Assay for detecting neurodegeneration

Abstract

To provide a method for measuring an amount of singly or multiply phosphorylated p217+tau protein in a sample, regarding compositions and methods for detecting neurodegeneration.SOLUTION: A method for measuring a p217+ tau peptide in a sample, comprises: (i) contacting the sample with a capture antibody against a p217+ tau epitope to capture the p217+ tau peptide in the sample; and (ii) contacting the captured p217+ tau peptide with at least one of a first detection antibody against an epitope comprising amino acid residues 119 to 126 of a tau protein and a second detection antibody against an epitope comprising amino acid residues 7 to 20 of the tau protein, and measuring at least one of an amount of the p217+ tau peptide and an amount of a long p217+ tau peptide, where amino acid numbering refers to a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for detecting neurodegeneration. METHOD FOR MEASURING THE AMOUNT OF SINGLY OR MULTIPLELY PHOSPHORYLATED p217+ TAU PROTEIN SPECIES IN BIOLOGICAL SAMPLES - Patent application The present invention relates to a method, its use, and antibodies and kits for use in the method. [Background technology]

[0002] Alzheimer's disease (AD) leads to gradual and severe mental decline and ultimately death. Clinically characterized by progressive loss of memory, cognition, reasoning, judgment, and emotional stability, including AD is a degenerative brain disorder that causes progressive mental dysfunction (dementia) in the elderly. AD is a common cause of death and is thought to represent the fourth leading medical cause of death in the United States. It is observed in people all over the world and represents a major current and future public health problem.

[0003] The brains of individuals with AD are characterized by senile (or amyloid) plaques, amyloid angiopathy (amyloid in blood vessels), and These lesions are characterized by characteristic lesions called neurofibromatosis (neurofibrillary tangles) and neurofibromatosis (neurofibrillary tangles). Amyloid plaques and paired helical filament neurofibrillary tangles, in particular, are commonly associated with memory and memory impairment in AD patients. It is found in several areas of the human brain that are important for cognitive function.

[0004] Neurofibrillary tangles are primarily composed of aggregates of hyperphosphorylated tau protein. The physiological function of tau is the polymerization and stabilization of microtubules. It is generated by ionic interactions between positive charges in the tube-binding region and negative charges in the microtubule lattice. (Butner and Kirschner, J Cell Biol. 115 (3):717-30, 1991). Tau protein has 85 potential phosphorylation sites. It contains tau-like proteins, and phosphorylation at many of these sites interferes with the primary function of tau. Tau bound to the tubular lattice is hypophosphorylated, whereas aggregated tau in AD is hyperphosphorylated. It is phosphorylated and has a unique epitope that is distinct from the physiologically active pool of tau. (Iqbal et al., Curr Alzheimer Res.7(8): 656-664,2010).

[0005] Tauopathy progression in AD brains follows a distinct pattern of expansion. Braak stages of tauopathy progression and after injection of tau aggregates in preclinical tau models Based on the spread of tauopathy in the Frost et al.,J Biol Chem.284:12845-52,20 09;Clavaguera et al., Nat Cell Biol.11:90 9-13, 2009). Tauopathies spread from one brain region to the next, like prions. This expansion process involves uptake by nearby neurons. This is accompanied by externalization of tau seeds that can lead to further tauopathy.

[0006] Fragments of tau protein in neurofibrillary tangles migrate into the cerebrospinal fluid (CSF), where they It can be obtained by lumbar puncture and measured by a highly sensitive assay. In order to identify neurological diseases, an assay that recognizes fragments derived from tau protein in CSF has been used. Such tau assays can detect the presence of tau, which is characteristic of neurodegenerative conditions. Multiphosphorylated tau is the major form of AD-associated tau protein. Therefore, an assay for detecting multiply phosphorylated tau protein in CSF is may be most effective in detecting the presence of AD.

[0007] Phosphorylation is not the only post-translational modification considered in measuring tau. In CSF, tau protein is mainly present as fragments rather than as full-length protein. It has been demonstrated that the ATPase activity is present in the ATP-dependent ATPase domain (Meredith et al. PLoS One. 8(10):e76523,2013). Furthermore, in pathological conditions, protein degradation is abnormal. Tau fragmentation patterns can be affected by disease, as they are often associated with Tau-based assays for degradation measure not only phosphorylation state (e.g., phosphorylation sites) but also It also provides information about the properties of the tau fragments measured (e.g., length, polarity of the tau fragments). However, this conceptual translation is particularly difficult to achieve from healthy subjects. This is precluded by the low endogenous levels of phosphorylated tau in previous samples. Summary of the Invention

[0008] In summary, we present a highly sensitive, accurate, and cost-effective method for detecting multiphosphorylated tau in biological fluids. There remains a need for a novel and accurate method for the treatment of AD and other tauopathies. To effectively detect, diagnose, stage, and track disease progression in neurodegenerative diseases such as Qi. The method may also be useful for determining the total, free, and multiple phosphates bound to therapeutic antibodies. It may also be useful as a pharmacodynamic marker for measuring the levels of multiphosphorylated tau. The ability to detect and measure tau fragments is important because propagating tau species can be one or more tau fragments. , which is of further importance to the field.

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

[0010] A highly sensitive enzyme-linked immunosorbent assay (ELISA) according to an embodiment of the present invention was developed. )R(pT)PSLPTPPTR (SEQ ID NO: 25), (217)RTPSLP(pT)P PTR (SEQ ID NO: 26), or (212 and 217)R(pT)PSLP(pT)PPT R (SEQ ID NO: 27) p217+ tau containing oxidized tau epitopes ("p217+ tau epitopes" or "pT3 The measurement of the epitope was certified.

[0011] Assays according to embodiments of the present invention can be performed on CSF, interstitial fluid (ISF), brain homogenate, blood Various fluid matrices, including but not limited to serum, plasma, and denatured or enriched forms thereof, The p217+ tau species in the sample can be measured. B) a first monoclonal antibody against the pT3 epitope of tau as a capture antibody; and A second monoclonal antibody directed against a second epitope of tau is used as the detection antibody. The assay is sensitive, precise, accurate, transferable between laboratories, linear in dilution, and compatible with many samples. In addition to measuring p217+ tau species in raw biological fluids, The assay can be used to measure denatured or non-denatured samples. or after immunoprecipitation, free p217+ tau or endogenous or therapeutically administered We used two complementary techniques to quantify the amount of p217+ tau bound to the antibody. The assay can be performed in parallel with reversed-phase high performance liquid chromatography (rpHPLC). The fragment profile of p217+ tau was determined by measuring fractionated CSF using This allows for analysis of the file.

[0012] In one general aspect, the invention provides a method for measuring the amount of p217+ tau peptide in a sample. The method comprises: (i) subjecting a sample to a capture antibody directed against a p217+ tau epitope; (ii) contacting the sample with a capture agent to capture p217+tau peptides in the sample; The captured p217+ tau peptide was isolated from amino acid residues 119-126 of the tau protein, e.g., For example, an epitope comprising amino acid residues 116 to 127, or amino acid residues of tau protein. 7 to 20, respectively, to detect the p217+ antigen. and measuring the amount of long p217+ tau peptide or the amount of long p217+ tau peptide, Amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO:1.

[0013] In one specific aspect, the present invention provides a method for detecting long p217+ tau peptides or short p217+ tau peptides in a sample. The present invention relates to a method for determining the relative amount of a p217 tau peptide fragment in a sample. is contacted with a capture antibody against the p217+ tau epitope to detect p21 in the sample. (ii) capturing the captured p217+ tau peptides; and (iii) eluting the captured p217+ tau peptides with tau. a first detection antibody against an epitope comprising amino acid residues 119 to 126 of the protein; (iii) contacting the captured p2 The 17+ tau peptide is directed against an epitope containing amino acid residues 7 to 20 of the tau protein. measuring the amount of long p217+ tau peptide by contacting the sample with a second detection antibody comprising the antibody; (iv) the amount of the p217+ tau peptide and the amount of the long p217+ tau peptide The relative amounts of long p217+ tau peptides or short p217+ tau peptides were determined based on and determining the amino acids, wherein the numbering of the amino acids refers to the amino acid sequence set forth in SEQ ID NO: 1. do.

[0014] In one embodiment of the present invention, the amount of short p217+ tau peptides in a sample can be determined by, for example, For example, subtract the amount of long p217+ tau peptide from the amount of p217+ tau peptide. The amount of p217+ tau peptides and long p217+ tau peptides in the sample can be determined by In another embodiment, the amount of short p217+ tau peptide is calculated based on the amount of The ratio of the amount of long p217+ tau peptide to the amount of p217+ tau peptide The ratio of the amount of long p217+ tau peptide to the amount of short p217+ tau peptide was The ratio of the amount of p217+ tau peptide to the amount of long p217 tau peptide in the sample was calculated. + tau peptides in the sample. The amount of 217+ tau peptide and / or the amount of long p217+ tau peptide, and and one or more of the above ratios. Information based on the determined amount can be used for one or more diagnostic purposes.

[0015] Thus, in one specific aspect, the present invention provides a method for detecting p217+ tau peptides in a sample. The present invention relates to a method for determining the ratio of tau peptides to total tau peptides, the method comprising: (i) separating a sample into p217 + tau epitope in the sample by contacting with a capture antibody against the p217 + tau epitope. The sample is then subjected to a tau protein analysis using an epitope of amino acids 150 to 250 of the tau protein. , preferably a phosphate group for an epitope comprising amino acids 159 to 163 of tau protein contacting the sample with a tyrosine-independent capture antibody to capture total tau peptides in the sample; (ii) (a) The captured p217+tau peptide is then ligated to amino acid residue 1 of the tau protein. contacting the p217+ taupe with a first detection antibody directed against an epitope comprising 19-126; and measuring the amount of total tau peptide captured, contacting the total captured tau peptide with the first detection antibody. (b) measuring the amount of tau peptide; and (b) determining the amount of captured p217+ tau peptide. and contacting the antibody with a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the protein. The amount of long p217+ tau peptide was measured, and the total captured tau peptide was calculated based on the corresponding and measuring the amount of total long tau peptides by contacting the sample with two detection antibodies, (iii) the amount of p217+ tau peptides in the total tau peptides The ratio of the amount of long p217+ tau peptide to the amount of total long tau peptide and determining a ratio of the amino acids to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the amount of short p217+ tau peptide is determined by the amount of p217+ tau peptide. The amount of long p217+ tau peptide was calculated by subtracting the amount of long p217+ tau peptide from the amount of long p217+ tau peptide. The amount of total short tau peptides is calculated by subtracting the amount of total long tau peptides from the amount of total tau peptides. The amount of short p217+ tau peptides was calculated by subtracting the total short tau peptides. The ratio is determined relative to the amount of peptide.

[0016] According to a particular embodiment, the method of the present invention comprises: (i) a biological sample from a subject, preferably The CSF sample is contacted with a capture antibody against the p217+ tau epitope to obtain the sample. (ii) capturing p217+ tau peptides in the pool; and (iii) (a) capturing the captured p217. + Tau peptide is targeted to an epitope containing amino acid residues 119-126 of the tau protein. measuring the amount of p217+ tau peptide; and b) The captured p217+tau peptide was analyzed by ELISA using a tau peptide containing amino acid residues 7-20 of the tau protein. The amount of long p217+ tau peptide is measured by contacting the antibody with a second detection antibody directed against an epitope containing the long p217+ tau peptide. (iii) measuring p217+ tau The amount of peptide, the amount of long p217+ tau peptide, and the amount of long p217+ tau peptide The amount of short p217+ tau peptide obtained by subtracting the amount of long p217+ tau peptide and determining whether the subject has tauopoietin, based on at least one of the amounts of tauopoietin and tauopoietin, and the ratio thereof. Determine if you have or are at risk for developing a tauopathy and wherein the amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the method comprises administering to a subject a therapeutic agent to treat or prevent a tauopathy. providing the

[0017] According to a particular embodiment, the method of the present invention comprises: (i) a biological sample from a subject, preferably The CSF sample is contacted with a capture antibody against the p217+ tau epitope to obtain the sample. The p217+ tau peptide in the sample was captured and the sample was analyzed by the amino acid sequence of the tau protein. An epitope of 150 to 250, preferably containing amino acids 159 to 163 of tau protein. The total tau protein in the sample is then contacted with a phosphorylation-independent capture antibody directed against an epitope containing the tau protein. (ii) (a) capturing the captured p217+tau peptides; and (ii) (a) transferring the captured p217+tau peptides to a tau antibody. A first detection antibody against an epitope containing amino acid residues 119 to 126 of the protein is then conjugated. The amount of p217+ tau peptide was measured, and the total captured tau peptide was calculated by the first (b) contacting the captured p with a detection antibody to measure the amount of total tau peptide; and 217+ tau peptides were targeted to epitopes containing amino acid residues 7 to 20 of the tau protein. The amount of captured long p217+ tau peptide is measured by contacting the antibody with a second detection antibody. The total tau peptides thus obtained are contacted with the second detection antibody to measure the amount of total long tau peptides. (iii) performing at least one of (a) determining whether a p217+ tag is present; (b) ratio of the amount of long p217+ tau peptide to the amount of total tau peptide; (c) the ratio of the amount of short p217+ tau peptide to the amount of total long tau peptide the ratio of the amount of short tau peptide to the amount of total short tau peptide. whether the subject is suffering from or at risk of developing a tauopathy and determining whether the amount of the short p217+ tau peptide is 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 peptides is calculated by subtracting the amount of the total tau peptides from the amount of the total short tau peptides. The amino acid numbering is obtained by subtracting the amount of total short tau peptides. See the amino acid sequence set forth in column number 1. In one embodiment, the method comprises treating a tauopathy. The method further includes administering to the subject a therapeutic agent for the treatment or prevention of the disease.

[0018] According to another specific embodiment, the method of the present invention comprises: (i) analyzing a biological sample from a subject undergoing treatment; Preferably, the CSF sample is contacted with a capture antibody directed against the p217+ tau epitope. (ii) capturing p217+ tau peptides in the sample; and (ii) (a) capturing the captured p217+ tau peptides. The p217+tau peptide was then purified by ELISA using an enzyme containing amino acid residues 119-126 of the tau protein. and measuring the amount of p217+ tau peptide by contacting the sample with a first detection antibody directed against the target peptide. and (b) isolating the captured p217+tau peptide by the amino acid residues of the tau protein. and contacting the long p217+ tau with a second detection antibody directed against an epitope comprising 7-20. (iii) measuring the amount of the peptide; and The amount of 217+ tau peptide, the amount of long p217+ tau peptide, The amount of the long p217+ tau peptide is obtained by subtracting the amount of the long p217+ tau peptide from the amount of the long p217+ tau peptide. and the amount of short p217+ tau peptide, and at least one of the ratios thereof. and determining the effectiveness of said treatment in said subject, refers to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the method comprises: The method further includes administering to the subject a therapeutic agent to treat or prevent the disease.

[0019] According to another specific embodiment, the method of the present invention comprises: (i) analyzing a biological sample from a subject undergoing treatment; Preferably, the CSF sample is contacted with a capture antibody directed against the p217+ tau epitope. The sample is then purified by HPLC to capture p217+ tau peptides in the sample, and the sample is purified by HPLC to capture p217+ tau peptides in the sample. an epitope between amino acids 150 and 250 of a protein, preferably amino acid 159 of tau protein The sample is contacted with a phosphorylation-independent capture antibody against an epitope containing 163. (ii) (a) capturing the total tau peptides in the sample; and (ii) (a) capturing the captured p217+ tau peptides. a first antibody directed against an epitope comprising amino acid residues 119 to 126 of tau protein; The amount of p217+ tau peptide is measured by contacting it with a detection antibody, and the total tau peptide captured is contacting the antibody with the first detection antibody to measure the amount of total tau peptide; and (b) The captured p217+tau peptide was then purified by ELISA using an enzyme containing amino acid residues 7–20 of the tau protein. The amount of long p217+ tau peptide is measured by contacting the cells with a second detection antibody directed against the target peptide. The captured total tau peptides are contacted with the second detection antibody to detect total long tau peptides. (iii)(a) measuring the amount of thiamin in the blood; and (iii)(a) measuring the amount of thiamin in the blood. (b) ratio of the amount of p217+ tau peptide to the amount of total tau peptide; (b) long p217+ (c) ratio of the amount of tau peptide to the amount of total long tau peptide, and (c) short p217 + at least one of the ratios of the amount of tau peptide to the amount of total short tau peptide and determining the effectiveness of the treatment in the subject based on the short The amount of p217+tau peptide is calculated by subtracting the amount of p217+tau peptide from the amount of long p21 The amount of total short tau peptides is obtained by subtracting the amount of 7+ tau peptides. is calculated by subtracting the amount of the total short tau peptides from the amount of the total tau peptides. The amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the method includes administering to a subject a therapeutic agent for treating or preventing a tauopathy. , further includes.

