (65 minutes vs. 35 minutes) and sample volume (100 μl vs. 150 μl) were also evaluated. The ideal reagent concentrations for the two assays were 150K capture beads + 200K auxiliary beads, 1.8 μg / ml detector, and 200 pM SBG, respectively. The sample volume and incubation time had the least impact on the assay, so the lower conditions of 100 μl sample and 35-minute incubation were selected.

[0266] Example 7: Technical validation of the pT3xhT43 assay and the pT3xpT82 assay

[0267] Linear range with calibration materials

[0268] Prepare the calibration peptides described in Example 1. The calibration peptides contain the core epitopes of pT3 and hT43 or pT3 and pT82 separated by a PEG4 linker, and these epitopes are used to generate a standard curve. Representative standard curves are shown in Figure 1 . The calibration peptides were titrated from 30 pg / ml to 0.041 pg / ml in a 1:3 jump with assay buffer and then measured with pT3xhT43 and pT3xpT82 assays. A calibration curve was generated using a 4-parameter curve fitting data reduction method (4PL, 1 / y2 weighting). The lower limit of detection (LLOD) was defined as the calculated calibrator level that produced an AEB equal to the mean of zero calibrator + 2.5 standard deviations (SD), including a 10% coefficient of variation (CV). Based on these criteria, representative data yielded an LLOD of approximately 0.002 pg / ml. The linear range, lower limit of quantification (LLOQ), and upper limit of quantification (ULOQ) of the assay were defined as the lowest and highest standard curve points that achieved a CV < 20% and an expected recovery of 80% - 120%. Based on these criteria, the linear range of the pT3xhT43 and pT3xpT82 assays was 0.041 pg / ml to 30 pg / ml ( Figure 1 , Table 2).

[0269] Table 2: Representative calibration curve of the optimized pT3xhT43 assay with LLOD calculation results

[0270]

[0271]

[0272] Dilution linearity of CSF

[0273] To assess dilution linearity and determine the ideal dilution for testing CSF samples, four CSF samples (high tau, low AB42) from AD subjects in a batch were titrated from a 1:2 dilution to a 1:4096 dilution with assay buffer and then measured using the p217+tau assay. Samples diluted beyond 1:512 were generally measured below the LLOQ. Samples from 1:4 to 1:512 were dilution linear, such that it was the defined range for measuring CSF samples. To confirm in cognitively normal subjects, CSF pools from subjects with low tau and high AB42 were similarly measured. Dilution linearity from 1:4 dilution to 1:256 dilution was again observed, and beyond this range, the measurements were below the LLOQ (Figure 2).

[0274] Precision

[0275] To assess the precision of the measurement, a standard curve of pT3xhT43 was prepared and measured on 3 separate days ( Figure 3 and Table 3). The calibration peptide was diluted from 30 pg / ml to 0.041 pg / ml in a series of 1:3 jumps and measured in duplicate in the pT3xhT43 assay. The process was repeated for 3 consecutive days by the same technician at the same location. Analysis of the 4 points in the middle of the curve (where CSF samples were measured) showed that the precision within a batch (intra-assay CV%) was consistently <10% and averaged 2.46 - 5.18% CV, and the inter-assay precision averaged 6.46% CV. These were well within the acceptable limit of 20% CV for assays intended for research use only (RUO), and were partly attributed to the automated nature of all ELISA steps in the Simoa HD-1 analyzer.

[0276] Table 3: Intra-assay precision and inter-assay precision of the pT3xhT43 assay

[0277]

[0278] Transferability between laboratories

[0279] To assess the precision of the p217+tau assay between test sites, the same batch of reagents was used to titrate the same AD CSF pool at Janssen Neuroscience Biomarkers and Quanterix. Figure 4 shows that the measurements of the pT3xhT43 assay and the pT3xpT82 assay at the two test sites are very similar.

[0280] Accuracy

[0281] To assess the accuracy of the assay, calibration peptides at known concentrations (0 pg / ml, 2 pg / ml, or 20 pg / ml) were incorporated into two different pools of HVCSF, diluted to the recommended 1:4 dilution, and then measured in the pT3xhT43 assay and the pT3xpT82 assay. This is a measure of the potential interference presented by the components of the sample matrix. The levels of the endogenous signal were subtracted from the measurements of the 2 pg / ml and 20 pg / ml incorporations, and then the concentration of the observed calibration material was compared with the expected concentration to calculate the recovery percentage. The measured concentration was compared with the expected concentration to calculate the incorporation recovery rate, and an average recovery rate of 114% was obtained (Table 4). This is well within the acceptable limit of 80%-120% recovery for the RUO assay, indicating no significant interference with CSF when tested at a ≥1:4 dilution.

[0282] Table 4: Spike recovery of the pT3xhT43 assay

[0283]

[0284] Signal competition of p217+-directed antibodies in CSF

[0285] To confirm the accuracy of the pT3xhT43 and pT3xpT82 assay signals in CSF and to evaluate their potential utility as pharmacodynamic assays in clinical studies of p217+tau-directed antibodies, titrations of the pT3 mAb or humanized pT3 mAb were incorporated into pools of AD CSF and measured in the pT3xhT43 assay and the pT3xpT82 assay after incubation at room temperature for 2 hours (Figure 5). The administration of soluble pT3 and humanized pT3 antibodies decreased the signal in the pT3-based assays in a dose-dependent manner. Incorporation of a comparable concentration of msIgG (negative control) did not affect any of the measurements. The lower ability of the humanized pT3 mAb to compete with pT3 can be attributed to the higher affinity of pT3 for p217+tau.