[0020] According to another specific embodiment, the method of the present invention comprises: (i) a biological sample from a subject, preferably or CSF sample is contacted with a capture antibody against the p217+ tau epitope, (ii) capturing p217+ tau peptides in the sample; and (iii) (a) capturing the captured p2 17+ tau peptide, an epitope containing amino acid residues 119-126 of the tau protein measuring the amount of p217+ tau peptide; and and (b) the captured p217+tau peptide was transferred to amino acid residues 7–20 of the tau protein. and contacting the long p217+ tau peptide with a second detection antibody directed against an epitope comprising (iii) measuring the amount of said p21 the amount of the long p217+ tau peptide, the amount of the long p217+ tau peptide, The amount of the long p217+ tau peptide is obtained by subtracting the amount of the long p217+ tau peptide from the amount of the long p217+ tau peptide. and the amount of short p217+ tau peptide, and at least one of the ratios thereof. and determining whether the subject is suitable for an anti-p217+ tau antibody; The amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, The method comprises administering to a subject an anti-p217+ tau antibody to treat or prevent a tauopathy. It further includes:

[0021] According to another specific embodiment, the method of the present invention comprises: (i) a biological sample from a subject, preferably or CSF sample is contacted with a capture antibody against the p217+ tau epitope, The p217+ tau peptide in the sample is captured and the sample is then purified by cleavage of the tau protein. an epitope of amino acids 150 to 250, preferably amino acids 159 to 163 of tau protein and contacting the sample with a phosphorylation-independent capture antibody against an epitope containing (ii) (a) capturing the captured p217+tau peptide; a first detection antibody against an epitope comprising amino acid residues 119 to 126 of tau protein; and measuring the amount of p217+ tau peptide, and the total captured tau peptide is (b) contacting the captured tau peptide with a first detection antibody to measure the amount of total tau peptide; and The p217+tau peptide is an epitope containing amino acid residues 7 to 20 of the tau protein. The amount of long p217+ tau peptide is measured by contacting the sample with a second detection antibody against the captured peptide. The captured total tau peptides are contacted with the second detection antibody to determine the amount of total long tau peptides. (iii) (a) measuring the p2 (b) the ratio of the amount of 17+ tau peptide to the amount of the total tau peptide; (c) the ratio of the amount of 7+ tau peptides to the amount of the total long tau peptides; and At least the ratio of the amount of p217+ tau peptide to the amount of total short tau peptide and determining whether the subject is suitable for anti-p217+ tau antibody therapy based on one or more of the above. and measuring the amount of the short p217+ tau peptide in the blood. the amount of the long p217+ tau peptide is obtained by subtracting the amount of the long p217+ tau peptide from the amount of the long p217+ tau peptide, The amount of the total short tau peptides is calculated by subtracting the amount of the total tau peptides from the amount of the total short tau peptides. The amino acid numbering is given by the number given in SEQ ID NO: 1. In one embodiment, the method comprises administering to a subject a subject having a tauopathy, the subject having a tauopathy, ... or a tauopathy-associated tauopathy. The method further comprises administering to the subject an anti-p217+ tau antibody for the treatment of rheumatoid arthritis.

[0022] In another specific embodiment, the present invention provides a method for monitoring treatment of a subject with an anti-p217+ tau antibody. 1. A method of monitoring a subject, comprising: (i) obtaining a biological sample from the subject; and (ii) The biological sample is subjected to p217 tau antibody assay, preferably IgG-derived, which does not contain anti-p217+ tau antibodies. a first sample containing tau and p217 bound to an anti-p217 tau antibody; (iii) separating the tau peptides into a second sample containing the tau peptides, preferably at rpH and extracting p217+ tau antibodies free from the second sample via PLC. (iv) obtaining a third sample containing the compound (c); and (iv) combining the first sample and the third sample. Each of the samples is contacted with a capture antibody against the p217+ tau epitope to identify the (v) capturing p217+tau peptides in each of the samples; and The captured p217+tau peptide was isolated from the tau protein containing amino acid residues 119-126. and contacting each of the samples with a first detection antibody directed to an epitope containing the (b) measuring the amount of captured p217+ tau peptide; and a second detection antibody directed against an epitope comprising amino acid residues 7 to 20 of the tau protein. and measuring the amount of long p217+ tau peptide in each of the samples. (vi) determining the identity of each of the samples; the amount of the p217+ tau peptide and the amount of the long p217+ tau peptide and monitoring treatment with the anti-p217+ tau antibody based on at least one of the following: and numbering the amino acids with reference to the amino acid sequence set forth in SEQ ID NO: 1. For example, treatment with an anti-p217+ tau antibody may be Amount of long p217+ tau peptide in the third sample the ratio of the amount of p217+ tau peptide in the first sample to the amount of p217+ tau peptide in the third sample the ratio of the amount of p217+ tau peptide to the amount of short p217+ tau peptide in the first sample The amount of p217+tau peptide (the amount of long p217+tau peptide) (This can be calculated by subtracting the short p217+ In one embodiment, the amount of tau peptide can be monitored based on the ratio of the amount of tau peptide to the amount of tau peptide. The method includes administering to a subject an anti-p217+ tau antibody to treat or prevent a tauopathy. The present invention further includes:

[0023] In another general aspect, the invention provides a method for monitoring treatment with an anti-p217+ tau antibody in a subject. 1. A method of monitoring a subject, comprising: (i) obtaining a biological sample from the subject; and (ii) obtaining a semi-denatured sample from a biological sample containing total p217+ tau, wherein the semi-denatured sample is heated to denature the antibody in the sample; and Non-denatured samples were obtained from biological samples containing p217+ tau without anti-p217+ tau antibodies. (iii) obtaining a sample of the semi-denatured sample and the non-denatured sample; , and contacting each of the samples with a capture antibody against the p217+ tau epitope. (v) capturing the p217+ tau peptide in (a) the captured p217+ tau peptide; The peptide is directed against an epitope containing amino acid residues 119 to 126 of the tau protein. contacting the first detection antibody with the p217+tau peptide in each of the samples; and (b) measuring the amount of captured p217+ tau peptides. and contacting the sample with a second detection antibody against an epitope containing amino acid residues 7 to 20 of the sample. measuring the amount of long p217+ tau peptide in each of the samples, vi) performing at least one of the p217 + at least one of the amount of tau peptide and the amount of the long p217+ tau peptide and monitoring treatment with the anti-p217+ tau antibody based on the results of The amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO: 1. For example, treatment with anti-p217+ tau antibodies reduces the number of long p217+ tau proteins in semi-denatured samples. Ratio of the amount of long p217+ tau peptide in the non-denatured sample to the amount of long p217+ tau peptide in the semi-denatured sample Amount of p217+ tau peptide in sample p217+ tau peptide in non-denatured sample or the amount of short p217+tau peptide in the semi-denatured sample (p21 Calculated by subtracting the amount of long p217+ tau peptide from the amount of 7+ tau peptide. (which can be calculated) relative to the amount of short p217+ tau peptide in the non-denatured sample. In one embodiment, the method can be used to monitor tauopathy based on a ratio of The method further comprises administering to the subject an anti-p217+ tau antibody for treating or preventing the disease.

[0024] According to a specific embodiment, the tauopathy is Alzheimer's disease (familial Alzheimer's disease). Alzheimer's disease and sporadic Alzheimer's disease), linked to chromosome 17 and causing parkinsonism Frontotemporal dementia with progressive supranuclear palsy (FTDP-17), corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia ), diffuse neurofibrillary tangle disease with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinson-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute Myotonic dystrophy, non-Guam motor neuron syndrome with neurofibrillary tangles Parkinson's disease, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (pugilistica) ) are not limited to.

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

[0026] Most preferably, the tauopathy is Alzheimer's disease (familial Alzheimer's disease and diffuse alveolar leukemia). (including onset Alzheimer's disease).

[0027] According to a specific embodiment, the lower limit of quantitation of the method of the present invention is approximately 40 fg / mL of p217+ tag. The lower limit of detection of the method of the present invention is about 2 fg / mL of p217+ tau peptide. It is Do.

[0028] According to a specific embodiment, the sample may be blood, brain homogenate, or the like from a subject in need thereof. Preferably, the sample is a biological sample such as a cerebrospinal fluid (CSF) sample. The sample may be used to diagnose a tauopathy, monitor the effectiveness of a tauopathy treatment, or to detect an anti-p A CSF sample from a subject in need of determining suitability for 217+ tau antibody therapy. is.

[0029] According to a specific embodiment, the capture antibody useful in the method of the present invention is a capture antibody that binds to the p217+ tau epitope. , preferably p217+tau containing the amino acid sequence of SEQ ID NO: 25, 26, or 27. In one embodiment, the capture antibodies useful in the methods of the invention are each directed against an epitope of the sequence Immunoglobulin heavy chain HCDR1 having the polypeptide sequences of numbers 32, 33, and 34; HCDR2 and HCDR3, and the polypeptides of SEQ ID NOs: 35, 36, and 37, respectively The immunoglobulin light chain comprises LCDR1, LCDR2, and LCDR3 having the sequence LCDR1, LCDR2, and LCDR3. Preferably, the capture antibody comprises 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 embodiment, the detection antibody useful in the method of the present invention is An epitope comprising residues 119 to 126, preferably a sequence such as the amino acid sequence of SEQ ID NO: 11. In one embodiment, the method of the present invention is directed to an epitope comprising the amino acid sequence of SEQ ID NO: 10. Useful detection antibodies include immunoglobulins having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively. Ig heavy chain HCDR1, HCDR2, and HCDR3, and SEQ ID NOs: 5 and 6, respectively and immunoglobulin light chain LCDR1, LCDR2, and Preferably, the detection antibody comprises a polypeptide having the polypeptide sequence of SEQ ID NO: 8. pT82 antibody comprising a light chain variable region and a light chain variable region having the polypeptide sequence of SEQ ID NO:9 is.

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

[0032] In another specific embodiment, the phosphorylation-independent capture antibody useful in the present invention is a capture antibody for tau protein. an epitope of amino acids 150 to 250 of a tau protein, preferably amino acids 2 to 3 of a tau protein; an epitope comprising amino acids 11 to 221 of the tau protein, or an epitope comprising amino acids 159 to 163 of the tau protein; More preferably, the epitope has the amino acid sequence of SEQ ID NO: 21. In embodiments, the phosphorylation-independent capture antibody useful in the present invention is an hT7 antibody.

[0033] According to another specific embodiment, the sample used in the method of the present invention is purified by reversed-phase high performance liquid chromatography. It is obtained after fractionation of a biological sample using rpHPLC.

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

[0035] In another general aspect, the invention provides: (a) a capture antibody against the p217+ tau epitope; and (b) a tau protein containing amino acid residues 7 to 20 or 116 to 127 of the tau protein. and a detection antibody against the protein epitope. Optionally, the kit comprises: Phosphorylation-independent capture of the tau epitope between amino acids 150 and 250 of the tau protein The kit further comprises a capture antibody. The kit can be used to measure, for example, the amount of p217+ tau peptide in a sample, Amount of short p217+tau peptide, amount of short p217+tau peptide, amount of short p21 Ratio of the amount of 7+ tau peptide to the amount of long p217+ tau peptide, short p21 It is used to measure the ratio of the amount of 7+ tau peptide to the amount of total short tau peptide. The kits can also be used for various diagnostic or monitoring purposes, e.g. Whether the subject has or is at risk of developing a tauopathy To determine whether or not treatment for tauopathy, such as anti-p217+ tau antibodies, is effective, The effectiveness of the treatment will be monitored to determine whether the subject is suitable for anti-p217+ tau antibodies. The purpose may be to determine the following.

[0036] According to a specific embodiment, the kit of the present invention comprises the nucleic acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having polypeptide sequences and immunoglobulins having the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively. The capture antibody comprises the phospho-light chains LCDR1, LCDR2, and LCDR3. The capture antibody comprises a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28 and a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 29. It has a light chain variable region having a polypeptide sequence.

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

[0038] According to another specific embodiment, the kit of the present invention comprises the nucleic acid sequences of SEQ ID NOs: 12, 13, and 14, respectively. Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR having the polypeptide sequence of R3, and immunoglobulins having the polypeptide sequences of SEQ ID NOs: 15, 16, and 17, respectively. The detection antibody comprises globulin light chain LCDR1, LCDR2, and LCDR3. The detection antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 The hT43 antibody comprises a light chain variable region having a polypeptide sequence of 9.

[0039] Other aspects, features, and advantages of the present invention are set forth in the detailed description of the invention, as well as in the preferred embodiments thereof. These and other aspects of the present invention will become apparent from the following disclosure, including the embodiments and appended claims. [Brief explanation of the drawings]

[0040] The above Summary of the Invention and the following Detailed Description of the Invention should be read in conjunction with the accompanying drawings. The invention will be better understood by reading the accompanying drawings. It should be understood that the present invention is not limited to the above. [Figure 1] Representative standard curves for the pT3xhT43 and pT3xpT82 assays generated using calibration peptides are shown, with each point representing the mean ± SD of duplicate measurements. [Figure 2A] Dilution linearity of the pT3xhT43 and pT3xpT82 assays in CSF samples with measurements shown in (A, C, and E) dilution-corrected pg / mL or (B and D) as dilution-corrected % of the 1:4 measurement, with dashed lines indicating ±20% of the 1:4 measurement. [Figure 2B]Dilution linearity of the pT3xhT43 and pT3xpT82 assays in CSF samples with measurements shown in (A, C, and E) dilution-corrected pg / mL or (B and D) as dilution-corrected % of the 1:4 measurement, with dashed lines indicating ±20% of the 1:4 measurement. [Figure 2C] Dilution linearity of the pT3xhT43 and pT3xpT82 assays in CSF samples with measurements shown in (A, C, and E) dilution-corrected pg / mL or (B and D) as dilution-corrected % of the 1:4 measurement, with dashed lines indicating ±20% of the 1:4 measurement. [Figure 2D] Dilution linearity of the pT3xhT43 and pT3xpT82 assays in CSF samples with measurements shown in (A, C, and E) dilution-corrected pg / mL or (B and D) as dilution-corrected % of the 1:4 measurement, with dashed lines indicating ±20% of the 1:4 measurement. [Figure 2E] Dilution linearity of the pT3xhT43 and pT3xpT82 assays in CSF samples with measurements shown in (A, C, and E) dilution-corrected pg / mL or (B and D) as dilution-corrected % of the 1:4 measurement, with dashed lines indicating ±20% of the 1:4 measurement. [Figure 3] Intra- and inter-assay precision of the (A) pT3xhT43 and (B) pT3xpT82 assays is shown. [Figure 4A] Figure 1 shows inter-laboratory precision of the pT3xhT43 and pT3xpT82 assays with data graphed as signal / noise (S / N). [Figure 4B] Figure 1 shows inter-laboratory precision of the pT3xhT43 and pT3xpT82 assays with data graphed as signal / noise (S / N). [Figure 5A] 1 shows competition of the signal of a pT3-based assay by antibodies targeting soluble p217+ tau in (A) pT3xhT43 and (B) pT3xpT82 assays. [Figure 5B]1 shows competition of the signal of a pT3-based assay by antibodies targeting soluble p217+ tau in (A) pT3xhT43 and (B) pT3xpT82 assays. [Figure 6] 1 shows the phosphorylation dependence of the pT3xhT43 and pT3xpT82 assays. [Figure 7] 1 shows the p217+ tau fragment profile of AD CSF measured using the pT3xhT43 and pT3xpT82 assays, with data graphed as signal minus noise. [Figure 8A] Temperature and freeze-thaw stability of p217+ tau signals in AD CSF samples using (A) pT3xhT43 and (B) hT7xpT82 assays. [Figure 8B] Temperature and freeze-thaw stability of p217+ tau signals in AD CSF samples using (A) pT3xhT43 and (B) hT7xpT82 assays. [Figure 9] Figure 1 shows the long-term stability of p217+ tau signal in CSF samples after storage at -70°C. No change in signal was detected. [Figure 10A] Correlations between p217+ tau and classical AD biomarkers Aβ42, ttau (total tau), and ptau181 as measured by (A-C) pT3xhT43 and (D-F) pT3xpT82 assays are shown. [Figure 10B] Correlations between p217+ tau and classical AD biomarkers Aβ42, ttau (total tau), and ptau181 as measured by (A-C) pT3xhT43 and (D-F) pT3xpT82 assays are shown. [Figure 10C] Correlations between p217+ tau and classical AD biomarkers Aβ42, ttau (total tau), and ptau181 as measured by (A-C) pT3xhT43 and (D-F) pT3xpT82 assays are shown. [Figure 10D]Correlations between p217+ tau and classical AD biomarkers Aβ42, ttau (total tau), and ptau181 as measured by (A-C) pT3xhT43 and (D-F) pT3xpT82 assays are shown. [Figure 10E] Correlations between p217+ tau and classical AD biomarkers Aβ42, ttau (total tau), and ptau181 as measured by (A-C) pT3xhT43 and (D-F) pT3xpT82 assays are shown. [Figure 10F] Correlations between p217+ tau and classical AD biomarkers Aβ42, ttau (total tau), and ptau181 as measured by (A-C) pT3xhT43 and (D-F) pT3xpT82 assays are shown. [Figure 11A] Correlation between brain biopsy IHC analysis and p217+ tau as measured by (A) pT3xhT43 and (B) pT3xpT82 assays. [Figure 11B] Correlation between brain biopsy IHC analysis and p217+ tau as measured by (A) pT3xhT43 and (B) pT3xpT82 assays. [Figure 12A] 1 shows the results of analysis of crude CSF from AD and HV patients for (A) pT3xhT43, (B) pT3xpT82, (C) hT7xpT82, and (D) the ratio of pT3xpT82 to hT7xpT82. [Figure 12B] 1 shows the results of analysis of crude CSF from AD and HV patients for (A) pT3xhT43, (B) pT3xpT82, (C) hT7xpT82, and (D) the ratio of pT3xpT82 to hT7xpT82. [Figure 12C] 1 shows the results of analysis of crude CSF from AD and HV patients for (A) pT3xhT43, (B) pT3xpT82, (C) hT7xpT82, and (D) the ratio of pT3xpT82 to hT7xpT82. [Figure 12D] 1 shows the results of analysis of crude CSF from AD and HV patients for (A) pT3xhT43, (B) pT3xpT82, (C) hT7xpT82, and (D) the ratio of pT3xpT82 to hT7xpT82. [Figure 13] 1 shows the predictive power of the pT3xhT43 ("343"), pT3xpT82 ("382"), and hT7xpT82 ("782") assays in distinguishing between AD and HV subjects. [Figure 14A] Shown are signals from (A, B) pT3xhT43 ("343"), (C, D) pT3xpT82 ("382"), and (E, F) hT7xpT82 ("782") assays performed on rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects. [Figure 14B] Shown are signals from (A, B) pT3xhT43 ("343"), (C, D) pT3xpT82 ("382"), and (E, F) hT7xpT82 ("782") assays performed on rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects. [Figure 14C] Shown are signals from (A, B) pT3xhT43 ("343"), (C, D) pT3xpT82 ("382"), and (E, F) hT7xpT82 ("782") assays performed on rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects. [Figure 14D] Shown are signals from (A, B) pT3xhT43 ("343"), (C, D) pT3xpT82 ("382"), and (E, F) hT7xpT82 ("782") assays performed on rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects. [Figure 14E] Shown are signals from (A, B) pT3xhT43 ("343"), (C, D) pT3xpT82 ("382"), and (E, F) hT7xpT82 ("782") assays performed on rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects. [Figure 14F]Shown are signals from (A, B) pT3xhT43 ("343"), (C, D) pT3xpT82 ("382"), and (E, F) hT7xpT82 ("782") assays performed on rp-HPLC fractions of CSF from (A, C, E) AD subjects and (B, D, F) HV subjects. [Figure 15A] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15B] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15C] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15D] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15E] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15F] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15G] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15H] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15I] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15J] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15K] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15L] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15M] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15N] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 15O] Signals from (AE) pT3xhT43, (FJ) pT3xpT82, and (KO) hT7xpT82 assays performed on rp-HPLC fractions of CSF from CDR0 and CDR0.5 subjects are shown. [Figure 16A] Results are shown for (A) analysis of the ratio of pT3xpT82 to hT7xpT82 (pTau short) or the ratio of pT3xhT43 to hT7xpT82 (pTau long) and (B) analysis of the ratio of pT3xpT82 to hT7xpT82 of rp-HPLC fractions of CSF compared with MMSE scores, all from blinded cohorts of CDR0 and CDR1 subjects. [Figure 16B] Results are shown for (A) analysis of the ratio of pT3xpT82 to hT7xpT82 (pTau short) or the ratio of pT3xhT43 to hT7xpT82 (pTau long) and (B) analysis of the ratio of pT3xpT82 to hT7xpT82 of rp-HPLC fractions of CSF compared with MMSE scores, all from blinded cohorts of CDR0 and CDR1 subjects. [Figure 17A] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17B](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17C] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17D](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17E] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17F](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17G] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17H](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17I] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17J](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17K] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17L](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17M] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17N](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17O] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17P](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17Q] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17R](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17S] (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 17T](A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 analysis in crude CSF; (D) correlation of two pT3 assays in crude CSF; (E) correlation of pT3xpT82 vs. hT7xpT82 in crude CSF; (F) correlation of pT3xhT43 vs. Innotest t-tau in crude CSF; (G) correlation of pT3xhT43 vs. Innotest p-tau181 in crude CSF; (H) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF; (I) correlation of pT3xhT43 vs. Innotest AB42 in crude CSF. Correlation of AB42 / 40 ratios; (J-I) pT3xhT43 signal, (M-P) pT3xpT82 signal, or (Q-T) hT7xpT82 signal in (J, M, Q) all rp-HPLC fractions, (K, N, R) all fractions combined, (O, S) early peak fraction combined (short tau fragments), or (L, P, T) late peak fraction combined (larger tau fragments), all from cohorts of HV, MCI, and AD subjects. [Figure 18A] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18B](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18C] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18D](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18E] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18F](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18G] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18H](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18I] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18J](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18K] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18L](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18M] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18N](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18O] (A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 18P](A) pT3xpT82 (p217+ short) vs. pT3xhT43 (p217+ long), (B) pT3xpT82 vs. hT7xpT82 (t-tau short), (C) pT3xpT82 vs. NFL, and (D) pT3xhT43, or (E) pT3xpT82 vs. amyloid status, and correlations of (F-I, N-P) pT3xhT43 or (J-M) pT3xpT82 with (F-M) various cognitive scores or (N-P) change 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 (and classified as AD) based on cognition, but upon biochemical evaluation (AB40 and AB42), 27 of the subjects were determined to be amyloid negative and therefore likely represented dementia not due to AD. These subjects were analyzed as a separate cohort in the figure above, designated amyloid negative = 0, while amyloid positive subjects = 1. [Figure 19] Shown are signals from pT3xhT43 assays performed on rp-HPLC fractions of AD CSF samples spiked with IgG, pT3 mAb, humanized pT3 mAb, or mock control, followed by immunoprecipitation to recover antibody-bound p217+ tau. [Figure 20A] Antibody dose dependence of the immunocapture / rpHPLC method for quantifying p217+ tau (A) without antibody and (B) bound to antibody is shown, with data graphed as the sum of the signal in rpHPLC fractions 12-16. [Figure 20B] Antibody dose dependence of the immunocapture / rpHPLC method for quantifying p217+ tau (A) without antibody and (B) bound to antibody is shown, with data graphed as the sum of the signal in rpHPLC fractions 12-16. [Figure 21A]Differential kinetics of damage of antibodies versus p217+ tau that were heat-denatured (A, C) or not (B) are shown: (A) humanized PT3 mAb / CSF mixture; (B) untreated CSF; (C) humanized PT3 mAb. [Figure 21B] Differential kinetics of damage of antibodies versus p217+ tau that were heat-denatured (A, C) or not (B) are shown: (A) humanized PT3 mAb / CSF mixture; (B) untreated CSF; (C) humanized PT3 mAb. [Figure 21C] Differential kinetics of damage of antibodies versus p217+ tau that were heat-denatured (A, C) or not (B) are shown: (A) humanized PT3 mAb / CSF mixture; (B) untreated CSF; (C) humanized PT3 mAb. [Figure 22A] (A) Heat-denatured and (B) immunocapture / rpHPLC methods for quantifying antibody-free versus antibody-bound p217+ tau are shown; (C) Comparison of methods is shown. [Figure 22B] (A) Heat-denatured and (B) immunocapture / rpHPLC methods for quantifying antibody-free versus antibody-bound p217+ tau are shown; (C) Comparison of methods is shown. [Figure 22C] (A) Heat-denatured and (B) immunocapture / rpHPLC methods for quantifying antibody-free versus antibody-bound p217+ tau are shown; (C) Comparison of methods is shown. [Figure 23] 1 shows the lack of pT3-based assay recognition of p217+ tau in cynomolgus monkey CSF. [Figure 24A] Measurements of p217+ tau in marmoset CSF determined using (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 assays are shown. [Figure 24B] Measurements of p217+ tau in marmoset CSF determined using (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 assays are shown. [Figure 24C] Measurements of p217+ tau in marmoset CSF determined using (A) pT3xhT43, (B) pT3xpT82, and (C) hT7xpT82 assays are shown. [Figure 25A] Measurements of (A, B) hT7xpT82 (t-tau) or (C, D) pT3xpT82 (p217+tau) in crude serum from four AD and four HV subjects are shown. Measurements were performed at a (A, C) 1:4 or (B, D) 1:16 dilution; note the lack of dilution linearity and sensitivity. [Figure 25B] Measurements of (A, B) hT7xpT82 (t-tau) or (C, D) pT3xpT82 (p217+tau) in crude serum from four AD and four HV subjects are shown. Measurements were performed at a (A, C) 1:4 or (B, D) 1:16 dilution; note the lack of dilution linearity and sensitivity. [Figure 25C] Measurements of (A, B) hT7xpT82 (t-tau) or (C, D) pT3xpT82 (p217+tau) in crude serum from four AD and four HV subjects are shown. Measurements were performed at a (A, C) 1:4 or (B, D) 1:16 dilution; note the lack of dilution linearity and sensitivity. [Figure 25D] Measurements of (A, B) hT7xpT82 (t-tau) or (C, D) pT3xpT82 (p217+tau) in crude serum from four AD and four HV subjects are shown. Measurements were performed at a (A, C) 1:4 or (B, D) 1:16 dilution; note the lack of dilution linearity and sensitivity. [Figure 26A] Measurements of (A) hT7xpT82 (t-tau) or (B) pT3xpT82 (p217+tau short) in NaOAc-pretreated, heat-denatured serum from the same four AD and four HV subjects evaluated in Figures 25A-25D are shown. [Figure 26B] Measurements of (A) hT7xpT82 (t-tau) or (B) pT3xpT82 (p217+tau short) in NaOAc-pretreated, heat-denatured serum from the same four AD and four HV subjects evaluated in Figures 25A-25D are shown. [Figure 27]Shown are measurements of pT3xpT82 (p217+tau short) in pT3 immunoprecipitations (IP) of sera from the same four AD and four HV subjects assessed in Figures 25A-25D and Figures 26A-26B. DETAILED DESCRIPTION OF THE INVENTION

[0041] Various publications, articles and patents are cited or referenced in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which is included in the specification is indicative of the context of this invention. Such discussion is intended to provide a basis for understanding whether any or all of these matters are disclosed or implied. is admitted to constitute prior art to any claimed invention. isn't it.

[0042] definition Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. Unless otherwise specified, certain terms used herein shall have the meanings ascribed to them. All patents, published patent applications and publications cited herein are hereby incorporated by reference. The present specification and the accompanying drawings are incorporated herein by reference in their entirety as if set forth herein. As used in the appended claims, the singular forms "a," "an," and "the" are used where appropriate, particularly where the context requires. It should be noted that unless otherwise clear, plural referents are included.

[0043] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein are the total In all cases, the terms "about" and "about" should be understood as being modified by the term "about." Therefore, numerical values ​​typically include ±10% of the stated value. For example, 1 mg The concentration of 1% to 10% (w / mL) is 0.9 mg / mL to 1.1 mg / mL. The concentration range of 0.9% (w / v) to 11% (w / v) is included. In this case, the use of a numerical range refers to the whole number within that range unless the context clearly indicates otherwise. All possible subranges, including fractions of numbers and values, and all individual numbers within the range are explicit Included.

[0044] As used herein, the terms "antibody" or "immunoglobulin" are used broadly. Immunoglobulin or antibody molecules, including polyclonal antibodies, murine, human, human-adapted, human This includes monoclonal antibodies, including homogenized and chimeric monoclonal antibodies and antibody fragments.

[0045] In general, an antibody is a protein or peptide chain that exhibits binding specificity to a specific antigen. The structure of an antibody is known. Immunoglobulins are composed of heavy chain constant domain amino acid sequences. They are divided into five major classes according to their antigenicity: IgA, IgD, IgE, IgG, and IgM. IgA and IgG can be assigned to different isotypes: IgA1, IgA2, and Ig IgG1, IgG2, IgG3, and IgG4. Antibodies can be of any of five major classes or corresponding subclasses: The antibody of the present invention is preferably IgG1, IgG2, IgG3, or IgG4. The antibody light chains of any vertebrate species can be classified into two groups based on the amino acid sequence of their constant domains. can be assigned to one of two distinct types: κ and λ. Thus, the antibodies of the present invention may contain either a kappa or a lambda light chain constant domain. According to an embodiment, the antibody of the present invention comprises heavy and / or light chain constant sequences of a murine or human antibody. Includes the area.

[0046] In addition to heavy and light chain constant regions, antibodies contain light and heavy chain variable regions. The light or heavy chain variable region consists of "framework" regions interrupted by "antigen binding sites". The antigen-binding site is defined using various terms and numbering schemes as follows: : (i) Kabat: "Complementarity-Determining Regions" or "CDRs" are based on sequence variability (W u and Kabat, J Exp Med. 132:211-50, 1970). one Generally, an antigen-binding site comprises three CDRs in each variable region (e.g., a heavy chain variable region (VH) and a heavy chain variable region (VH)). CDR1, HCDR2, and HCDR3, and LCDR1, L in the light chain variable region (VL). CDR2, and LCDR3). (ii) Chothia: The term "hypervariable region", "HVR" refers to the esk(Chothia and Lesk,J Mol Biol.196:901- As defined by [17, 1987], the antibody variable domain is hypervariable in structure. Generally, an antigen-binding site is composed of each VH (H1, H2, H3) and VL (L It has three hypervariable regions (L1, L2, L3). The annotations are based on Abhinandan and Martin (Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008) It has been revised. (iii) IMGT: Another definition of the region that forms the antigen-binding site is the region of an immunoglobulin and Based on a comparison of V domains from T cell receptors, Lefranc (Dev Comp Immunol. 27:55-77, 2003) It has been proposed that the International ImmunoGeneTics (IM The Imgt (GT) database (http: / / www_imgt_org) provides information on these areas. Provides standardized numbering and definitions for all CDR, HVR, and IMGT descriptions. The reaction is described in Lefranc et al., 2003, supra. (iv) The antigen-binding site also has a "specificity-determining residue usage" (SDRU) (Almagr o, Mol Recognit.17:132-43,2004) In this case, the SDRs are the amino acid residues of the immunoglobulin that are directly involved in antigen contact. Refers to...

[0047] "Framework" or "framework sequence" is defined as the sequence of the antigen-binding site. The precise definition of the antigen-binding site is The exact framework sequence can be determined by various delineations as described above. The framework regions (FR) are more highly conserved and likely The variable domains of naturally occurring heavy and light chains each contain three hypervariable loops. Four FRs (FR1, FR2, FR3, FR4, FR5, FR6, FR7, FR8, FR9, FR10, FR11, FR12, FR13, FR14, FR15, FR16, FR17, FR18, FR19 ...0, FR12, FR13, FR14, The hypervariable loops of each chain are tightly folded together by the FRs. These, together with the hypervariable loops of the other chains, contribute to the formation of the antigen-binding site of the antibody. Structural analysis has revealed 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.21 5:175-182, 1990). Despite these high sequence variabilities, six loci Only five of the groups adopt a small repertoire of main-chain structures, called "canonical structures." These structures are primarily determined by the length of the loops, followed by folding, hydrogen bonding, and or loops and frameworks whose structure is determined by their ability to predict unusual main-chain structures. It is determined by the presence of key residues at specific positions in the region.

[0048] As used herein, the term "antigen-binding fragment" refers to, for example, a diabody, a Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (d sFv), bispecific dsFv (dsFv-dsFv'), disulfide-stabilized dimers diabodies (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab ), scFv dimers (bivalent diabodies), and fragments formed from portions of antibodies containing one or more CDRs. Multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, bivalent Such as a domain antibody or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. An antigen-binding fragment refers to an antibody fragment that binds to the same antigen as the parent antibody or parent antibody fragment. According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and a According to another particular embodiment, the antigen binding The combined fragments include Fab and F(ab').

[0049] As used herein, the term "epitope" refers to an antigen-binding domain of an immunoglobulin, antibody, or its antigen. An epitope is a site on an antigen to which an antigen-binding fragment specifically binds. Formed both from juxtaposed consecutive amino acids by tertiary folding, and from non-consecutive amino acids. Epitopes formed from consecutive amino acids are typically soluble in denaturing solvents. epitopes formed by tertiary folding are typically kept exposed upon denaturation. Epitopes are typically found in a unique spatial conformation and are lost by treatment with solvents. At most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 meshes Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography. and 2D nuclear magnetic resonance. For example, Epitope Mapping Pr otocols in Methods in Molecular Biology, See Vol. 66, GEMorris, Ed. (1996).

[0050] As used herein, the term "tau" or "tau protein" refers to multiple isoforms of tau. It refers to a number of proteins in the central and peripheral nervous system that have the same structure. In the central nervous system (CNS), alternative splicing produces a sequence of 352–441 amino acids. There are six major tau isoforms ranging in size (Hanger et al., 2013). Rends Mol Med.15:112-9,2009). These isoforms The nucleotide sequence is regulated by 0 to 2 N-terminal insertions and 3 or 4 tandemly arranged microtubule-binding repeats. They differ from each other by being controlled by the As used herein, the term "control tau" refers to a tau mutant of the tau family. refers to the tau isoform of SEQ ID NO: 1 that is not phosphorylated or otherwise post-translationally modified. As used herein, the term "tau" refers to a mutation of full-length wild-type tau (e.g., a point mutation). This includes proteins containing fragments, insertions, deletions, and splice variants. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Examples of such reactions include, but are not limited to, phosphorylation.

[0051] Unless otherwise indicated, as used herein, in tau protein or fragments thereof Amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO:1.

[0052] As used herein, the terms "p217+ tau peptide," "p217+ tau," or "p217+ tau protein" refers to the tau protein at residue 217 (pT217) and residue human tau protein phosphorylated at one or both of residues 212 (pT212); or It refers to a tau fragment, and the position numbering follows that of SEQ ID NO: 1.

[0053] As used herein, the term "p217+ tau epitope" refers to the epitope that binds to phosphorylated T217 and and phosphorylated T212, and The numbers follow the numbering of SEQ ID NO: 1. Examples of p217+ tau epitopes include, for example, pT As used herein, the term "pT3 epitope" refers to a human pT3 epitope. Human tau protein phosphorylated at at least one residue of T217 and T212 of tau The position numbering is as in SEQ ID NO: 1. Examples of pT3 epitopes include those listed in SEQ ID NOs: 25, 26, and 27. Examples include:

[0054] As used herein, the terms "long p217+ tau peptide," "long p217 + tau,” “long form p217+ tau peptide,” or “long p217+ tau peptide.” The terms "p217+ tau epitope" and "tau protein fragment" have the same meaning, respectively. It refers to a p217+tau peptide containing an epitope comprising amino acid residues 7 to 20 of the present invention. "Long p217+ tau peptides" according to embodiments may have different lengths, for example: The amino terminus of the "long p217+ tau peptide fragment" is the amino acid residue of the tau protein. It can be 1, 2, 4, 5, 6, or 7.

[0055] As used herein, the terms "short p217+tau peptide," "short p2 17+ tau,” “short form p217+ tau peptide,” or “short p217+ "Tau peptide fragment" and "p217+ tau epitope" have the same meaning, and "tau protein" and "tau protein fragment" have the same meaning, and "p217+ tau epitope" and "tau protein fragment" have the same meaning, and It contains an epitope containing amino acid residues 119-126 of the protein, but This refers to the p217+ tau peptide that does not contain the epitope containing amino acid residues 7 to 20. "Short p217+ tau peptides" according to certain embodiments may have different lengths. For example, the amino terminus of the "short p217+tau peptide" is the amino acid An epitope containing residues 7-20 and amino acid residues 119-126 of the tau protein It may be any amino acid residue between the epitope.