[0286] Phosphorylation dependence

[0287] To confirm that the signals in CSF obtained with pT3xhT43 assay and pT3xpT82 assay were indeed based on phosphorylated epitopes, AD CSF was treated with alkaline phosphatase to dephosphorylate all residues. The samples were then analyzed in the pT3 assay and two hT7-based assays (hT7xpT82 or hT7xBT2). Since hT7 is known to be phosphorylation-independent, it was used as a negative control.

[0288] Pooled CSF from AD patients was treated with increasing amounts of alkaline phosphatase (AP) for 4 h at 37 °C in a buffer containing zinc and magnesium chloride. The pT3xhT43 assay and pT3xpT82 assay were used to measure the effect on the pT3-directed epitopes. Treatment with alkaline phosphatase decreased the pT3xhT43 and pT3xpT82 signals in a dose-dependent manner. However, the phosphorylation-independent assays hT7xpT82 or hT7xBT2 did not show a decrease in signal; instead, they showed an increase in signal, as expected since phosphorylation reduces pT7 binding ( Figure 6 ).

[0289] p217+tau fragment profile

[0290] To explore the nature of the p217+ tau signal derived from measuring crude CSF, samples of AD CSF were fractionated by rpHPLC via a method similar to that described by Meredith et al., PLoS One. 8(10):e76523, 2013. Fractions were collected and measured using the pT3xhT43 assay and pT3xpT82 assay ( Figure 7)。In this chromatographic form, the smaller tau fractions elute earlier (smaller fraction numbers), while the larger fractions elute later (larger fraction numbers). Full-length tau elutes in fraction 19. According to previous reports (Meredith et al., PLoS One. 8(10):e76523, 2013, Barthelemy et al., J Alzheimers Dis. 51(4):1033 - 43, 2016), the tau fragment profiles indicate that full-length tau is hardly detected by either of these two assays. The pT3xpT82 assay detects two major peaks (tau species) smaller than full-length tau (fractions 12 and 14), while the pT3xhT43 assay detects only one of these major peaks (fraction 14). This indicates that p217+ tau in CSF exists in at least two fragments, namely a larger fragment encoding at least the region from hT43 to pT3 (aa 7 - 220 of tau) and a smaller fragment encoding at least the region from pT82 to pT3 (aa 116 - 220 of tau), but does not fully reach the hT43 epitope. That is, there may be a proteolytic cleavage site between aa 20 and aa 116, which is cleaved only in a subset of tau molecules at any given time. This profile is not specific to p217+ as measurements with other tau assays that recognize similar regions of tau but are not phosphorylation-specific yield similar results (data not shown).

[0291] Analyte stability

[0292] The stability of the endogenous p217+ tau epitope was evaluated at various temperatures. Pooled AD CSF was aliquoted, and each aliquot was stored at 4 °C, 22 °C, or 37 °C for 1 hour, 2 hours, or 4 hours. Additionally, a subset of the aliquots was freeze-thawed (-80 °C to 22 °C) 2 or 3 times. Then all samples were diluted 1:20 and analyzed using the pT3xhT43 assay and the hT7xpT82 assay (Figure 8). No significant change in signal was observed under any of the tested conditions, indicating that all 4 epitopes recognized by these assays are stable enough to withstand standard storage / test procedures. Finally, CSF was prospectively collected from 4 donors, then aliquoted and frozen at -70 °C, and samples were removed every 3 months for measurement using the pT3xpT82 assay. No significant change in signal was observed at the 3-, 6-, or 9-month time points ( Figure 9 )。

[0293] Example 8: Clinical validation of the pT3xhT43 assay and the pT3xpT82 assay

[0294] To evaluate the utility of the pT3xhT43 assay and the pT3xpT82 assay in the diagnosis and staging of AD, three cohorts of CSF samples were obtained for p217+tau measurement. The measured values were analyzed for their correlations with cognitive scores and with other classical AD biomarkers.

[0295] Cohort 1: "Inter-assay correlation cohort"

[0296] CSF samples, VF and LF, and brain biopsies (ventricular) were obtained from 10 subjects with the neurodegenerative disorder normal pressure hydrocephalus (NPH), a condition characterized by the production of excessive interstitial fluid in the brain and presenting a high AD incidence. Crude CSF was assayed for p217+tau and its correlations with traditional AD biomarkers were analyzed.

[0297] The levels of Aβ42 ( Figure 10A 、 Figure 10D ), tTau ( Figure 10B 、 Figure 10E ), and pTau181 ( Figure 10C 、 Figure 10F ) in VF were assayed by Innotest ELISA (classical measurement). The same samples were measured using the pT3xhT43 ( Figure 10A 、 Figure 10B 、 Figure 10C ) assay and the pT3xpT82 ( Figure 10D 、 Figure 10E 、 Figure 10F ) assay, and the correlations were evaluated. Both the pT3xhT43 assay and the pT3xpT82 assay showed negative correlations with CSF Aβ42 (r 2 = 0.609, p = 0.0077 and r 2 = 0.590, p = 0.0095 respectively) and positive correlations with CSF tTau (r 2 = 0.525, p = 0.0177 and r 2 = 0.435, p = 0.0381 respectively), but they were not significantly correlated with CSF pTau181 (Figure 10).

[0298] Brain biopsies from the same 10 NPH subjects were analyzed by IHC and rated amyloid positive / negative and tau positive / negative by a pathologist. When both were positive, the sample was designated "biopsy +" and was a classic diagnosis of AD. When both were negative, the sample was designated "biopsy –" and was a classic diagnosis of non-AD. Samples designated "biopsy + (amyloid)" were positive for amyloid but negative for tau. CSF obtained from ventricular puncture (VF = black dots) or lumbar puncture (LF = red dots) was measured using the pT3xhT43 assay and the pT3xpT82 assay, and correlations were evaluated (Figure 11). Both the pT3xhT43 assay and the pT3xpT82 assay were able to separate brain biopsy-negative samples (amyloid- / tau-) from positive samples (amyloid+ / tau+) (p = 0.04 and p = 0.02, respectively). Samples that were positive for amyloid but not positive for tau were generally measured between biopsy + samples and biopsy - samples. Amyloid plaques in the brain are believed to precede tau tangles, so amyloid+ / tau- samples may represent early AD or another disease.

[0299] Cohort 2: "HV vs. AD cohort"

[0300] CSF samples (LF) from biochemically defined AD subjects and HV subjects (n = 20 per group) were obtained from the Sahlgrenska University. The levels of Aβ42 and tTau were measured by Innotest ELISA (classic measurement) to subdivide the groups (AD = CSF Aβ42 < 400 pg / ml and CSF tTau > 600 pg / ml, HV = CSF Aβ42 > 400 pg / ml and CSF tTau < 600 pg / ml). Subsets of crude CSF and rpHPLC-fractionated CSF were measured using the pT3xhT43, pT3xpT82, and hT7xpT82 assays. The results were analyzed for correlation with traditional AD biomarkers (Figure 12). The data in Panels A and B of Figure 12 confirm that the pT3 epitope is an indicator of high risk for patients to rapidly progress to incipient AD. The pT3 epitope is highly elevated in patients showing high total tau and low Aβ42. Conversely, the pT3 epitope is present at low levels in subjects with low total tau and high Aβ42. Figure 12C It was confirmed that the elevated tau containing the pT3 epitope was at least partially driven by the elevated level of total tau, as confirmed by the hT7xpT82 total tau assay, but not completely ( Figure 12D ). This indicates that both the amount of tau in AD and the degree of its phosphorylation at the p217+ epitope are elevated.

[0301] Data from Figure 11 were used to create ROC curves for the ability of the pT3XHT43, pT3xpT82, and hT7xpT82 assays to distinguish AD samples from HV samples. All three assays showed excellent specificity and sensitivity. However, the two pT3-based assays (pT3xhT43 and pT3xpT82, which detect p217+tau) had higher diagnostic ability than the hT7-based assay ( Figure 13 ).

[0302] A subset (n = 11 per group) of the same CSF samples measured in Figure 12 was fractionated by rpHPLC and then measured with the pT3xhT43, pT3xpT82, and hT7xpT82 assays (the latter measures the same tau fragment in a phosphorylation-independent manner) (Figure 14). The profiles of the observed tau fragments were similar to those shown in Example 7 and Figure 7 . That is, two major species were observed in both pT82-based assays (pT3xpT82 and hT7xpT82), while only one peak in the peak was observed in the pT3xhT43 assay. The concentrations of both major species were higher in the AD group than in the HV group. In addition, the pT3-based assay (p217+tau) showed a greater difference between groups than the hT7-based assay (total tau), as detected in the crude CSF analysis. The larger p217+tau species (fractions 13 to 14) provided the greatest AD-HV difference (Figure 14).

[0303] The sum of all major tau fragments (fractions 11 to 14) in Figure 14 was calculated and then compared between the AD and HV subgroups. The percentage increase in tau containing the pT3-epitope (pT3xhT43 or pT3xpT82) in AD was twice the percentage increase shown for tau containing the non-pT3-epitope (hT7xpT82) (Table 5).

[0304] Table 5: Total tau signal in pT3 assays and non-pT3 assays

[0305]

[0306] The signals in each fraction of Figure 14 were analyzed independently and the sums were tabulated in Table 5 to show which fraction produced the greatest AD-versus-HV signal. The most informative fragment pools were detected on fragment pools 13 and 14 using the pT3 antibody (Table 6).

[0307] Table 6: Tau signal in different tau fragment pools in pT3 assays and non-pT3 assays

[0308]

[0309]

[0310] Cohort 3: "HV vs. ARAD vs. early AD cohort"

[0311] CSF samples (LF) from clinically defined normal (CDR 0) subjects and mildly memory impaired (CDR 0.5) subjects (n = 20 per group) were obtained from the Janssen study ALZ100 / 5 / 2002. Levels of Aβ42, tTau, and pTau181 were measured by Innotest ELISA. Subjects were classified into (a) HV = CDR 0 and Aβ42 > 600 pg / ml, (b) ARAD = CDR 0 and Aβ42 < 600 pg / ml, (c) potential non-AD dementia = CDR 0.5 and Aβ42 > 600 pg / ml, and (d) early AD = CDR 0.5 and Aβ42 < 600 pg / ml based on CDR and CSF Aβ42 scores.

[0312] CSF samples were also fractionated by rpHPLC and measured using pT3-based assays (pT3xhT43, Figures 15A to 15E , and pT3xpT82, Figures 15F to 15J ) and total tau assays (hT7xpT82, Figures 15K to 15O ). All pT3-based assays and hT7-based assays showed elevated signals in samples with CDR 0 vs CDR 0.5 ( Figure 15A , Figure 15F and Figure 15K ) and with Aβ42 < 600 pg / ml vs Aβ42 > 600 pg / ml ( Figure 15B , Figure 15G and Figure 15L ). Decomposition of CDRxAβ42 levels is shown in Figure 15C , Figure 15D , Figure 15H , Figure 15I , Figure 15M and Figure 15N , and the summed signals in all fractions are shown in Figure 15E , Figure 15J and Figure 15O . Signal levels were highest in the Aβ42 < 600 pg / ml + CDR 0.5 subgroup, which is consistent with elevated p217+tau signals in early AD vs HV or ARAD. Separation between subgroups was better in pT3-based assays than in hT7-based assays, indicating that hyperphosphorylation of pT3 epitopes is particularly enriched in disease (above simple total tau elevation).