[0056] As used herein, the terms "long tau peptide," "long tau," and "long form" refer to "tau peptide in a form" or "long tau peptide fragment" have the same meaning, Tau epitopes recognized by oxidation-independent capture antibodies and amino acids of tau protein and an epitope comprising residues 7 to 20. The "long tau peptide fragment" can have different lengths. For example, The amino terminus of the fragment is amino acid residue 1, 2, 4, 5, 6, or 7 of the tau protein. obtain.

[0057] As used herein, the terms "short tau peptide," "short tau," "short "short form of tau peptide," or "short tau peptide fragment" have the same meaning. The tau epitopes recognized by the phosphorylation-independent capture antibodies and the tau protein It contains amino acid residues 119 to 126, but not amino acid residues 7 to 20 of the tau protein. The term "tau peptide" refers to a tau peptide comprising an epitope and an epitope that does not contain an epitope. "Short tau peptide fragments" can have different lengths. The amino terminus of the tau peptide binds to an epitope containing amino acid residues 7 to 20 of the tau protein. The amino acid residues between the epitope containing amino acid residues 119 to 126 of the protein There may be a deviation.

[0058] As used herein, the term "capture antibody" refers to an antibody that binds to an antigen of interest and is directly attached to a solid support. Examples of solid supports include microparticles or magnetic beads. Examples of capture antibodies include, but are not limited to, beads such as p2 monoclonal antibodies that bind to the 17+ tau epitope, According to an embodiment of the present invention, the capture antibodies are SEQ ID NOs: 32, 33, and 34, respectively. an immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the polypeptide sequence 3, and immunoglobulin light chains having the polypeptide sequences of SEQ ID NOs: 35, 36 and 37. It may be a monoclonal antibody that comprises LCDR1, LCDR2, and LCDR3. In certain embodiments, the capture antibody is pT3. As used herein, the term "pT3" binds to p217+tau peptide and has the heavy chain variable region amino acid sequence of SEQ ID NO: 28 and It refers to an antibody having the light chain variable region amino acid sequence of SEQ ID NO: 29. In one embodiment, the pT3 motif In another embodiment, the monoclonal antibody is expressed by a mouse hybridoma. The capture antibody has 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. It is a humanized antibody having the amino acid sequence

[0059] According to another embodiment of the present invention, the capture antibody is capable of phosphorylation-independently capturing tau protein. amino acids 150 to 250, preferably amino acids 211 to 221 or It may be a monoclonal antibody that binds to an epitope at amino acids 159 to 163, Numbering follows that of SEQ ID NO: 1. In a specific embodiment, the capture antibody is hT7. As used herein, the term "hT7" refers to the amino acids 159-160 of the human tau protein. Refers to publicly available monoclonal antibodies that bind to epitopes including 63, and position numbering The numbers follow the numbering of SEQ ID NO: 1. The hT7 monoclonal antibody can be obtained from, for example, ThermoFilm It is commercially available from Sher (eg, catalog number: MN1000).

[0060] As used herein, the term "detection antibody" refers to a molecule that binds to an antigen of interest and is detectably labeled. or linked to a secondary detection system. Examples of detectable labels include: These include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of detection antibodies include, but are not limited to, antibodies against tau protein, preferably human A monoclonal antibody that binds to an epitope containing amino acids 7-20 or 116-127 of the tau protein The position numbering may be the same as that of SEQ ID NO: 1. The sequence follows: Monoclonal antibody that binds to tau protein in an epitope containing amino acids 7 to 20 When a local antibody is used as the detection antibody for the captured p217+ tau peptide, The tau fragment is detected in the epitope containing amino acids 116-127. A monoclonal antibody that binds to the protein was used as a detection antibody for the captured p217+ tau peptide. When used as a tau fragment detection assay, short and long tau fragments are detected.

[0061] In a specific embodiment, the detection antibody is hT43. hT43 is a molecule that binds to an epitope containing amino acids 7 to 20 of the human tau protein. The position numbering is in accordance with the numbering of SEQ ID NO: 1, and the antibody is It has the heavy chain variable region amino acid sequence of SEQ ID NO: 8 and the light chain variable region amino acid sequence of SEQ ID NO: 9. In another specific embodiment, the detection antibody is pT82. The term "pT82" refers to the amino acids 119 to 126, preferably 116 to 126, of the human tau protein. 127, and the position numbering is SEQ ID NO: 1, the antibody has a heavy chain variable region amino acid sequence of SEQ ID NO: 18 and a heavy chain variable region amino acid sequence of SEQ ID NO: 1 It has a light chain variable region amino acid sequence of 9.

[0062] As used herein, the term "pT3-based assay" refers to a method for detecting a pT3 antibody using a capture antibody. As used herein, refers to an assay according to an embodiment of the present invention in which the The term "pT3xhT43" refers to the use of pT3 antibody as the capture antibody and hT43 antibody as the detection antibody. As used herein, refers to an assay according to an embodiment of the present invention in which the The term "pT3xpT82" refers to the use of pT3 antibody as a capture antibody and pT82 antibody as a detection antibody. It refers to an assay according to an embodiment of the present invention that uses an antibody.

[0063] As used herein, the term "hT7-based assay" refers to an assay in which the hT7 antibody is used as a capture antibody. As used herein, refers to an assay according to an embodiment of the present invention in which the The term "hT7xpT82" refers to the use of the hT7 antibody as the capture antibody and the pT82 antibody as the detection antibody. "Assays" refer to assays according to embodiments of the present invention that use a marker as a template.

[0064] As used herein, the term "subject" refers to an animal, preferably a mammal. According to specific embodiments, the subject is a non-primate (e.g., camel, donkey, zebra, cow, , pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, marmosets , or mouse), or mammals, including primates (e.g., monkeys, chimpanzees, or humans) In a specific embodiment, the subject is a human.

[0065] As used herein, "tauopathy" refers to any disorder involving pathological aggregation of tau in the brain. In addition to familial and sporadic AD, other exemplary tauopathies include: is a type of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). , progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurogenic Tangle-only dementia, diffuse neurofibrillary tangles with calcification Disease, argyrophoric 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, Lyon protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystonia Rhophy, non-Guamanian motor neuron disease with neurofibrillary tangles, encephalitis Post-Parkinsonism syndrome and chronic traumatic encephalopathy such as dementia pugilistica (boxer's disease) (Morris et al., Neuron, 70:410-26, 2011).

[0066] As used herein, the terms "determine," "measure," "assess," and "assess" are used interchangeably. The terms "assess" and "assess" are used interchangeably and include both quantitative and qualitative determinations. refers to any form of measurement to determine whether a property, trait, or characteristic is present or not. The evaluation can be relative or absolute. "Evaluate the existence of" means This includes determining the amount of something that occurs, as well as determining whether it is present or absent.

[0067] As used herein, the term "diagnosis" refers to detecting a disease or disorder, or It is intended to determine the stage or degree of a disease or disorder such as a tauopathy. Diagnosis of a disease or disorder typically involves the evaluation of one or more factors and / or symptoms indicative of the disease. Diagnosis is based on factors indicative of the presence or absence of a disease or condition, such as the presence or absence of p217+ tau. The diagnosis can be based on the amount or quantity of each factor or factor that is considered to be indicative of a particular disease. Symptoms do not have to be related exclusively to the particular disease, i.e., they may be diagnostic factors or symptoms. Similarly, there may be different diagnoses that can be inferred from a particular disease. In some cases, the diagnosis may be present in individuals who do not have a specific disease. For example, to determine the therapeutic effect of anti-p217+tau antibody therapy, or to determine the therapeutic effect of drug therapy, e.g., anti-p The diagnostic method also includes predicting the pattern of response to 217+ tau antibody therapy. It may be used independently or in combination with other compounds to treat a specific disease or disorder, such as Alzheimer's disease. Use in combination with other diagnostic and / or staging methods known in the medical arts You may do so.

[0068] As used herein, the terms "increase" and "decrease" refer to a decrease in a control or baseline level. It refers to the difference in the amount of a particular biomarker in a sample compared to the amount of a particular peptide. The amount of the compound is determined by an increased or decreased amount in samples from patients with the disease compared to the reference level. In one embodiment, "increased levels" or "decreased levels" refers to the amount of a protein present in a given molecule that is present in a controlled amount. and the difference between the levels of the biomarkers present in the samples is at least about 1 when compared to %, 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%, It may be at least about 75%, at least about 80%, or more. In this context, "increased levels" or "decreased levels" refers to the presence or absence of a specific marker in a sample when compared to a control. The difference between the levels of biomarkers present can be statistically significant. For example, The level of the measured biomarker is less than about 1.0 standard deviation from the mean of any control or reference group. difference, if it is outside by about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 standard deviations The difference may be statistically significant. A reference or control may be, for example, a sample from a healthy individual, or at an earlier time point, such as before administration of the therapeutic agent or at an earlier time point during the treatment regimen. The samples may be taken from the same individual.

[0069] As used herein, the term "isolated" refers to a substance that is free from biological components (e.g., nucleic acids, peptides, or proteins) from other organisms in which these components naturally occur biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins) ) or produced separately from these other components, or This means that the nucleic acid, peptide, or other component has been purified from other components. The peptides and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition, which Such compositions are not part of the natural environment of the nucleic acid, peptide, or protein. The term also refers to a strain of a plant that has been isolated by recombinant expression in a host cell. It also includes synthesized nucleic acids, peptides, and proteins, as well as chemically synthesized nucleic acids.

[0070] As used herein, an "isolated antibody that binds to tau protein" or "isolated "Anti-tau antibodies" specifically bind to tau protein and are different from other antibodies with different antigen specificities. It is intended to refer to an antibody that is substantially free of antibodies (e.g., an isolated anti-tau detecting antibody). (The isolated antibody is substantially free of antibodies that specifically bind to antigens other than tau.) Anti-tau detection antibodies may be directed against other related antigens (e.g., homologs of tau species) from other species. There may be cross-reactivity.

[0071] As used herein, the term "specifically bind" or "specific binding" refers to the binding of Anti-tau antibodies were approximately 1 × 10 -6 M or tighter, e.g., about 1 x 10 -7 M and below, Approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1 x 10M -10 Below, approximately 1×10 - 11 M or less, approximately 1×10 -12 M or less, or about 1 x 10 -13 The dissociation constant (K D ) KD refers to the ability of an antibody to bind to a given target at a specific binding site. KD is the ratio of Kd to Ka (i.e., The KD value of an antibody is obtained from the KD / Ka (Kd / Ka) and expressed as a molar concentration (M). This can be determined using methods known in the art, taking into consideration the effects of anti-tau antibodies. KD values ​​can be determined by using surface plasmon resonance, for example, in a biosensor system, e.g. For example, Biacore® system, Proteon instrument (BioRad), Ki nExA instrument (Sapidyne), ELISA, or competitive binding assays known to those skilled in the art Typically, anti-tau antibodies can be determined using, for example, Protein Nonspecific as measured by surface plasmon resonance using a BioRad instrument K against target D At least 10 times lower than D to a given target (i.e., tau) However, anti-tau antibodies that specifically bind to tau may bind to other related targets, e.g., other species. (homologue), for example, a given homologue from mouse, rat, marmoset, dog, or pig There may be cross-reactivity to one target.

[0072] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid molecule." ", "nucleotides," or "nucleic acids," and may be unmodified RNA or DNA or modified Any polyribonucleotide or polydeoxyribonucleotide, which may be RNA or DNA. "Polynucleotide" refers to a nucleotide. "Polynucleotide" includes, but is not limited to, However, there are single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, RNA that is a mixture of single-stranded and double-stranded regions, single-stranded or double-stranded RNA, DNA and RNA, which may typically be double-stranded or a mixture of single- and double-stranded regions, In addition, "polynucleotide" includes RNA or hybrid molecules containing the same. The term polynucleotide refers to a triple-stranded region containing DNA or both RNA and DNA. is DNA or RNA that contains one or more modified bases and that are modified for stability or other reasons. Also included are DNAs or RNAs with modified backbones. Various modifications to DNA and RNA include modified and unusual bases, e.g., inosine. Thus, a "polynucleotide" is a polynucleotide that is typically found in nature. Chemically, enzymatically or metabolically modified forms of oligonucleotides, as well as viruses and cells "Polynucleotide" also refers to chemical forms that have the characteristics of DNA and RNA. , also encompasses relatively short nucleic acid chains (often called oligonucleotides).

[0073] As used herein, the term "vector" refers to a vector that operably carries another nucleic acid segment. A replicon into which a segment can be inserted to replicate or express that segment. And so.

[0074] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the present invention. A "host cell" refers to, for example, a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell that contains a nucleic acid molecule of the invention. In another embodiment, a "host cell" is a cell transfected with such a transfectant. The progeny or potential progeny of the infected cell. The progeny of a cell are, for example, mutations or environmental influences that may occur over generations, or integration of the nucleic acid molecule into the host cell genome Depending on the circumstances, the clone may not have identity with the parent cell.

[0075] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the transcription of a gene into RNA. This includes translation into polypeptides, and also includes all naturally occurring post-transcriptional and post-translational modifications. The expressed detection antibody or antigen-binding fragment thereof that binds to tau is located within the cytoplasm of the host cell. They may enter the extracellular environment, such as the growth medium of cell cultures, or they may become anchored to the cell membrane. In some cases, this is the case.

[0076] Anti-tau antibody In one general aspect, the present invention relates to a method for detecting tau protein that is immobilized by a capture antibody. The present invention relates to an isolated detection antibody or antigen-binding fragment thereof that binds to tau. , which bind to a phosphorylated epitope in tau or a non-phosphorylated epitope in tau The anti-tau detection antibody may have the property of binding to tau. It may be useful as a research or diagnostic reagent.

[0077] According to a specific embodiment, the present invention relates to a tau protein comprising amino acid residues 119 to 126, preferably Preferably, the epitope comprises amino acid residues 116 to 127 of the tau protein. The present invention relates to an isolated detection antibody or antigen-binding fragment thereof.

[0078] According to a specific embodiment, an epitope comprising amino acid residues 116 to 127 of tau protein is The isolated detection antibody or antigen-binding fragment thereof that binds to tau protein in the antibody fragment is a) immunoglobulin heavy chain H having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively (b) CDR1, HCDR2 and HCDR3 of SEQ ID NOs: 5, 6 and 7, respectively Immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences Includes.

[0079] According to a specific embodiment, an epitope comprising amino acid residues 116 to 127 of tau protein is The isolated detection antibody or antigen-binding fragment thereof that binds to tau protein in the target peptide is Row No. 8 and at least 80%, preferably at least 85% or 90%, more preferably Heavy chain variable regions having polypeptide sequences that are at least 95%, and most preferably 100%, identical and SEQ ID NO: 9, at least 80%, preferably at least 85% or 90%, more preferably Preferably, the polypeptide has at least 95%, and most preferably 100% identical polypeptide sequence. and a chain variable region.

[0080] Preferably, in an epitope comprising amino acid residues 116 to 127 of tau protein The isolated detection antibody or antigen-binding fragment thereof that binds to tau protein is a pT82 antibody. is.

[0081] According to a specific embodiment, the present invention provides an epitope comprising amino acid residues 7 to 20 of tau protein. and an isolated detection antibody or antigen-binding fragment thereof that binds to tau protein at the target polypeptide. Regarding.

[0082] According to a specific embodiment, the present invention relates to an epitope comprising amino acid residues 7 to 20 of tau protein. The isolated detection antibody or antigen-binding fragment thereof that binds to tau protein in the presence of the antibody comprises: (a) Immunoglobulin heavy chain H having the polypeptide sequences of SEQ ID NOs: 12, 13, and 14, respectively CDR1, HCDR2 and HCDR3, and (b) SEQ ID NOs: 15, 16, and 17, respectively. Immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having 17 polypeptide sequences Includes DR3.

[0083] According to a specific embodiment, the present invention relates to an epitope comprising amino acid residues 7 to 20 of tau protein. The isolated detection antibody or antigen-binding fragment thereof that binds to tau protein is represented by SEQ ID NO: 18 and at least 80%, preferably at least 85% or 90%, more preferably at least and heavy chain variable regions having polypeptide sequences that are at least 95%, and most preferably 100%, identical. , at least 80%, preferably at least 85% or 90%, more preferably at least 80% identical to SEQ ID NO: 19. or light chains having at least 95%, and most preferably 100%, identical polypeptide sequences. and a variable region.

[0084] Preferably, the epitope comprises amino acid residues 7 to 20 of the tau protein. The isolated detection antibody or antigen-binding fragment thereof that binds to the protein is an hT43 antibody. .

[0085] The antibodies of the present invention can be produced by various techniques, for example, by the hybridoma method (Kohler and Milstein, Nature. 256:495-7, 1975). The light chain and the antibody fragments derived from the acceptor antibody (typically from another mammalian species such as a human) can be used. Light and heavy chain constant regions derived from a donor antibody (typically murine) associated with a heavy chain constant region. Chimeric mAbs containing variable regions can be produced using the methods described in U.S. Pat. No. 4,816,567. They can be prepared from non-human donor immunoglobulins (typically murine). and the remaining immunoglobulin-derived portion of the molecule is derived from one or more human immunoglobulins. CDR-grafted mAbs derived from These can be prepared by techniques known to those skilled in the art, including those that contain a complete human polypeptide lacking any non-human sequences. The mAb was (Lonberg et al., Nature. 368:856-9, 1 994;Fishwild et al., Nat Biotechnol.14:84 5-51, 1996;Mendez et al., Nat Genet.15:146 -56, 1997) to produce human immunoglobulin trans. Human mAbs can be prepared from transgenic mice. It can also be prepared and optimized from a library (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).

[0086] The functional activity of detection antibodies and antigen-binding fragments thereof that bind to tau has been described in the art. Antibodies and antigen-binding fragments thereof that bind to tau can be characterized by known methods. Characterization methods include Biacore, ELISA, and FACS analysis, immunohistochemistry. These include, but are not limited to, affinity and specificity assays, including assays such as ELISA.

[0087] Several well-known methods can be used to determine the binding epitope of an antibody of the present invention. For example, protein-protein hybridization can be performed in silico if the structures of both individual components are known. Protein docking can be performed to identify compatible interaction sites. By using hydrogen-deuterium (H / D) exchange with fusion, the region of the antigen to which the antibody binds can be identified. By using segment and point mutagenesis of the antigen, it is possible to map the region. This allows the identification of amino acid positions that are important for antibody binding. The crystal structure can be used to identify residues that contribute to the epitope and paratope.

[0088] In another general aspect, the invention provides a method for detecting a denatured antibody or antigen-binding fragment thereof, comprising: The present invention relates to an isolated polynucleotide that is capable of producing a protein without changing the amino acid sequence of the protein. The protein coding sequence can be altered (e.g., substituted, deleted, inserted, etc.). It will be understood by those skilled in the art that the amino acid sequence of a protein can be changed. without altering the nucleic acid sequence encoding the detection antibody or antigen-binding fragment thereof of the present invention. It will be understood by those skilled in the art that exemplary isolated polynucleotides are The immunoglobulin heavy chain CDRs HCDR1, HCDR2, and and HCDR3, or the immunoglobulins shown in SEQ ID NOs: 5, 6, and 7, respectively. A polypeptide comprising globulin light chain CDRs LCDR1, LCDR2, and LCDR3. Other exemplary isolated polynucleotides are Immunoglobulin heavy chain CDRs HCDR1, HCDR2 shown in SEQ ID NOs: 12, 13, and 14 2, and HCDR3, or polypeptides comprising SEQ ID NOs: 15, 16, and 17, respectively A polypeptide comprising immunoglobulin light chain CDRs LCDR1, LCDR2, and LCDR3. Other exemplary isolated polynucleotides are The polynucleotides encoding the antibody variable regions of the present invention are Considering the codon preferences in a given expression system, other fragments encoding the antibodies of the present invention may be used. The isolated nucleic acids of the present invention may be prepared by any known recombinant or The DNA encoding the monoclonal antibody can be produced using synthetic techniques. They are easily isolated and sequenced using methods known in the art. If new genes are produced, such cells can serve as a source of their DNA. Alternatively, display technologies in which the coding sequence and the translation product are linked, such as phage Alternatively, a ribosome display library can be used.

[0089] In another general aspect, the invention provides a method for detecting a denatured antibody or antigen-binding fragment thereof, comprising: The present invention relates to vectors containing the isolated polynucleotides. vector, or any vector known to those of skill in the art in light of the present disclosure, such as a viral vector. In some embodiments, the vector may be a recombinant vector such as a plasmid. A recombinant expression vector. A vector contains, for example, a promoter, a ribosome-binding element, expression vectors, including promoters, terminators, enhancers, selectable markers, and replication origins. The promoter may contain any element necessary to establish the conventional function of the promoter. The promoter may be constitutive, inducible, or reconfigurable. Numerous expression vectors are known in the art that can produce antibodies or can be used herein to generate antigen-binding fragments thereof. Genetic engineering or artificial gene synthesis may be used to produce recombinant expression vectors according to embodiments of the present invention. It is possible to generate a

[0090] In another general aspect, the invention provides a method for detecting a denatured antibody or antigen-binding fragment thereof, comprising: In view of the present disclosure, any host cell known to those of skill in the art that contains the isolated polynucleotide. Any host cell known to those skilled in the art may be used for recombinant expression of the antibodies or antigen-binding fragments of the invention. Such host cells may be eukaryotic, bacterial, plant or archaeal cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. As mammalian eukaryotic cells, SP2 / 0 (American Type Culture Collection (ATC)) may be used. C), Manassas, Va., CRL-1581), NS0 (European Cell Culture Collection) ECACC, Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (A Hybridoma or myeloma cells, such as the TCC CRL-1580 murine cell line. Immortalized cell lines such as human myeloma cell lines are also included. An exemplary human myeloma cell line is U266 (ATT Other useful cell lines include CHO-K1 SV (Lonza Biologics), CHO-K1 (ATCC CRL-61, Inv rogen), or Chinese hamster ovary (CHO) cells such as DG44. The origins include:

[0091] In another general aspect, the invention provides a method for producing a detection antibody or antigen-binding fragment thereof of the invention. under conditions that produce the detection antibody or antigen-binding fragment thereof of the present invention, Culturing cells containing polynucleotides encoding the antibodies or antigen-binding fragments thereof. and extracting the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). and recovering the expressed antibody or antigen-binding fragment thereof from the cell. It can be recovered and purified according to techniques conventional in the art.

[0092] Diagnostic methods The present invention provides, for example, reporter elements that allow for the detection of captured p217+ tau species. This method selectively immobilizes p217+ tau species in combination with an anti-tau detection antibody labeled with a thiol. By using capture antibodies such as pT3, which is highly abundant in AD, The methods of the present invention relate to the measurement of tauopathies, for example, for diagnosing AD or other tauopathies in a subject. To identify suitable targets for anti-p217+ tau treatment, to monitor the efficacy of treatment, It can be used for various diagnostic purposes, such as to identify elephants.

[0093] According to an embodiment of the present invention, the p217+ tau peptide in the sample of interest is p217+ tau epitopes, such as epitopes having a sequence of 25, 26 or 27 amino acids The captured p217+ tau peptides were all p217+ Contains the tau epitope but may have different lengths, which bind to different epitopes It can be detected by a detection antibody. For example, the amino acid residues 7-2 of the tau protein Detection antibodies against epitopes containing 0 still exist between amino acid residues 7 and 8 of the tau protein. 20, a captured p217+ tau peptide or fragment thereof ("long p217+" Only the amino acid residue 11 of the tau protein can be detected. The detection antibody against the epitope containing 9-126 was only found in the long p217+ tau peptide. The captured p217+ tau peptides can also be detected. The tau peptide is an epitope containing amino acid residues 7 to 20 or 116 to 127 of the tau protein. and contacting the sample with a detection antibody against the tau peptide to detect long p217+ tau peptide or Detect and quantify the amount of p217+ tau peptides (long and short p217+ tau peptides) The amount of short p217+ tau peptide in a sample can be measured by Calculated by subtracting the amount of long p217+ tau peptide from the amount of + tau peptide will be done.

[0094] According to another embodiment of the present invention, the amount of p217+ tau peptide in a sample is captured and In addition to measuring the tau protein, An antibody, preferably an antibody against an epitope comprising amino acids 159 to 163 of tau protein. The total tau peptides in the sample are captured with a phosphorylation-independent capture antibody, such as a phospho-antibody. The total tau peptides obtained were analyzed by analyzing the total tau peptides containing amino acid residues 7 to 20 or 116 to 127 of the tau protein. The total long tau peptide or the total long tau peptide in the sample is contacted with a detection antibody against an epitope containing the The amount of tau peptides (long and short tau peptide fragments) can be detected and measured. The amount of short total tau peptides in a sample can be calculated by subtracting the amount of long total tau peptides from the amount of total tau peptides. The amount of α-peptide is calculated by subtracting the amount of α-peptide.

[0095] According to an embodiment of the present invention, a value related to p217+ tau peptide in a sample, e.g. For example, the amount of p217+ tau peptide and the amount of long p217+ tau peptide in the sample, Optionally, the amount of total tau peptides and the amount of total long tau fragments, and information based on the measured amounts; For example, calculated short p217+ tau peptides and short total tau peptides, or Ratios relating to p217+ tau peptides, e.g., the amount of short tau peptide fragments relative to the amount of long The ratio of the amount of short p217+ tau peptide to the amount of short tau peptide fragments. Ratio of the amount of long p217+ tau peptide to the total amount of long tau fragments. The ratios, etc., can be used for one or more diagnostic purposes.

[0096] The diagnosis is made by comparing the values ​​associated with p217+ tau peptide in a sample from a subject with the corresponding baseline values. This is done by comparing the baseline values ​​obtained in a population of healthy individuals. The baseline value may represent the average level determined in the same subject. In one embodiment, the p217+ tau peptide in a biological sample from a subject is expressed as a tau peptide at a level prior to the assay. values ​​related to the peptide, such as the amount of long or short p217 tau peptide, or p217 + ratios related to tau peptides, e.g., short p217 + the amount of long p If the ratio of 217+ to the amount of tau peptide is significantly higher than the corresponding baseline value If the subject has a tauopathy, the subject is determined to have a tauopathy. "High" means a statistically significant higher value that is not due to chance alone, with a p-value of 0.05 or less. "Significantly high" refers to values ​​of 0.05, 0.04, 0.03, 0.01, and 0.005. , with a p-value of less than 0.001, or at least about 1% greater than that seen in healthy volunteers %, 2%, 5%, or 10% higher.

[0097] In one embodiment, the method of the present invention comprises: (i) collecting a biological sample, preferably a CSF sample; The sample is contacted with a capture antibody directed against an epitope containing phosphorylated p217+ tau. (ii) capturing the p217+ tau peptide in the solution; and The peptide is contacted with a detection antibody directed against an epitope comprising amino acid residues 7 to 20 to form a nucleotide sequence. measuring the amount of p217+ tau peptide and / or the amino acid residues of tau protein; The long chain fragments in the sample are contacted with a detection antibody directed against an epitope containing residues 119-126. and (iii) measuring the amount of p217+ tau peptides. The amount of long p217+tau peptides or the amount of short p217+tau peptides Based on the ratio of the amount of tauopathy to the amount of tauopathy, the subject is diagnosed as having or having a tauopathy. and determining whether the subject is at risk of developing the disease. The amount or concentration of p217+ tau peptide in the sample is compared with the corresponding baseline value. The diagnosis can also be performed by detecting short p21 in a sample derived from a subject. The ratio of the amount of 7+ tau peptide to the amount of long p217+ tau peptide was calculated as the corresponding baseline. This can also be done by comparing with the Sline value.

[0098] In another embodiment, the method of the present invention comprises the steps of: (i) a biological sample, preferably a CSF sample; is contacted with a capture antibody against the p217+ tau epitope to detect p21 in the sample. Capture 7+ tau peptides, or tau peptides of amino acids 150-250 of tau protein. The total taupe in the sample is then contacted with a phosphorylation-independent capture antibody directed against the epitope. (ii) capturing the captured p217+ tau peptide or the captured total peptide; The tau peptide is directed against an epitope containing amino acid residues 116 to 127 of the tau protein. and contacting the sample with a detection antibody to detect long and short p217+ tau peptides. or the amount of total short tau protein; and (iii) measuring the amount of the biological sample. The ratio of the amount of short p217+ tau peptides to the amount of total short tau peptides in Based on this, the subject is diagnosed as having a tauopathy or as being at risk of developing a tauopathy. and determining whether the patient has pT 3 antibody, i.e., the amino acids 211 to 221 of tau are recognized by the same The amount of p217+ tau peptides in total short tau peptides containing the region of tau protein This can be done by comparing the ratio of the amount of α-tocopherol to the corresponding baseline value.

[0099] In another embodiment, the method of the present invention comprises the steps of: (i) a biological sample, preferably a CSF sample; is contacted with a capture antibody against the p217+ tau epitope to detect p21 in the sample. (ii) capturing the p217+ tau peptide; and (iii) separating the captured p217+ tau peptide from the amplified tau peptide. Long p217+ was contacted with a detection antibody against an epitope containing amino acid residues 7-20. Measuring the amount of tau peptide and / or measuring the amount of amino acid residues 116-117 of tau protein 27, and the long and short (iii) measuring the amount of p217+tau peptide; or the amount of short p217+ tau peptide relative to the amount of long p217+ tau peptide and determining the efficacy of the treatment in the subject based on the ratio.

[0100] In yet another embodiment, the method of the present invention comprises: (i) a biological sample, preferably a CSF sample; The sample is contacted with a capture antibody against the p217+ tau epitope to detect p217+ tau in the sample. Capture of 217+ tau peptides, or amino acids 150-250 of the tau protein The total tau in the sample is then contacted with a phosphorylation-independent capture antibody against a tau epitope. (ii) capturing the captured p217+tau peptide or the captured p217+tau peptide; The total tau peptide was targeted to an epitope containing amino acid residues 116 to 127 of the tau protein. The long and short p217+ taupeptides in the sample are contacted with a detection antibody against the (iii) measuring the amount of short tau protein or the amount of total short tau protein in the biological sample; Ratio of the amount of short p217+ tau peptides in the pool to the amount of total short tau peptides and determining the effectiveness of the treatment in the subject based on the amount of

[0101] In yet another embodiment, the efficacy of treatment in a subject is determined by measuring 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 total short of the amount of short p217+ tau peptide before, during, or after treatment. The amount of tau peptide in the blood is determined by monitoring the ratio of the amount of tau peptide in the blood to the amount of baseline tau peptide. A decrease in the value for tau indicates a positive response to treatment. may temporarily increase in biological fluids when cleared from the body.

[0102] According to a specific embodiment, the tauopathy is Alzheimer's disease (familial Alzheimer's disease). Alzheimer's disease and sporadic Alzheimer's disease), linked to chromosome 17 and causing parkinsonism Frontotemporal dementia with progressive supranuclear palsy (FTDP-17), corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia ), diffuse neurofibrillary tangle disease with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinson-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute Myotonic dystrophy, non-Guam motor neuron syndrome with neurofibrillary tangles Parkinson's disease, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (pugilistica) ) are not limited to.

[0103] Preferably, the tauopathy is Alzheimer's disease (familial Alzheimer's disease and sporadic Alzheimer's disease). Alzheimer's disease), FTDP-17, or progressive supranuclear palsy.

[0104] Most preferably, the tauopathy is Alzheimer's disease (familial Alzheimer's disease and diffuse alveolar leukemia). (including onset Alzheimer's disease).

[0105] According to one embodiment, the method of the present invention comprises: (i) extracting a biological sample, preferably a CSF sample; The sample is contacted with a capture antibody against the p217+ tau epitope to identify p217+ tau in the sample. (ii) capturing the p217+ tau peptide; and (iii) separating the captured p217+ tau peptide into Long p217 was contacted with a detection antibody directed against an epitope containing amino acid residues 7-20. + Measuring the amount of tau peptide and / or amino acid residues 116 to 118 of the tau protein 127 in the sample. (iii) measuring the amount of short p217+ tau peptide; The amount of long p217+ tau peptides or the amount of short p217+ tau peptides Based on the ratio of tau to p217, the subject is determined to be suitable for anti-p217+ tau antibody therapy. This includes:

[0106] According to a specific embodiment, the amount of p217+ tau peptide in a biological sample or the amount of p217+ tau peptide in a biological sample is measured. The amount of short p217+ tau peptides in the pool was compared with the amount of long p217+ tau peptides. If the ratio of p217 to p217+ tau is significantly higher than the corresponding baseline value, the subject is are deemed suitable for therapy.

[0107] According to another specific embodiment, the method of the present invention comprises: (i) collecting a biological sample, preferably CSF; The sample is contacted with a capture antibody against the p217+ tau epitope, The capture of p217+ tau peptide, or amino acids 150-25 of the tau protein 0 tau epitope in the sample, (ii) capturing total tau peptides; and (iii) capturing p217+tau peptides or captured p217+tau peptides. The total tau peptides were analyzed by the epitope containing amino acid residues 116 to 127 of the tau protein. The sample is contacted with a detection antibody against long and short p217+ tau. (iii) measuring the amount of the peptide or the total amount of short tau protein in the living body; The amount of short p217+ tau peptides in the sample relative to the amount of total short tau peptides Based on the amount of the ratio, whether the subject is suitable for anti-p217+ tau antibody therapy is determined. This includes:

[0108] According to one embodiment, the amount of short p217+ tau peptides is If the ratio of the amount of anti-p217+ The patient is deemed suitable for tau antibody therapy.

[0109] The present invention also relates to p217+ tau complexed with an antibody in a biological sample, and It also relates to the measurement of free p217+ tau in a sample that is not bound to the antibody. Total antibodies are captured using affinity techniques, followed by chaotrope, heat inactivation, or p217+ tau is subjected to denaturing conditions, including other protein disruption techniques. and measured using the method of the present invention, thereby This allows for quantification of bound p217+ tau.

[0110] According to a general aspect, the present invention provides a method for monitoring treatment of a subject with an anti-p217+ tau antibody. 1. A method of monitoring a biological sample, comprising: (i) obtaining a biological sample from a subject; and (ii) detecting the biological sample. The biological sample is separated into an IgG-enriched sample containing p217+ tau bound to the antibody, and and an IgG-depleted sample containing p217+ tau without antibody, and (i ii) Purify the p217+ tau from IgG by rpHPLC to obtain antibody-free (iv) obtaining a p217+ tau sample; and (iv) combining the IgG-enriched sample and the antibody. Each of the p217+ tau-free samples was subjected to capture assays against the p217+ tau epitope. contacting the sample with an antibody to capture p217+tau peptides in each of the samples; and (v) analyzing the captured p217+ tau peptides in each of the samples. Long p217 was contacted with a detection antibody directed against an epitope containing amino acid residues 7-20. + Measuring the amount of tau peptide, or amino acid residues 116-127 of the tau protein and contacting each of the samples with a detection antibody directed against an epitope comprising (vi) measuring the amount of long and short p217+ tau peptides bound to the antibody; Calculate the ratio of the amount of p217+ tau present to the amount of p217+ tau without antibody. and (vii) a response to anti-p217+ tau antibodies in the subject based on the calculated ratio. and monitoring the treatment.