[0313] Cohort 4 ("CDR 0 vs. CDR 1 cohort")

[0314] LF CSF samples from clinically defined normal (Clinical Dementia Rating stage 0; CDR 0) subjects and mildly memory impaired (CDR 1) subjects (n = 5 per group) were obtained from the University of Washington. CDR and MMSE determined by Innotest, and CSF Aβ42, tTau, and pTau181 measurements were obtained at the University of Washington. Samples were coded prior to shipment such that Janssen was blinded to sample identity or characteristics. rpHPLC and Simoa tTau&p217+tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla and the measurements were sent to the University of Washington for analysis.

[0315] Crude CSF samples or CSF samples after rpHPLC separation were measured using two pT3-based assays (pT3xhT43 and pT3xpT82) and tTau (hT7xpT82). Data were presented as the ratio between the two pT3 assays (Table 7) to assess the relative impact of short tau species, or as the ratio between ( Figures 16A to 16B ) a pT3 assay and tTau to assess the relative impact of that phosphorylation event. In both cases, the results accurately predicted the CDR status of 9 out of 10 subjects. One abnormal subject determined by Innotest also had abnormally low Tau and thus may represent dementia from non-tauopathies. Interestingly, a correlation was observed between the p217+Tau / tTau ratio and MMSE, suggesting that the signal detected by the pT3 assays may be related to cognition.

[0316] Table 7: Ratio of pT3xpT82 (p217+ short) analysis to pT3xhT43 (p217+ long) analysis of crude CSF

[0317]

[0318] *CDR 1 and Aβ positive, but low Tau and pTau in Innotest and Simoa

[0319] Cohort 5 ("HV vs. MCI vs. AD cohort")

[0320] Low-flow CSF samples from HV (n = 7) defined by clinical and biochemistry (Innotest AB42 > 600 pg / ml) were obtained from Precision Medicine in San Diego, CA. Low-flow CSF samples from MCI (n = 28) and AD (n = 12) defined by clinical and biochemistry (Innotest AB42 < 600 pg / ml) were obtained from the University of Antwerp. RpHPLC and Simoap217 + tau measurements were performed at Janssen Neuroscience Biomarkers in La Jolla.

[0321] Crude CSF samples or CSF samples after rpHPLC separation were measured using two pT3-based assays (pT3xhT43 and pT3xpT82) and tTau (hT7xpT82). All pT3-based assays and hT7-based assays showed elevated and increasing signals in the HV group vs. the MCI group vs. the AD group ( Figures 17A to 17C ) and were well correlated with each other (as shown in cohort 1 and Figure 9 ). ( Figure 17D and Figure 17E ). The pT3 assays were also somewhat correlated with Innotest tTau and pTau181 ( Figure 17F and Figure 17G ), but not with Innotest AB42 or the AB42 / 40 ratio ( Figure 17H and Figure 17I ). Similar diagnostic grading results were observed in crude CSF measurements ( Figures 17A to 17C ) or rpHPLC fractionated material ( Figures 17J to 17T ). As shown in cohort 3, the separation of HV vs. MCI vs. AD using the pT3-based assay was more pronounced (more statistically significant) than using the tTau assay, highlighting the pathological relevance measured by this pT3 assay.

[0322] Cohort 6 ("Disease severity and progression cohort")

[0323] CSF samples (LF) from subjects with clinically defined AD (Clinical Dementia Rating stage 1+) (n = 235) were obtained from the Janssen study ELN115727301 / 302. These samples were baseline (pre-dose) samples from all subjects in the trial. In addition, CSF samples from placebo subjects (n = 90) at 78-week follow-up were included to evaluate biomarkers of disease progression. Cognitive assessments (ADAS-COG, MMSE, NTB, and CDR.SOB), ApoE genotype, gender, and age were obtained from the trial. Innotest AB42, Innotest AB40, Simoa Neurofilament Light (NFL), pT3xpT82, pT3xhT43, and hT7xpT82 assays were performed at Janssen Neuroscience Biomarkers in La Jolla. Subjects were identified as amyloid positive or negative based on an AB42 / 40 ratio cutoff of 0.09 (e.g., subjects with ratio < 0.09 = amyloid positive = AD, and those with ratio > 0.09 = amyloid negative = dementia of non-AD origin). Twenty-seven of the 235 subjects were determined to be amyloid negative, and thus each group was analyzed separately.

[0324] Signals from crude CSF measurements again showed good correlation between the two pT3 assays and the tTau assay ( Figure 18A and Figure 18B ), but no correlation with NFL (a putative marker of general neurodegeneration) ( Figure 18C ), suggesting that the pT3 assays can identify specific forms or stages of neurodegeneration.

[0325] Assays based on pT3 again showed higher signals in amyloid-positive subjects than in amyloid-negative subjects ( Figures 18D to 18E ).

[0326] Assays based on pT3 showed moderate correlation with several cognitive scores (ADAS-COG, MMSE, NTB, CDR.SOB, Figures 18F to 18M ), confirming the findings in cohort 4 ( Figure 16B ). Interestingly, the signal from the baseline pT3 assay was also moderately correlated with changes in cognitive scores during the 18-month follow-up period, suggesting the ability to predict cognitive decline ( Figures 18N to 18P ).