[0111] According to another general aspect, the present invention provides a method for treating a subject with an anti-p217+ tau antibody. 1. A method for monitoring a subject, comprising: (i) obtaining a biological sample from a subject; and (ii) A semi-denatured sample was obtained from the biological sample containing total p217+ tau, and antibody-free p2 obtaining a non-denatured sample from a biological sample containing 17+ tau, (iii) heating the semi-denatured sample to denature the antibodies in the sample; ) each of the semi-denatured sample and the non-denatured sample is subjected to p217+ tau epitope and contacting the sample with a capture antibody against p217+tau peptides. and (iv) capturing the captured p217+ tau in each of the samples. The peptide is contacted with a detection antibody directed against an epitope containing amino acid residues 7 to 20 to detect the peptide. measuring the amount of tau peptide, or the amount of tau protein at amino acid residue 1 Each of the samples is contacted with a detection antibody against an epitope including 16 to 127. (v) measuring the amount of long and short p217+ tau peptides in the The amount of antibody-free p217+ tau was subtracted from the amount of p217+ tau. (vi) calculating the amount of p217+ tau bound to the antibody in the sample; and Calculate the ratio of antibody-bound p217+ tau to antibody-free p217+ tau (vii) determining whether or not the subject has an anti-p217+tau antibody response based on the calculated ratio; and and monitoring the bodily treatment.

[0112] In particular embodiments, the effectiveness of a treatment in a subject can be assessed by measuring the efficacy of the treatment before, during, or after the treatment. , monitoring the amount of antibody-bound and antibody-free p217+ tau peptide. Antibody-free p217+ tau values ​​relative to baseline are determined by a decrease in or an increase in antibody-bound p217+ tau levels relative to baseline, Therefore, the antibody containing p217+ tau binding to the antibody relative to baseline An increase in the ratio of p217+ tau to non-p217+ tau indicates a positive response to treatment. The level of p217+ tau that is not detected is the biological level when pathological tau is cleared from the brain. There may also be a temporary increase in the fluid.

[0113] According to a specific embodiment, the capture antibody of the method of the present invention is conjugated to beads, such as magnetic beads. According to another specific embodiment, the detection antibody is biotinylated.

[0114] According to a specific embodiment, the amount of p217+ tau peptide measured by the method of the present invention is E Any known in the art, including LISA and single molecule array platforms According to a particular embodiment, the method of the present invention comprises: High-sensitivity array platforms such as Quanterix Simoa or MSD S-plex The form is used to measure the amount of p217+ tau peptide in a sample. According to the method of the present invention, the lower limit of quantitation is about 40 fg / mL, and the lower limit of detection of the method is It is approximately 2 fg / mL.

[0115] According to a specific embodiment, the sample used in the method of the present invention is blood, brain homogenate, or a biological sample such as a cerebrospinal fluid (CSF) sample. Preferably, the sample is According to a specific embodiment, the sample is a crude CSF sample. According to a specific embodiment, the sample contains full-length tau protein and tau fragments of differing sizes. and CSF using reversed-phase high-performance liquid chromatography (rpHPLC) to separate obtained after fractionation of a biological sample.

[0116] According to a specific embodiment, the capture antibodies of the method of the present invention are SEQ ID NOs: 32, 33, and and immunoglobulin heavy chain HCDR1, HCDR2, and H having polypeptide sequences of 34. CDR3 and the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively. Preferably, the capture antibody comprises an immunoglobulin light chain LCDR1, LCDR2, and LCDR3. The antibody comprises a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO:28 and a polypeptide of SEQ ID NO:29. The pT3 antibody contains a light chain variable region having a nucleotide sequence.

[0117] According to a specific embodiment, the detection antibodies of the method of the present invention are those of SEQ ID NOs: 2, 3, and 4, respectively. an immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the polypeptide sequence 3, and immunoglobulins having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7, respectively. Preferably, the detection antibody comprises the light chain LCDR1, LCDR2, and LCDR3. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and a The antibody is a pT82 antibody containing a light chain variable region comprising:

[0118] According to another specific embodiment, the detection antibodies of the method of the present invention are those of SEQ ID NOs: 12 and 13, respectively. and immunoglobulin heavy chain HCDR1, HCDR2, and and HCDR3, and the polypeptide sequences of SEQ ID NOs: 15, 16, and 17, respectively. Preferably, the immunoglobulin light chain LCDR1, LCDR2, and LCDR3 are The antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a polypeptide sequence of SEQ ID NO: 19. The antibody is an hT43 antibody containing a light chain variable region having a polypeptide sequence.

[0119] kit In another general aspect, the invention provides: (a) a capture antibody against the p217+ tau epitope; , optionally a phospho-antibody against a tau epitope between amino acids 150 and 250 of the tau protein (b) an oxidation-independent capture antibody; and (b) an antibody targeting amino acid residues 7-20 or 116-1 of tau protein. and at least one detection antibody against a tau protein epitope, including 27. The kit was used to measure the amount of p217+ tau peptide, which was measured in the samples. The amount of short p217+ tau peptides in the pool was compared with the amount of long p217+ tau peptides. the ratio of the amount of short p217+ tau peptides to the amount of total short tau peptides, and / or the amount of short p217+ tau peptides Used as a ratio to

[0120] The detection antibody may be directly detectable or may undergo a secondary reaction (e.g., reaction with streptavidin). Any detectable label (e.g., fluorescent label, biotin, etc.) that is detectable via a Alternatively, a second reagent containing a detectable label may be used. In this case, the second reagent has binding specificity for the primary antibody. In a diagnostic kit suitable for measuring p217+ tau in It may be supplied pre-bound to a solid phase such as a well in a food dish or beads.

[0121] According to a specific embodiment, the capture antibodies of the kit of the present invention are those of SEQ ID NOs: 32, 33, and 34 polypeptide sequences of immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3, and the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively. It comprises immunoglobulin light chain LCDR1, LCDR2, and LCDR3. The antibody comprises a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO:28 and a polypeptide of SEQ ID NO:29. The pT3 antibody comprises a light chain variable region having a peptide sequence.

[0122] According to a specific embodiment, the detection antibodies of the kit of the present invention are SEQ ID NOs: 2, 3, and Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR having the polypeptide sequence of R3, and immunoglobulins having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7, respectively. Preferably, the detection antibody comprises the sequence A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and a polypeptide variable region having the polypeptide sequence of SEQ ID NO: 9. The antibody is a pT82 antibody containing a light chain variable region that

[0123] According to another specific embodiment, the detection antibodies of the kit of the present invention are those of SEQ ID NOs: 12, 1 immunoglobulin heavy chain HCDR1, HCDR2, having polypeptide sequences of 3 and 14; and HCDR3, and the polypeptide sequences of SEQ ID NOs: 15, 16, and 17, respectively. Preferably, the immunoglobulin light chain comprises LCDR1, LCDR2, and LCDR3. The detection antibody comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19. The hT43 antibody comprises a light chain variable region having a polypeptide sequence:

[0124] According to another specific embodiment, the kit of the present invention is adapted to detect in a sample using the method of the present invention. The amount of p217+ tau peptides, which is the amount of short p217+ tau peptides the ratio of the amount of p217+ tau peptide to the amount of short p217+ tau peptide The ratio of the amount of short tau peptides to the amount of total short tau peptides is used to measure the ratio.

[0125] All cited references (including article references, issued patents, published The contents of the above-referenced patent application, including any co-pending patent applications, are expressly incorporated herein by reference. Be absorbed.

[0126] Embodiment The present invention also provides the following non-limiting embodiments.

[0127] Embodiment 1 is a method for measuring the amount of p217+ tau peptide in a sample, comprising: (i) contacting the sample with a capture antibody against the p217+ tau epitope; capturing p217+ tau peptides in the sample; (ii) The captured p217+tau peptide was ligated to amino acid residue 119 of the tau protein. an epitope comprising amino acid residues 116-126, e.g., amino acid residues 116-127, or an epitope comprising amino acid residues 116-127 of the tau protein and contacting the antibody with a detection antibody against an epitope containing amino acid residues 7 to 20, respectively. measuring the amount of p217+ tau peptide or the amount of long p217+ tau peptide; The method includes:

[0128] Embodiment 2 is directed to detecting long p217+ tau peptides or short p217 tau peptides in a sample. 1. A method for determining relative amounts of peptide fragments, comprising: (i) contacting the sample with a capture antibody against the p217+ tau epitope; capturing p217+ tau peptides in the sample; (ii) The captured p217+tau peptide was ligated to amino acid residue 119 of the tau protein. contacting the p217+ tau peptide with a first detection antibody directed against an epitope comprising .about.126; measuring the amount of (iii) cleaving the captured p217+tau peptide by the amino acid residues of the tau protein; and contacting the long p217+ tau with a second detection antibody directed against an epitope comprising 7-20. measuring the amount of the peptide; (iv) the amount of the p217+tau peptide and the amount of the long p217+tau peptide The relative amounts of long p217+ tau peptides or short p217+ tau peptides based on and determining:

[0129] In a third embodiment, the capture antibody is conjugated to a bead and the detection antibody is conjugated to a bead. 3. The method of embodiment 1 or 2, wherein the antibody is oxaliplatinized.

[0130] In a fourth embodiment, the amount of p217+ tau peptide in the sample is determined using a highly sensitive platform. The method according to any one of embodiments 1 to 3, wherein the measurement is performed using a fluoroscopy.

[0131] In embodiment 5, the lower limit of quantitation 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. The method according to any one of the first to fourth embodiments.

[0132] Embodiment 6 is a method for detecting a CSF sample from a subject, the method comprising: and the method comprises determining the amount of the p217+ tau peptide in the biological sample, The ratio of the amount of p217+ tau peptide to the amount of long p217+ tau peptide, or short based on the ratio of the amount of short p217+ tau peptide to the amount of total short tau peptides The subject may be diagnosed with or at risk of developing a tauopathy. 6. The method of any one of embodiments 1 to 5, further comprising determining whether .

[0133] Embodiment 7 is a method for determining the amount of the p217+ tau peptide in the biological sample, the short the ratio of the amount of p217+ tau peptide to the amount of long p217+ tau peptide, or The ratio of the amount of short p217+ tau peptides to the amount of total short tau peptides is baseline values, e.g., significantly higher than the corresponding mean values ​​of healthy volunteers the subject is diagnosed as suffering from or at risk of developing a tauopathy, 7. The method of embodiment 6, wherein the

[0134] Embodiment 8 is a biological sample from a subject undergoing treatment for a tauopathy, preferably Preferably, the biological sample is a CSF sample, and the method comprises detecting the p217+ tau in the biological sample. The amount of the short p217+tau peptide and the amount of the long p217+tau peptide the ratio of the amount of short p217+ tau peptide to the amount of total short tau peptides, or determining the efficacy of the treatment in the subject based on the ratio of the amount of ribonucleotide to the amount of ribonucleotide. The method according to any one of embodiments 1 to 5 further comprises:

[0135] Embodiment 9 is a method for determining whether the amount of the p217+ tau peptide in the biological sample increases over the course of treatment. In the method of embodiment 8, the treatment is determined to be effective if the measured value of the IL-1 signal is reduced over a period of time. be.

[0136] In embodiment 10, the tauopathy is Alzheimer's disease (familial Alzheimer's disease and and sporadic Alzheimer's disease), frontal locomotion syndrome with parkinsonism linked to chromosome 17 Temporal dementia (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease , progressive subcortical gliosis, tangle-only dementia, calcification Diffuse neurofibrillary tangle disease with aging, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkin Son dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Halaf Wolden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Roman-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing angiopathy Panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles , postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (pugilist's disease). The method according to any one of embodiments 6 to 9, wherein the compound is selected from the group consisting of:

[0137] Embodiment 11 is the method of embodiment 10, wherein the tauopathy is Alzheimer's disease. This is the method.

[0138] Embodiment 12 is a method for treating a subject, the method comprising administering to a subject a biological sample from a human subject, preferably a CSF sample. the method comprises determining the amount of the p217+ tau peptide in the biological sample, the ratio of the amount of the short p217+ tau peptide to the amount of the long p217+ tau peptide or the amount of said short p217+ tau peptide relative to the amount of total short tau peptides Based on the ratio, it is possible to determine whether the subject is suitable for anti-p217+ tau antibody therapy. The method according to any one of embodiments 1 to 5, further comprising:

[0139] Embodiment 13 is a method for determining the amount of the p217+ tau peptide in the biological sample, ... 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 the short p217+ tau peptide to the amount of the total short tau peptide is If the baseline value is significantly higher than the corresponding mean value of healthy volunteers, e.g. 13. The method of claim 12, wherein the subject is determined to be suitable for anti-p217+ tau antibody therapy if This is the method described above.

[0140] Embodiment 14 is a method for monitoring treatment with an anti-p217+ tau antibody in a subject. It is a law, i. obtaining a biological sample from the subject; ii. The biological sample is subjected to IgG enrichment containing p217+ tau bound to the antibody. and an IgG-depleted sample containing p217+ tau without antibody. And, iii. Each of the IgG-enriched sample and the IgG-depleted sample is subjected to the tau assay. capture of an epitope containing phosphorylated T212 and / or phosphorylated T217 of a protein The sample is contacted with a capture antibody to capture p217+ tau peptide in each of the samples. And, iv. The captured p217+tau peptide is then purified by cleaving the captured p217+tau peptide from amino acid residues 7-20 of the tau protein. or 116 to 127, and contacting the biological sample with a detection antibody against an epitope including the amount of p217+ tau bound to the antibody in the and measuring the amount of v. p217+ tau bound to the antibody versus p217+ tau not containing the antibody Calculating the ratio of vi. Based on the calculated ratio, determining whether the subject is treated with the anti-p217+ tau antibody. and monitoring the treatment.

[0141] Embodiment 15 is a method for monitoring treatment with an anti-p217+ tau antibody in a subject. It is a law, i. obtaining a biological sample from the subject; ii. Obtain a semi-denatured sample from a biological sample containing total p217+ tau and obtaining a non-denatured sample from a biological sample containing p217+ tau that is not denatured, heating the semi-denatured sample to denature the antibodies in the sample; iii. Each of the semi-denatured sample and the non-denatured sample is subjected to the analysis of the tau protein. a capture antibody against an epitope containing phosphorylated T212 and / or phosphorylated T217 of the protein; and capturing the p217+ tau peptide in each of the samples. And, iv. The captured p217+tau peptide is then purified by cleaving the captured p217+tau peptide from amino acid residues 7-20 of the tau protein. or 116 to 127, and contacting the biological sample with a detection antibody against an epitope including The amount of total p217+ tau and the amount of antibody-free p217+ tau in the And, v. Subtract the amount of p217+ tau that does not contain the antibody from the amount of total p217+ tau. and calculate the amount of p217+ tau bound to the antibody in the sample. and, vi. p217+ tau bound to the antibody to p217+ tau not containing the antibody Calculating the ratio of vii. Based on the calculated ratio, the anti-p217+ tau antibody in the subject and monitoring the treatment.

[0142] Embodiment 16 is a method for preparing a capture antibody comprising the steps of: immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the nucleotide sequence Immunoglobulin light chains having the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively and LCDR1, LCDR2, and LCDR3, and preferably the capture antibody has SEQ ID NO: A heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28 and a polypeptide sequence of SEQ ID NO: 29. 16. The method according to any one of embodiments 1 to 15, wherein the antibody has a light chain variable region that is

[0143] Embodiment 17 is a method for detecting a polypeptide comprising the steps of: immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having sequences immunoglobulin light chain LCDR1 having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7; and LCDR2 and LCDR3, and preferably the detection antibody is a polypeptide of SEQ ID NO: 8. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10 17. The method of any one of embodiments 1 to 16, comprising the steps of:

[0144] Embodiment 18 is a method for detecting a denatured ... immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the nucleotide sequence Immunoglobulin light chains having the polypeptide sequences of SEQ ID NOs: 15, 16, and 17, respectively and LCDR1, LCDR2, and LCDR3, and preferably the detection antibody is a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a polypeptide sequence of SEQ ID NO: 19 17. The method of any one of embodiments 1 to 16, wherein the light chain variable region comprises:

[0145] Embodiment 19 is a method for detecting a subject in which the sample is blood, a brain homogenate, or a cerebrospinal fluid (CSF) sample. 19. The method according to any one of embodiments 1 to 18, which is a simple method.

[0146] Embodiment 20 is a method for determining whether the sample is purified by reversed-phase high performance liquid chromatography (rpHPLC). 20. The method of any one of embodiments 1 to 19, wherein the method is obtained after fractionating a biological sample using This is the method described above.

[0147] Embodiment 21 is directed to an epitope comprising amino acid residues 116 to 127 of tau protein. An isolated detection antibody or antigen-binding fragment thereof that binds to tau protein in a subject, a. immunoglobulin heavy chain H having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively CDR1, HCDR2, and HCDR3, b. Immunoglobulin light chain L having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7, respectively An isolated detection antibody or its antigen binding domain, comprising CDR1, LCDR2, and LCDR3. It is a combined fragment.

[0148] Embodiment 22 preferably comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8 and and a light chain variable region having the polypeptide sequence of SEQ ID NO: 9. The isolated detection antibody or antigen-binding fragment thereof.

[0149] Embodiment 23 is directed to a method for identifying a tau protein in an epitope comprising amino acid residues 7 to 20 of the tau protein. 1. An isolated detection antibody or antigen-binding fragment thereof that binds to a protein, a. Immunoglobulins having the polypeptide sequences of SEQ ID NOs: 12, 13, and 14, respectively heavy chain HCDR1, HCDR2, and HCDR3; b. Immunoglobulins having the polypeptide sequences of SEQ ID NOS: 15, 16, and 17, respectively an isolated detection antibody or its derivatives, comprising light chains LCDR1, LCDR2, and LCDR3; It is an antigen-binding fragment.

[0150] Embodiment 24 preferably 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. or an antigen-binding fragment thereof.

[0151] Embodiment 25 is a method for detecting a detection antibody or an antigen-binding fragment thereof according to any one of Embodiments 21 to 24. An isolated nucleic acid encoding a fragment thereof.