[0327] The ratio of the pT3-based signal to the tTau signal (p217_tau / tTau) yielded similar results, which are not shown.

[0328] Correlations with cognition and cognitive change were observed in both the amyloid-positive and amyloid-negative groups, although the latter was a small sample set. If confirmed, this would suggest that the link between p217+ and cognition may not be specific to AD.

[0329] Example 9: Quantification of free p217+ tau and antibody-bound p217+ tau

[0330] The assays described in Example 3 were performed as follows.

[0331] Assay 1: Quantification of free p217+ tau and bound p217+ tau in biological fluids by immunocapture / depletion followed by rpHPLC Figure 7

[0332] The assay was tested by incorporating the antibody into CSF samples. 10 μg of pT3 mAb, humanized pT3 mAb, msIgG, or an equivalent volume of PBS (mock) was incorporated into pooled AD CSF, then incubated at 4 °C for 24 h, followed by immunocapture. The samples, as well as the parental CSF that had not undergone immunocapture, were fractionated on rpHPLC, and each fraction was measured using the pT3xhT43 assay to assess the amounts of total p217+ tau and bound p217+ tau. Substantial signals were observed in one of the major peaks similar to those shown in Example 7 and Figure 19 in the parental samples (total p217+ tau) and pT3 mAb or humanized pT3 mAb immunocaptures (bound p217+ tau), but not in mock or IgG immunocaptures ( Figure 20A ).

[0333] Titrations of the humanized pT3 mAb were incorporated into pooled AD CSF, then incubated at 22 °C for 2 h, followed by immunocapture, rpHPLC, and the pT3xhT43 assay to assess bound p217+ tau ( Figure 20B ). The IgG-depleted supernatants were also fractionated and measured to assess free p217+ tau ( Figure 19 ). Incorporation of the humanized pT3 mAb increased the amount of measured bound p217+ tau in a dose-dependent manner and decreased the amount of free p217+ tau.

[0334] In summary, the results show that this method is a direct measure of target engagement, is specific for antibodies targeting the p217+ tau epitope ( Assay 2: Quantification of free p217+ tau and bound p217+ tau in biological fluids by selective denaturation of antibodies ), and is target antibody dose-dependent (Figure 20).

[0335] Figure 21A Figure 21B

[0336] A biological sample (e.g., CSF) is heated at a temperature close to boiling for 4 minutes, then cooled on ice, and subsequently measured using pT3xhT43 and / or pT3xpT82 assays. It was determined that the exact time of this process irreversibly destroys the antibodies in the sample such that these antibodies do not interfere with the assay (Figure 21), but does not affect the p217+tau signal itself (Figure 21). It is believed that this is due to the particular lack of a tertiary structure in the tau protein, making the tau protein particularly stable at high temperatures. This sample is referred to as total p217+tau, while parallel measurements of samples not subjected to heat treatment are referred to as free p217+tau. The bound p217+tau measurement is obtained by subtracting the free concentration from the total concentration.

[0337] The effect of heating on the assay was determined as follows.

[0338] Effect of heating on CSF / humanized pT3 mAb mixtures: Humanized pT3 mAb was incorporated into aliquots of pooled AD CSF to 1 μg / ml, incubated at 22 °C for 2 hours, heated at 95 °C for 0 to 20 minutes, cooled to 4 °C, and then measured using the pT3xpT82 assay at a 1:10 dilution ( Figure 21C ). The p217+tau signal was lower after approximately 2 minutes of heating treatment and then recovered to the level shown in unincorporated CSF, remained stable after approximately 10 minutes of heating, and then decreased.

[0339] Effect of heating on initial CSF: Aliquots of pooled AD CSF were heated at 95 °C for 0 to 20 minutes, then cooled to 4 °C, and then measured using the pT3xpT82 assay at a 1:10 dilution ( Figure 21B ). The p217+tau signal remained stable after approximately 10 minutes of heating and then decreased.

[0340] Effect of heating on the ability of humanized pT3 mAb to interfere with the pT3xpT82 assay: Aliquots of 10 μg / ml humanized pT3 mAb in PBS were heated at 95 °C for 0 to 20 minutes, then cooled to 4 °C. These samples were then mixed with pooled AD CSF (to a final concentration of 1 μg / ml of humanized pT3 mAb) and incubated at 22 °C for 2 hours, and then measured using the pT3xpT82 assay at a 1:10 dilution ( Figure 22A ). The p217+tau signal was lower after approximately 2 minutes of JNJ heating treatment and then recovered to the level shown in unincorporated CSF (see Figure 22B ), and remained stable after at least 20 minutes of heating.

[0341] Humanized pT3 mAb was titrated against parallel aliquots of pooled AD CSF, incubated at 22 °C for 2 h, and then subjected to a heat denaturation process (heating for 4 min)( Figure 22C ) or immunocapture / rpHPLC( Example 10: p217+ tau signal in preclinical animal models ) and then measured using the pT3xpT82 assay. Both methods showed that the humanized pT3 mAb dose-dependently increased in binding to the p217+tau signal, decreased in the free p217+tau signal, and showed no change in the total p217+tau signal. In addition, the heat-mediated denaturation method produced a humanized pT3 mAb dose-dependence comparable to that obtained using the more laborious immunocapture / rpHPLC method of Assay 1, as well as relative free-versus-bound-versus-total p217+ measurements( Cynomolgus macaque ). Thus, the heating method is recommended for standard sample analysis.

[0342] Figure 23

[0343] To support preclinical studies, pT3-based assays and / or sequence alignments were used to evaluate initial samples from various common laboratory animals to predict cross-reactivity.

[0344] Common marmoset

[0345] CSF from two cynomolgus monkeys was measured at various dilutions using pT3-based assays and hT7-based assays( Figure 24B ). For comparison, the same detection antibody was paired with each of the two capture antibodies. In some cases, the CSF of two individuals (Cyno 1 or Cyno 2) was tested separately, and in other cases, the CSF samples were pooled to save volume. Regardless of the detection antibody, substantial signals (AEB) were observed in all assays using hT7 as the capture antibody, but no signal was detected in any assay using pT3 as the capture antibody. In addition, plate-based assays using pT3 have shown that pT3-based signals are almost absent or absent even in homogenates of cynomolgus monkey brains, although large signals are present in AD human brains (data not shown). This indicates that although there is a high level of tau in this species, the pT3 epitope is not retained in this species. In fact, analysis of the published protein sequences shows that there is one amino acid difference between the pT3 core epitopes of humans and cynomolgus monkeys, and structural modeling based on the crystal structure of the humanized pT3 mAb with tau indicates that this change can eliminate pT3 binding (data not shown).

[0346] Figure 24C

[0347] CSF from common marmosets was tested using pT3-based assays and hT7-based assays (Figure 24). CSF from three common marmosets was measured at various dilutions using the pT3xhT43, pT3xpT82, and hT7xpT82 assays. For comparison, pooled cynomolgus macaque CSF (negative control) and pooled AD human CSF (positive control) were tested in parallel. Substantial signals (AEB) were observed in marmoset CSF using the pT3xpT82 ( Figure 24A ) and hT7xpT82 ( Mouse, rat, dog, pig ) assays, but not using the pT3xhT43 assay ( Example 11: p217+ tau signal in blood ).

[0348] This indicates the absence of the hT43 epitope in this species, and indeed, protein sequence alignment does show a single amino acid difference between the human and common marmoset hT43 epitopes, while the pT3, hT7, and pT82 epitopes are retained. Measurement of marmoset brain homogenates using the same assays confirmed the presence of substantial signals using the pT3xpT82 and hT7xpT82 assays, but little signal using the pT3xhT43 assay (data not shown). Thus, analysis of p217+ tau signal in marmosets was achieved using the pT3xpT82 assay.

[0349] Figures 25A to 25D

[0350] Alignment of the predicted tau protein sequences in mouse, rat, dog, or pig (NCBI accession numbers: NP_001033698.1, NP_058908.2, NP_001104271.1, and AGJ26517.1 respectively) with the human sequence indicates that pT3 is 100% conserved in these species. However, the hT43 and pT82 sequences in mouse, rat, dog, and pig differ from the human sequence, and thus the pT3xhT43 and pT3xpT82 assays are required to evaluate samples from these animals.

[0351] In summary, the data presented here indicate that the pT3xhT43 and pT3xpT82 assays developed on the Simoa platform for CSF measurement are highly sensitive, with femtogram-level sensitivity, and are precise, accurate, dilution-linear, and analyte-stable. These assays appear to correlate well with classical AD biomarkers and dementia scores and may be superior to those metrics for identifying and staging AD subjects.

[0352] These assays can be used to measure total p217+tau levels in CSF, or to evaluate the fragment profile of p217+ in rpHPLC-fractionated CSF. These assays can also be combined with pre-analytical manipulations to measure the levels of p217+tau bound by endogenously or exogenously administered antibodies versus p217+tau without antibody. Thus, these assays can be used as predictive biomarkers to identify subjects suitable for anti-p217+tau antibody therapy by identifying subjects with high levels of the p217+tau target. By measuring the levels of total, free, and therapeutically antibody-bound p217+tau, these assays can also be used as pharmacodynamic biomarkers.

[0353] Figures 26A to 26B

[0354] Although measurements of Tau in CSF have shown excellent utility in the diagnosis and staging of neurodegenerative disorders, collection of CSF has limitations (e.g., patient burden, clinical site experience, collection volume and frequency constraints). Accordingly, there is great interest in making Tau measurements applicable to blood products (e.g., serum, plasma). However, recent literature has shown that tau measurements in crude serum or plasma do not exhibit ideal diagnostic performance and can be plagued by sensitivity and matrix interference hurdles. However, the pT3-based assay may represent a new opportunity due to its high sensitivity and specificity.

[0355] Using the pT3xpT82 and hT7xpT82 assays, sera from clinically defined AD subjects and HV subjects (n = 4 per group) were measured in crude samples ("crude", Figure 27 ) at various dilutions, in samples treated with acid (NaOAc pH5) and denatured (as described in D'Abramo et al., 2016 to remove most matrix interferences) ("boiled", Figure 25B ), and in samples after heat denaturation of the eluate after immunoprecipitation (IP) with pT3 beads ("pT3 IP", Figure 25D ).

[0356] Measurements in crude serum showed that most samples were below the limit of quantification (LOQ), while a few abnormal samples reported much higher levels. However, the signal disappeared after moderate dilution and was thus considered an interfering artifact. Evaluation of the highest dilutions tested ( Figures 26A to 26B and Figure 27 ) and thus least affected by interference, showed that the pT3xpT82 assay could detect slightly more signal in AD samples, but all signals detected were below the LOQ, and thus the assay may be inaccurate and / or imprecise.