[0152] Embodiment 26 is a vector comprising the nucleic acid of embodiment 25.

[0153] Embodiment 27 is a host cell comprising the nucleic acid of embodiment 25.

[0154] Embodiment 28 is a method for detecting a detection antibody or its antigen binding agent according to any one of embodiments 21 to 24. and a method for producing an antibody or antigen-binding fragment, the method comprising: culturing the cells under conditions to produce the antibody or antigen-binding fragment; or recovering the antibody or antigen-binding fragment from the cell culture.

[0155] Embodiment 29 is a. single or multiple tau proteins, including phosphorylated T212 and / or phosphorylated T217 of tau protein; a capture antibody against a multiply phosphorylated tau protein epitope; b. Tau protein containing amino acid residues 7 to 20 or 116 to 127 of tau protein and a detection antibody against the epitope, A kit used to measure the amount of p217+ tau peptide in a sample. .

[0156] Embodiment 30 is a method for preparing a capture antibody comprising the steps of: immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the nucleotide sequence Immunoglobulin light chains having the polypeptide sequences of SEQ ID NOs: 35, 36, and 37, respectively and LCDR1, LCDR2, and LCDR3, and preferably the capture antibody has SEQ ID NO: A heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 28 and a polypeptide sequence of SEQ ID NO: 29. 30. The kit of embodiment 29, wherein the kit has a light chain variable region corresponding to

[0157] Embodiment 31 is a method for detecting an isolated antibody according to any one of embodiments 20 to 23. 31. The kit of embodiment 29 or 30, wherein the detection antibody is a [Example]

[0158] The following examples of the present invention are intended to further illustrate the principles of the present invention. The examples do not limit the invention, the scope of which is defined by the appended claims. I would like you to understand this.

[0159] Example 1. Highly sensitive assay for detecting p217+ tau Assay-specific reagents were: Simoa Homebrew kit (Quanterix, Cat. No. 101351), helper beads (Quanterix rix, Catalog No. 101732), pT3 mouse monoclonal antibody (mAb), h T43 mAb, pT82 mAb, and hT7 mAb. pT3 inhibits p217+ tau. The parent antibody, developed at Janssen, recognizes the humanized version of which is described herein. Herein, this is referred to as humanized pT3 mAb.

[0160] Samples were buffered in 50 mM Tris, 50 mM NaCl, 5 mM EDTA, 2% bovine serum albumin (BSA). serum albumin, 0.1% Tween 20, 0.05% ProClin 300; Diluted to pH 7.8.

[0161] Three custom peptides created by New England Peptide The assay was calibrated using (calibration peptide).

[0162] The peptide pT3xhT43 is composed of hT43, PT5, and PEG linked by a PEG linker. 1 and pT3 epitopes and has a molecular weight of 6893 g / mol. The amino acid sequence of 3xhT43 is PRQEFEVMEDHAGTYGLGDR(dPEG 4)GKTKIATPRGAAPPGQKG(dPEG4)GSRSR(pT)PSLP (pT)PPTREPKKV-amide (SEQ ID NO: 22).

[0163] The peptide pT3xpT82 is composed of pT82 and pT3 linked by a PEG4 linker. Peptide pT3xpT82 contains 3 epitopes and has a molecular weight of 4551 g / mol. The amino acid sequence is Ac-SLEDEAAGHVTQARMVSK(dPEG4)GSR SR(pT)PSLP(pT)PPTREPKKV-amide (SEQ ID NO: 23).

[0164] The peptide hT7xpT82 consists of pT82 and hT linked by a PEG4 linker. Peptide hT7xpT82 contains 7 epitopes and has a molecular weight of 3619 g / mol. The amino acid sequence is Ac-SLEDEAAGHVTQARMVSK(dPEG4)PRG AAPPGQKGQANA-amide (SEQ ID NO: 24).

[0165] Preparation of reagents Follow the protocol provided in the Quanterix manual to separate the capture beads into 0. The coated capture beads were coated with 0.3 mg / mL capture Ab. Dilute to 200,000 beads / mL with dilution buffer for a total bead concentration of 400,000 beads. Helper beads were added at 200,000 beads / mL to achieve a total volume of 200,000 beads / mL.

[0166] Following the protocol provided in the Quanterix manual, the detection antibody was added for 60 min. Biotinylated at 1:200 and diluted to 1.8 μg / mL in Homebrew Detection Agent / Sample Diluent did.

[0167] Reconstitute the calibration peptide to 5 mg / mL in 0.1% phosphoric acid / water and aliquot into 20 µL When ready for use, thaw an aliquot of the calibration peptide and mix 1:1 Dilute the peptide by 100% (e.g., from 1.5 μL to 1498.5 μL) until the final concentration of the peptide is 5 The dilutions were diluted 1:1000 to give a total of 3000 pg / mL. A standard curve with a 3-fold jump was prepared.

[0168] CSF samples were diluted at least 1:4 with sample diluent. V) Samples were diluted 1:5 or 1:10, and AD samples were diluted at least 1:20. .

[0169] Simoa assay Capture Ab, sample, and detection Ab for 35 minutes, followed by washing and streptavidin. A two-step protocol including 5 min with bisphosphonate β-galactosidase (SBG) was used. A standard Simoa assay was created. Each reaction contained 25 μL of bead solution, 100 μL of Each tube contained 20 μL of sample or calibrator, 20 μL of detection solution, and 100 μL of SBG. You can assign names and load up to five capture antibodies and five detection antibodies at once. The reaction was carried out in a Simoa cuvette by the instrument, and one final wash was performed before the β-galactosidase reaction. After loading the measuring disc with the ribosomal enzyme substrate (RGP), the measurement was carried out by the instrument. went.

[0170] Example 2. Separation of native tau fragments by rpHPLC The reagents were as follows: trifluoroacetic acid (HPLC grade), water (HPLC grade), acetonitrile (HPLC grade), phosphoric acid (analytical grade), and HP LC binary gradient system, immunoassay buffer (100 mM TrisHCl, 100 mM NaCl, 0.05% Tween, and BSA, pH 7.8).

[0171] The protocol was as follows: 500 μL of frozen CSF was thawed on ice for 30 minutes. The thawed CSF was dissolved in 100 mM sodium phosphate buffer containing 100 mM sodium chloride. The resulting mixture (1.8 mL) was added to 1.5 mL of sodium (pH 2.5) and mixed. C18 or similar reversed-phase chromatography column equilibrated in 0.1% trifluoroacetic acid in water The HPLC column was then developed with an increasing gradient of acetonitrile. Fractions were collected during elution. The fractions were adjusted to 10 mM with guanidine HCl and then The dried fraction was resuspended in immunoassay buffer and purified using the method of the present invention. Based on the anti-tau capture and detection antibody pair, the tau peptides in the fractions were measured.

[0172] Example 3. Quantification of antibody-free or antibody-bound p217+ tau Additional upstream sample manipulations can be used to extract the protein from the patient, whether produced within the patient or administered exogenously. Binding of p217+ tau by any antibody, e.g., humanized pT3 mAb, is measured. A highly sensitive pT3-based assay can be used to identify and characterize the pT3-mediated HIV infection. Pharmacokinetic assays to study anti-p217+ tau antibodies, e.g., humanized pT3 mAb For example, the following method can be used to detect antibody-free antibodies. p217+ tau bound to the IL-1 receptor can be measured.

[0173] Assay 1: Release in biological fluids using immunocapture / depletion followed by rpHPLC Quantification of pair-bound p217+ tau Biological fluids (e.g., CSF) are transferred to magnetic beads coated with Protein A / G. The beads were then mixed with the PBS (15 μL of bead slurry per 0.5 mL of CSF) at room temperature with rocking. The beads were incubated for 2 hours to capture immunoglobulins in the sample. The supernatant was transferred to a second tube (sample = "IgG-depleted supernatant"). The tube was washed four times with 1 mL of cold phosphate-buffered saline (PBS). of 6 M GuHCl was added to (a) the washed beads and (b) the tube containing the IgG-depleted supernatant. The tube was then incubated at room temperature with rocking for 20 minutes. The beads were then precipitated using a magnet, and the resulting supernatant was transferred to a third tube (sample = "Ig"). Finally, the eluate was separated by rpHPLC in the same manner as in Example 2. Before this, 0.1 M phosphoric acid (pH 2) was added to the two solutions (1.0 mL of phosphoric acid was added to the denatured I Add 1.5 mL of phosphoric acid to the IgG-depleted supernatant and 1.5 mL of phosphoric acid to the IgG-enriched supernatant to complete the final step of the sample. The resulting rpHPLC fraction was purified as described in Example 2. Constructed and measured using the Simoa p217+ tau assay described in Example 1. IgG depletion The signal from the supernatant represents free p217+ tau (i.e., not bound to the antibody). , whereas the signal from IgG-enriched supernatant was higher than that from bound p217+ tau (i.e., humanized pT (3) represents the amount of antibody bound to an antibody such as mAb. As a normalization factor, we performed an immunocapture / depletion process to assess total p217+ tau signal. rpHPLC separation and Si of the same parent biological fluid (e.g., CSF) that was not subjected to The moa p217+ measurements were analyzed simultaneously.

[0174] Assay 2: Free vs. bound p217+ tau in biological fluids using heat denaturation of antibodies quantitative An aliquot of the biological fluid of interest (e.g., CSF) is heated at 95°C for 4 minutes, followed by , and cooled on ice for 4 minutes (sample = "semi-denatured fluid"). In parallel, a second assay of the same fluid was performed. The recoat was cooled on ice for 8 minutes (sample = non-denatured fluid). Both samples were measured using the imoa p217+ tau assay. The former represents total p217+ tau, whereas the latter represents free p217+ tau. Subtracting the latter from gives a measure of bound tau. The exact heating time and temperature are determined by Si Any smears in the fluid so that they cannot further interfere with the moa p217+ tau assay. Although we decided to irreversibly modify the antibody, the p217+ tau signal itself had no effect. This assay was used to determine whether the antibody was binding to p217+ tau. The assay demonstrates that competing antibodies are present, but is not a direct measure of This assay produced results similar to those of the more laborious Assay 1.

[0175] Example 4. Biological samples Samples used in assay development and technical qualification The assays of Examples 1-3 were performed using pooled CSF from human subjects with high tau levels. Some experiments have also been conducted in healthy volunteers in phase 1 trials. To ensure the assay sensitivity required for this study, samples were prepared from human subjects with low tau levels. The study was carried out using purified CSF. Cynomolgus monkeys (Macaca fascicularis) and com- mands obtained from CSF derived from marmosets (Callithrix jacchus) was also used in Examples 1 to 3. In addition, cognitively normal human subjects and common marmosets were also measured using the assay. Snap-frozen brain samples from the mice were homogenized and assayed using the assays described in Examples 1 and 3. Several experiments were performed using individual sera from clinically defined HV and AD subjects. This was carried out using

[0176] Samples used for preliminary clinical qualification Cohort 1 ("Inter-assay correction cohort"): Subjects with normal pressure hydrocephalus (NPH) ( Ventricular fluid (VF) and lumbar fluid (LF) CSF samples were obtained from 11 patients (Uni (Varsity of Kuopio, Professor Ville Lenoinen). Samples were collected from the University of Sahlgrenska (Kaj Blen CSF Aβ4 determined by Innotest assay performed by Professor now 2, total tau (t-tau), and p-tau181 measurements, as well as brain biopsy amyloid and Tau immunohistochemistry (IHC) assays were used to separate the tau-positive cells. a p217+ tau assay was performed using the Janssen Neuroscience Biomarkers I went to kers (La Jolla).

[0177] Cohort 2 ("overt HV vs. overt AD cohort"): biochemically defined Alzheimer's disease LF C from Zheimer's disease (AD) vs. healthy volunteer (HV) subjects (n=20 / group) SF samples were collected from the University of Sahlgrenska (KaJ Bl CSF Aβ42, t Tau and p-tau measurements were performed at the University of Sahlgrenska A large panel of samples was analyzed based on separation to a predetermined AD vs. HV cutoff measurement. Samples were selected from the 100% NIH (AD = CSF Aβ42 < 400 pg / mL and CSF t Tau > 600 pg / mL, HV = CSF Aβ42 > 400 pg / mL and CSF t-TA (<600 pg / mL). rpHPLC and Simoa p217+ tau assays were performed ssen Neuroscience Biomarkers (La Jolla) It was.

[0178] Cohort 3 ("HV vs. ARAD vs. early AD cohort"): Clinically defined normal (clinical Subjects with clinical dementia rating of 0 (CDR 0) vs. mild memory complaints (CDR 0.5) (n=20 / group) Previous LF CSF samples were obtained from JANSSEN studies ALZ1005 / 1002. CSF Aβ42, t-tau, and p-tau181 measurements by Innotest assay CDR and CSF were performed at the University of Sahlgrenska. Based on the Aβ42 score, subjects were classified as: (a) HV=CDR0 and Aβ42>600pg / mL, (b) risk of AD (ARAD) = CDR0 and Aβ42 < 600 pg / mL, (c) Potential non-AD dementia = CDR 0.5 and Aβ42 > 600 pg / mL, and (d ) Initial AD = CDR 0.5 and Aβ42 < 600 pg / mL. The Simoa p217+ tau assay was funded by Janssen Neuroscience B I did it at iomarkers (La Jolla).

[0179] Cohort 4 ("CDR0 vs. CDR1 cohort"): Clinically defined normal (clinical LF C from subjects with dementia rating scale 0 (CDR0) vs. mild memory complaints (CDR1) (n=5 / group) SF samples were obtained from Washington University. and MMSE, and CSF Aβ42, t-tau, and p by Innotest assay Tau-181 measurements were obtained at Washington University. , and the samples so that Janssen does not know the identity or characteristics of the samples. Coded. Janssen Neuroscience Biomarkers( La Jolla) and Simoa t tau and p217+ tau measurements. and sent to Washington University for analysis.

[0180] Cohort 5 ("HV vs. MCI vs. AD cohort"): Clinically and biochemically (Innot est AB42>600pg / mL) LF CSF samples from defined HVs (n=7) Samples were obtained from Precision Medicine (San Diego). Clinically and biochemically defined (Innotest AB42 < 600 pg / mL) LF CSF samples from MCI (n=28) and AD (n=12) were collected from the University of California obtained from the city of Antwerp. rpHPLC and Simoa p217 +Tau measurements were performed using Janssen Neuroscience Biomarkers (L a Jolla).

[0181] Cohort 6 ("Disease Severity and Progression Cohort"): Clinically defined AD (clinical LF CSF samples from dementia scale 1+ subjects (n=235) were analyzed using the Janssen study These samples were obtained from all clinical trials. Baseline (pre-dose) samples were from all subjects. In addition, placebo subjects (n = 10) 90) CSF samples from 78 weeks of follow-up were included to assess biomarkers of disease progression. Cognitive assessment (ADAS-COG, MMSE, NTB, CDR.SOB), ApoE Genotype, sex, and age were obtained from the clinical trial. Innotest AB42, Innote st AB40, Simoa NFL, pT3xpT82, pT3xhT43, and hT The 7xpT82 assay was obtained from Janssen Neuroscience Biomark The study was conducted at the University of California, San Diego, La Jolla. Subjects were randomly selected based on an AB42 / 40 ratio cutoff of 0.09. were confirmed as amyloid positive or negative based on the ratio (e.g., with a ratio of <0.09). Subjects with a >0.09 score = amyloid positive = AD, whereas subjects with a >0.09 score = amyloid negative = non-AD Of the 235 subjects, 27 were determined to be amyloid-negative, and the two groups were separated. were analyzed individually.

[0182] Samples used to assess target engagement after treatment with anti-p217+ agents HV subjects (n=40) treated with placebo or JNJ63733657 (single IV injection) ) derived LF CSF from Janssen clinical trial JNJ63733657EDI1001 The pT3xpT82 assay was obtained from Janssen Neuroscience The pT3xhT43 assay was performed at Q Biomarkers (La Jolla). This was performed at uanterix Corporation (Lexington MA).

[0183] Example 5. Screening of capture and detection antibody pairs using the Simoa platform G Previous reports from Janssen Neuroscience Discovery and literature (e.g., Meredith et al. PLoS One.8(10): e76523,2013;Barthelemy et al.,J Alzheimer rs Dis.51(4):1033-43,2016;Russell et al. ,J Alzheimers Dis.55(1):303-313,2017;Han ger et al.J Biol Chem.282(32):23645-54,2 007) is a tau fragment containing amino acids 200-220, particularly amino acids 212 and 214. , suggesting that some combination of phosphorylation at 217 is enriched in AD. Therefore, we developed an assay to measure this specific tau species ("p217+ tau"). By developing a novel antibody against AD, improved biomarkers for AD diagnosis and / or staging may be obtained. In addition to the carcinoma, potential predictions and / or pharmacological agents for new drugs targeting this tau region are also available. However, tau was not found to be a ubiquitous protein in healthy volunteers. It can be present at low levels (<200 pg / mL), and p217+ tau is a minor component of total tau. Therefore, p217+ tau assays require optimal antibody pairs and high sensitivity.

[0184] To this end, we developed a set of anti-tau mAbs discovered at Janssen, as well as Several high-affinity commercially available anti-tau mAbs were found to produce sandwich ELs when paired with pT3. The ability to generate a signal in an ISA (sELISA) format was evaluated. Antibody pairs were analyzed using the Simoa HD-1 Analyzer platform (Quanter ix Corporation) and pooled CSF from AD subjects. A serial dilution of 1000 mg / mL was used to provide the required sensitivity. Assay performance was evaluated as a signal / neutral = average enzyme per bead (AEB) / assay for sample diluted in sample diluent The optimal detection antibody to pair with pT3 was based on AEB of diluent only. In order of intensity, hT43, pT82, Quanterix Tau 2.0 detection reagent, and BT2 The results were as follows (Table 1). hT43 and Quanterix Tau 2.0 detection reagents detect the N-terminus of tau. pT82 and BT2 recognize sequences closer to the central region of tau. The best N-terminal (hT43) and central region (pT82) mAbs were selected for further optimization. Janssen Neuroscience Biomarkers and Q Parallel screening was conducted at uanterix Corporation. , similar results were obtained.