[0357] Measurements in serum after acid treatment (to dissociate protein-protein interactions) and heating (to denature most non-tau proteins) reduced all pT3xpT82 and hT7xpT82 signals to near or below the LOQ( Reference ). Similarly, the pT3xpT82 assay detected slightly more signal in AD samples, but all detected signals were near the LOQ, so the assay may be inaccurate and / or imprecise.

[0358] Measurements in serum after pT3-IP and denaturation (to remove most interfering substances and concentrate p217+ tau) showed much higher levels in AD samples than in HV samples( ​ ). The p217+ levels were approximately 4-fold those of the p217+ levels in the crude or boiling measurements, so the HV samples are now at the LOQ and the AD samples are now all within the linear range.

[0359] These results suggest that the pT3-based assay described herein can be used as a blood-based measurement of pathological tau, especially when paired with an enrichment strategy such as IP.

[0360] Although the present invention has been described in detail in connection with its specific embodiments, it will be apparent to those of ordinary skill in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

[0361] ​

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[0367] Chothia et al., J.Mol.Biol. 227:799-817, 1992

[0368] Clavaguera et al., Nat Cell Biol. 11:909-13, 2009

[0369] D'Abramo et al., Neurobiol Aging. 37:58-65, 2016

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Claims

1. An isolated antibody or antigen-binding fragment thereof, comprising: (a) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:18; and (b) immunoglobulin light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region, which comprise the amino acid sequences of SEQ ID NO:15, 16, and 17, respectively; wherein the isolated antibody or antigen-binding fragment thereof binds to tau protein at an epitope comprising amino acid residues 7 to 20 of tau protein, the numbering of the amino acid residues being referenced to the amino acid sequence shown in SEQ ID NO:

1.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises heavy chain complementarity determining regions (HCDR) 2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13 and 14, respectively.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

19.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the LCDR1, LCDR2, and LCDR3 are each composed of the amino acid sequences of SEQ ID NO:15, 16, and 17, respectively.

5. A kit, comprising: (a) a composition comprising a capture antibody, the capture antibody comprising (i) immunoglobulin heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3, which comprise the amino acid sequences of SEQ ID NO:32, 33, and 34, respectively, and (ii) immunoglobulin light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, which comprise the amino acid sequences of SEQ ID NO:35, 36, and 37, respectively; and (b) a composition comprising a detection antibody, the detection antibody comprising (i) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO:18; and (ii) immunoglobulin LCDR1, LCDR2, and LCDR3 of the light chain variable region, which comprise the amino acid sequences of SEQ ID NO:15, 16, and 17, respectively, wherein the detection antibody binds to tau protein at an epitope comprising amino acid residues 7 to 20 of tau protein, the numbering of the amino acid residues being referenced to the amino acid sequence shown in SEQ ID NO:

1.

6. The kit according to claim 5, wherein the capture antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:28 and a light chain variable region having the amino acid sequence of SEQ ID NO:

29.

7. The kit according to claim 5 or 6, wherein the heavy chain variable region of the detection antibody comprises HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO:13 and 14, respectively.

8. The kit according to any one of claims 5-7, wherein the light chain variable region of the detection antibody comprises an amino acid sequence having 95% identity with SEQ ID NO:

19.

9. Use of a capture antibody against the p217+tau epitope and a detection antibody against the epitope comprising amino acid residues 7 to 20 of the tau protein in the manufacture of a kit for a method of measuring the p217+tau peptide in a sample, the method comprising: (a) contacting the sample with the capture antibody to capture the p217+tau peptide in the sample, and (b) contacting the captured p217+tau peptide with the detection antibody, thereby measuring the amount of the p217+tau peptide, wherein the capture antibody comprises (i) immunoglobulin heavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO: 32, 33 and 34, respectively, and (ii) immunoglobulin light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NO: 35, 36 and 37, respectively; wherein the detection antibody comprises (i) an immunoglobulin heavy chain variable region which comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 18; and (ii) immunoglobulin LCDR1, LCDR2 and LCDR3 of the light chain variable region, which comprise the amino acid sequences of SEQ ID NO: 15, 16 and 17, respectively, and wherein the numbering of the amino acids refers to the amino acid sequence shown in SEQ ID NO:

1.

10. The use according to claim 9, wherein the capture antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

29.

11. The use according to claim 9 or 10, wherein the heavy chain variable region of the detection antibody comprises HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO: 13 and 14, respectively.

12. The use according to any one of claims 9-11, wherein the light chain variable region of the detection antibody comprises an amino acid sequence having 95% identity with SEQ ID NO:

19.

13. The use according to any one of claims 9-12, wherein the sample is a biological sample from a human subject.

14. The use according to claim 13, wherein the biological sample is selected from blood, brain homogenate or cerebrospinal fluid (CSF) from the subject.

15. The use according to claim 13 or 14, wherein the biological sample has been fractionated using reverse phase high performance liquid chromatography (rpHPLC).

16. Use according to claim 13 or 14, wherein the method further comprises determining whether the subject has a tauopathy or is at risk of developing a tauopathy, which comprises comparing the amount of p217+ tau peptide from the subject with a corresponding baseline value, wherein an increase in the amount as compared to the baseline indicates that the subject has a tauopathy or is at risk of having a tauopathy.

17. Use according to claim 13 or 14, wherein the method further comprises determining the effectiveness of treatment of a tauopathy in the subject, which comprises comparing the amount of p217+ tau peptide from the subject with a corresponding baseline value, wherein a decrease in the amount as compared to the baseline indicates that the treatment is effective.

18. Use according to claim 16 or 17, wherein the tauopathy is selected from familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam type motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (punch-drunk disease).

19. Use according to claim 18, wherein the tauopathy is Alzheimer's disease.

20. Use of at least one of a capture antibody against a p217+ tau epitope and a first detection antibody against an epitope comprising amino acid residues 119 to 126 of tau protein and a second detection antibody against an epitope comprising amino acid residues 7 to 20 of tau protein in the manufacture of a kit for a method of measuring p217+ tau peptide in a sample, the method comprising: (a) contacting the sample with the capture antibody to capture the p217+ tau peptide in the sample, and (b) contacting the captured p217+ tau peptide with at least one of the first detection antibody and the second detection antibody, thereby measuring at least one of the amount of the p217+ tau peptide and the amount of the long p217+ tau peptide, wherein the capture antibody comprises immunoglobulin heavy chain complementarity determining regions (HCDRs) 1, 2, and 3 respectively containing the amino acid sequences of SEQ ID NOs: 32, 33, and 34 and immunoglobulin light chain complementarity determining regions (LCDRs) 1, 2, and 3 respectively containing the amino acid sequences of SEQ ID NOs: 35, 36, and 37; Wherein the first detection antibody comprises immunoglobulin HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 2, 3, and 4, respectively, and immunoglobulin LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 5, 6, and 7, respectively, and wherein the second detection antibody comprises an immunoglobulin heavy chain variable region containing an amino acid sequence having at least 95% identity with SEQ ID NO: 18 and immunoglobulin LCDR1, LCDR2, and LCDR3 of the light chain variable region containing the amino acid sequences of SEQ ID NO: 15, 16, and 17, respectively, and wherein the numbering of the amino acid residues is referenced to the amino acid sequence shown in SEQ ID NO:

1.

21. The use according to claim 20, wherein the capture antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

29.

22. The use according to claim 20 or 21, wherein the heavy chain variable region of the second detection antibody comprises HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NO: 13 and 14, respectively.

23. The use according to any one of claims 20-22, wherein the light chain variable region of the second detection antibody comprises an amino acid sequence having 95% identity with SEQ ID NO:

19.

24. The use according to any one of claims 20-23, wherein the obtained sample is a biological sample from a human subject.

25. The use according to claim 24, wherein the biological sample is selected from blood, brain homogenate, or cerebrospinal fluid (CSF) from the subject.

26. The use according to claim 24 or 25, wherein the biological sample has been fractionated using reverse-phase high-performance liquid chromatography (rpHPLC).

27. The use according to any one of claims 20-26, wherein the method further comprises contacting the captured p217+ tau peptide with the first detection antibody and the second detection antibody, thereby measuring the amount of the p217+ tau peptide and the amount of the long p217+ tau peptide, respectively.

28. The use according to claim 27, wherein the method further comprises determining the ratio of the amount of the long p217+ tau peptide to the amount of the p217+ tau peptide.

29. The use according to claim 27, wherein the method further comprises determining the amount of the short p217+ tau peptide by subtracting the amount of the long p217+ tau peptide from the amount of the p217+ tau peptide.

30. The use according to claim 29, wherein the method further comprises determining the ratio of the amount of the short p217+ tau peptide to the amount of the p217+ tau peptide, or the ratio of the amount of the long p217+ tau peptide to the amount of the short p217+ tau peptide.

31. Use according to claim 29, wherein the method further comprises determining whether the subject has a tauopathy or is at risk of developing a tauopathy, wherein the determination comprises at least one of the following from the subject: a. the amount of the p217+tau peptide, b. the amount of the long p217+tau peptide, c. the amount of the short p217+tau peptide, and d. its ratio is compared with a corresponding baseline value, wherein an increase in the amount of the p217+tau peptide, the amount of the long p217+tau peptide, the amount of the short p217+tau peptide, or the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide as compared to the baseline indicates that the subject has a tauopathy or is at risk of developing a tauopathy.

32. Use according to claim 29, wherein the method further comprises determining the effectiveness of treatment of a tauopathy in the subject, wherein the determination comprises at least one of the following from the subject: a. the amount of the p217+tau peptide, b. the amount of the long p217+tau peptide, c. the amount of the short p217+tau peptide, and d. its ratio, is compared with a corresponding baseline value, wherein a decrease in the amount of the p217+tau peptide or the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide as compared to the baseline indicates that the treatment is effective.

33. Use according to claim 29, wherein the method further comprises determining whether the subject is suitable for treatment with an anti-p217+tau antibody, wherein the determination comprises at least one of the following from the subject: a. the amount of the p217+tau peptide, b. the amount of the long p217+tau peptide, c. the amount of the short p217+tau peptide obtained, and d. the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide, is compared with a corresponding baseline value, wherein an increase in the amount of the p217+tau peptide or the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide as compared to the baseline indicates that the subject is suitable for treatment with an anti-p217+tau antibody.

34. Use according to claim 29, wherein the method further comprises monitoring treatment with an anti-p217+tau antibody, wherein the monitoring comprises at least one of the following from the subject: a. the amount of the p217+tau peptide, b. the amount of the long p217+tau peptide, c. the amount of the short p217+tau peptide, and d. its ratio, is compared with a corresponding baseline value, wherein a decrease in the amount of the p217+tau peptide or the ratio of the amount of the short p217+tau peptide to the amount of the long p217+tau peptide as compared to the baseline indicates a positive response to the treatment.

35. Use according to claim 31 or 32, wherein the tau proteinopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcification, argentophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam type motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and pugilistic dementia (punch drunk disease).

36. Use according to claim 35, wherein the tau proteinopathy is Alzheimer's disease.

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