[0185] [Table 1] Tau antibody epitopes and signals measured in pooled CSF from AD subjects Signal to noise (S / N) ratio is indicated. NT = Not tested.

[0186] Example 6. Optimization of pT3xhT43 and pT3xpT82 assays A series of optimization experiments was performed together with optimization assays on the Simoa platform. Based on general Quanterix experience, 10% mouse serum or 500 μg / m Addition of L mouse IgG to the detector diluent did not improve assay sensitivity. Ab concentrations (0.15, 0.3, 0.6, 1.2, and 1.8 μg / mL), SβG concentrations ( 100, 200, or 300 pM), and capture mAb bead concentration (300K / well, 1 The dose setting of the 50K + 200K helper beads was evaluated. incubation time (65 min vs. 35 min) and sample volume (100 vs. 150 μL) The ideal reagent concentrations for both assays were 150K capture biotin and 150K HCl, respectively. 1.8 μg / mL detection agent, and 200 pM SBG The sample volume and incubation time had minimal effect on the assay. The lower conditions of 100 μL sample and 35 minutes of incubation did not affect the The item was selected.

[0187] Example 7. Technical qualification of the pT3xhT43 and pT3xpT82 assays Calibrator Linear Range The calibration peptides were prepared as described in Example 1. The calibration peptides were linked by a PEG4 linker. containing the core epitopes of pT3 and pT43 or pT3 and pT82 isolated by These were used to generate a standard curve. A representative standard curve is shown in Figure 1. Calibration peptide from 30 pg / mL to 0.041 pg / mL in a 1:3 jump with buffer The doses were titrated and measured in the pT3xhT43 and pT3xpT82 assays. Create a calibration curve using a curve-fit data reduction method (4PL, 1 / y2 weighting) The lower limit of detection (LLOD) was defined as the calculated calibration level, with a 10% coefficient of variation (C The AEB was obtained equal to the mean + 2.5 standard deviations (SD) of the zero calibrators, including the V. By this standard, a LLOD of approximately 0.002 pg / mL was obtained from representative data. The linear range, lower limit of quantitation (LLOQ), and upper limit of quantitation (ULOQ) of the assay are calculated based on the expected CV. Defined as the lowest and highest standard curve points achieving <20% and 80-120% recovery. According to these criteria, both the pT3xhT43 and pT3xpT82 assays The linear range for all measurements was 0.041 to 30 pg / mL (Figure 1, Table 2).

[0188] [Table 2]

[0189] Dilution linearity with CSF To assess the linearity of dilutions and determine the ideal dilution for testing CSF samples First, a panel of four CSF samples from AD subjects (high tau, low AB42) was assayed. Titrate from 1:2 to 1:4096 dilutions in buffer and test for p217+ tau assay. Samples diluted more than 1:512 typically had concentrations below the LLOQ. The measurements of the 1:4 to 1:512 samples were linear in dilution, resulting in a C The SF samples were measured within the prescribed range. A pool of CSF from tau- and AB42-high subjects was similarly assayed. Again, a 1:4 to 1:5 ratio was used. Dilution linearity was observed for a 1:256 dilution, and above this range, measurements were below the LLOQ. The level fell to less than 100% (Figure 2).

[0190] accuracy To assess the accuracy of the measurements, a standard curve of pT3xhT43 was prepared and run on three separate plates. The calibration peptides were measured on consecutive days from 30 to 0.041 pg / mL (Figure 3 and Table 3). 1:3 jump dilutions were run in duplicate in the pT3xhT43 assay. The procedure was repeated by the same technician on three consecutive days. The assay (in which CSF samples are measured) consistently demonstrated within-run precision (intra-assay CV%) of <10 %, with an average CV of 2.46–5.18%, and the inter-assay precision averaged 6.46 These are for research use only (RU). O) Well within the 20% CV acceptance limit for the assay, and in part, All ELISA steps are automated in the imoa HD-1 Analyzer. This is due to the nature of

[0191] [Table 3]

[0192] Transferability between labs To assess the accuracy of the p217+ tau assay between laboratories, the same AD CSF pool was used. The data were collected from Janssen Neuroscience Biomarkers and Quan The same lot of reagents were used at Terix Corporation to determine the appropriate dosage. Figure 4 shows the results of the pT3xhT43 and pT3xpT82 assays at two testing facilities. The results show that the measurements are very similar for the assays.

[0193] Accuracy To assess the accuracy of the assay, two different pools of HV CSF were diluted with known concentrations of of calibration peptide (0, 2, or 20 pg / mL) and diluted to the recommended 1:4 dilution. The nucleotides were diluted and then measured in the pT3xhT43 and pT3xpT82 assays. It is a measure of the potential interference presented by components of the sample matrix. and 20 pg / mL spike measurements, then subtracting the level of endogenous signal. Recovery was calculated by comparing the observed concentrations of the calibrators with the expected concentrations. The spike recovery was calculated and an average recovery of 114% was obtained (Table 4). Recovery for the RUO assay is within the acceptable limits of 80-120%, which is ≥ Shows no significant interference in CSF when tested at a 1:4 dilution.

[0194] [Table 4]

[0195] Signal competition by antibodies against p217+ in CSF To confirm the accuracy of the pT3xhT43 and pT3xpT82 assay signals in CSF This study confirms its potential as a pharmacodynamic assay in clinical trials of antibodies against p217+ tau. To assess the potential benefit of AD CSF pools, we administered an appropriately dosed pT3 mAb. or humanized pT3 mAb spiked into the cells and incubated for 2 hours at room temperature followed by pT3x The results were measured in the pT43 and pT3xpT82 assays (Figure 5). Administration of T3 antibodies resulted in a dose-dependent decrease in the signal in pT3-based assays Spiking with msIgG (negative control) at equivalent concentrations did not affect either assay. The lower competitive ability of humanized pT3 mAb versus pT3 was not related to p217+ tau. This may be due to the higher affinity of pT3 for

[0196] Phosphorylation Dependence Confirmation of signals in CSF obtained with pT3xhT43 and pT3xpT82 assays The idea is actually based on phosphorylated epitopes and is based on alkaline phosphatase activity in CSF of AD. The sample was then treated with phospholipase A to dephosphorylate all residues. and two hT7-based assays, hT7xpT82 or hT7xBT2. hT7 was used as a negative control because it is known to be phosphorylation-independent.

[0197] Pooled CSF from AD patients was collected in a zinc chloride and magnesium chloride-containing buffer. and treated with increasing amounts of alkaline phosphatase (AP) for 4 hours at 37°C. Using the pT3xhT43 and pT3xpT82 assays, the epitopes for pT3 were identified. The effect of pT3xhT43 and pT3xpT82 on the expression of alkaline phosphatase was measured. Phosphatase treatment reduced the activity of the enzyme in a dose-dependent manner. Assays hT7xpT82 or hT7xBT2 did not show a signal reduction, and in fact, pT 7 binding was reduced by phosphorylation, so the increase was as expected (Figure 6).

[0198] p217+ tau fragment profile To investigate the nature of the p217+ tau signal obtained from crude CSF measurements, Dith et al. PLoS One. 8(10):e76523, 2013 AD CSF samples were fractionated by rpHPLC via a method similar to that of Fractions were collected and assayed using the pT3xhT43 and pT3xpT82 assays (Figure 1). 7) In this chromatographic format, smaller tau fractions elute earlier. (lower fraction numbers), while larger fractions elute later (higher fraction numbers). Full-length tau elutes in fraction 19. The tau fragment profile is consistent with previous reports. (Meredith et al.PLoS One.8(10):e76523, 2013, Barthelemy et al., J Alzheimers Dis. 51(4):1033-43, 2016), detected by either assay The pT3xpT82 assay showed that very little full-length tau was detected. Two major peaks (tau species) smaller than pT3 (fractions 12 and 14) were detected. The xhT43 assay detected only one of these major peaks (fraction 14). This indicates that p217+ tau in CSF exists in at least two fragments, the larger The large fragment encodes at least the region from hT43 to pT3 (aa 7 to 220 of tau). The smaller fragment is located at least in the region from pT82 to pT3 (aa of tau). 116-220), but does not reach the farther hT43 epitope. i.e., cleaved in only a subset of tau molecules at any given time, There is a likely proteolytic cleavage site between a20 and aa116. Ile is not p217+ specific; it recognizes a similar region of tau but is phosphorylated. Similar findings were obtained from other non-specific tau assays (data not shown). is not shown).

[0199] Analyte stability The stability of the endogenous p217+ tau epitope was assessed at various temperatures. The solution was aliquoted and each aliquot was incubated at 4°C, 22°C, or 37°C for 1, 2, or 4 hours. A subset of the aliquots was frozen and thawed (-80°C to 22°C) two or three times. All samples were then diluted 1:20 to obtain pT3xhT43 and hT7xpT82. The assay was analyzed (Figure 8). No changes were observed, which indicates that all four epitopes recognized by these assays is sufficiently stable to allow standard storage and testing procedures. SF was collected prospectively from four donors, then aliquoted and frozen at -70°C. Samples were taken every 3 months for measurement by pT3xpT82 assay. 3, 6 No significant changes in signal were observed at 1 or 9 months (Figure 9).

[0200] Example 8. Clinical validation of the pT3xhT43 and pT3xpT82 assays pT3xhT43 and pT3xpT82 assays in the diagnosis and staging of AD To evaluate the utility of this method, we performed three analyses of CSF samples to measure p217+ tau. We obtained data on the correlation between cognitive scores and other classical AD biomarkers. analyzed.

[0201] Cohort 1: "Inter-assay correlation cohort" CSF samples, VF and LF, and brain biopsies (ventricles) were collected to identify excess interstitial fluid production in the brain. Normal pressure hydrocephalus (NP), a neurodegenerative disorder that is frequently present in AD, is characterized by p217+ tau measurements were performed on crude CSF obtained from 10 subjects with HIV (H). , and correlations with traditional AD biomarkers were analyzed.

[0202] Aβ42 (Figures 10A and 10D), t-tau (Figures 10B and 10E), and p-tau181 in VF The levels of α-glucan (Figures 10C and 10F) were measured by Innotest ELISA (classical measurement). The same sample was analyzed using pT3xhT43 (Figs. 10A, 10B, and 10C) and pT3 pT82 (Figures 10D, 10E, 10F) assay and correlation was assessed. Both hT43 and pT3xpT82 assays showed a negative correlation with CSF Aβ42 ( r respectively 2 = 0.609, p = 0.0077, and r 2 =0.590, p=0.009 5), and showed a positive correlation with CSF t-tau (r 2 =0.525, p=0.0177 , and r 2 = 0.435, p = 0.0381), but was not significantly correlated with CSF pTau181. It was revealed that there was no relationship between the two (Figure 10).

[0203] Brain biopsies from the same 10 NPH subjects were analyzed by IHC and pathologists were found to be amyloid-positive. If positive for both, the sample was scored as positive / negative and tau positive / negative. If both were negative, the diagnosis was considered to be "biopsy positive" and was a classic diagnosis of AD. The sample was designated "Biopsy-" and was a classic non-AD diagnosis. "Biopsy+ (amyloid) The sample, named "," tested positive for amyloid but negative for tau. CS obtained from ventricular puncture (VF = black dot) or lumbar puncture (LF = red dot) F was measured with the pT3xhT43 and pT3xpT82 assays to assess correlation ( Figure 1 1) Both the pT3xhT43 assay and the pT3xpT82 assay were positive in brain biopsies. Separating negative samples (amyloid- / tau-) from negative samples (amyloid+ / tau+) (p=0.04 and 0.02, respectively). However, samples that are not positive for tau are often biopsy + samples and biopsy - samples. The data were collected between samples. Amyloid plaques are thought to precede tau tangles in the brain. , amyloid+ / tau- samples may represent early AD or another disease.

[0204] Cohort 2: "HV vs. AD cohort" CSF samples (LF) from biochemically defined AD subjects vs. HV subjects (n=20 / Aβ42 (group) was obtained from the University of Sahlgrenska. and t-tau levels were determined by Innotest ELISA (classical measurement) and were subdivided (AD = CSF Aβ42 < 400 pg / mL and CSF t-tau > 600 pg / mL, HV = CSF Aβ42 > 400 pg / mL and CSF t-tau < 600 pg / mL pT3xhT43, pT3xpT82, and hT7xpT for crude CSF. Measurements were performed using 82 assays as well as a subset of rpHPLC-fractionated CSF. The results were analyzed for correlation with traditional AD biomarkers (Figure 12). The data in panels A and B of Figure 2 demonstrate that the pT3 epitope rapidly increases in insipient AD. demonstrated that the pT3 epitope is an indicator of patients at high risk of progressing to glioma. Conversely, the pT3 epitope was significantly elevated in patients with low total Aβ42. It was present at low levels in subjects with tau and high Aβ42. Elevated pT3 epitope-containing tau as demonstrated by pT82 total tau assay was at least partly, but not entirely, driven by elevated total tau levels. This confirmed the presence of tau in the IL-16 cells (Fig. 12D). Both of these levels are shown to be elevated in AD.

[0205] Using the data from Figure 11, pT3xhT43, pT3xpT82, and hT7x ROC curve for the ability of the pT82 assay to distinguish AD samples from HV samples All three assays showed excellent specificity and sensitivity. Two pT3-based assays (pT3xhT43, pT3xp) detect p217+ tau T82) had improved diagnostic power over the hT7-based assay (Figure 13).

[0206] A subset of the same CSF samples (n=11 / group) measured in Figure 12 was subjected to rpHPLC. Then, pT3xhT43, pT3xpT82, and hT7xpT82 were isolated. assay (the latter was a phosphorylation-independent measurement of the same tau fragment) (Fig. 14 The observed profile of tau fragments was similar to that seen in Example 7 and Figure 7. Both pT82-based assays, pT3xpT82 and hT7xpT Two major species were observed in 82, whereas only one of the peaks was observed in the pT3xhT43 assay. Both major species were present in higher concentrations in the AD group than in the HV group. Furthermore, the pT3-based assay (p217+ tau) was detected in crude CSF analysis. showed a larger difference between groups than the hT7-based assay (total tau). The larger p217+ tau species (fractions 13–14) resulted in the largest AD vs. HV difference (Figure 1). 14).

[0207] The sum of all major tau fragments (fractions 11-14) in Figure 14 was calculated and then analyzed by AD subgroup analysis. The pT3 epitope-containing tau ( The increase rate of pT3xhT43 or pT3xpT82 was higher than that of non-pT3 epitope-containing tau (hT43 or pT3xpT82). T7xpT82) was more than double that observed in the control group (Table 5).

[0208] [Table 5]

[0209] The signal fractions in each of Figure 14 were analyzed as a total as in Table 5. Independent analyses were performed to determine which fractions yielded the greatest AD vs. HV signal. The most informative fragment pools were selected from fragment pools 13 and 14, which contained pT3 antibodies. were used to detect (Table 6).

[0210] [Table 6]

[0211] Cohort 3: "HV vs. ARAD vs. early AD cohort" Clinically defined normal (CDR0) vs. mild memory complaints (CDR0.5) subjects (n=20 CSF samples (LF) from each group were analyzed in the Janssen study ALZ1005 / 2002. The levels of Aβ42, t-tau, and p-tau181 were measured using Innotest EL Based on CDR and CSF Aβ42 scores, subjects were classified as follows: (a) HV = CDR0 and Aβ42 > 600 pg / mL, (b) ARAD = CDR0 and Aβ4 2<600pg / mL, (c) potentially non-AD dementia = CDR0.5 and Aβ42>60 0 pg / mL, and (d) initial AD = CDR 0.5 and Aβ42 < 600 pg / mL. Similar.

[0212] In addition, CSF samples were fractionated by rpHPLC to isolate pT3-based (pT3xhT4 3, Figures 15A-15E, and pT3xpT82, Figures 15F-15J) and total tau (hT7 pT3-based and hT7-based assays were performed. All assays were performed for CDRs 0 vs. 0.5 (Figures 15A, 15F, and 15K), and Aβ42 < 600 pg / mL vs. >600 pg / mL samples (Figures 15B, 15G, and 15L). The results of the analysis by CDR×Aβ42 level are shown in Figure 15C. The signals shown in 15D, 15H, 15I, 15M and 15N are summed over all fractions. The signal levels were <600 pg / Aβ42. The highest rate was in the mL+CDR0.5 subgroup, and the rate was higher in early AD vs. HV or ARAD. The separation between the subgroups was consistent with the increase in p217+ tau signal in the hT7-based The pT3-based assay was superior to the pT3-based assay, which Hyperphosphorylation of epitopes is particularly prevalent in disease (rather than simply increasing total tau). This indicates that the

[0213] Cohort 4 ("CDR0 vs. CDR1 cohort") Clinically defined normal (Clinical Dementia Scale 0; CDR0) vs. mild memory complaints (CDR1 LF CSF samples from subjects (n=5 / group) were collected from the Washington University The CDR and MMSE, as well as the Innotest assay, were obtained from the University of Tokyo. Measurements of CSF Aβ42, t-tau, and p-tau181 were performed at the Washington Before shipping, Janssen will confirm the identity of the sample. Or the samples were coded to hide their characteristics. Janssen Neuros Science Biomarkers (La Jolla) tau and p217+ tau measurements were performed and submitted to the Washington University sent to iversity.

[0214] pT3-based assays (pT3xhT43 and pT3xpT82) and t-tau (hT 7xpT82) were used to prepare CSF samples either crude or after rpHPLC fractionation. These data were used to assess the relative influence of the two short tau species. as an inter-assay ratio (Table 7) or to assess the relative impact of this phosphorylation event. The results were expressed as the ratio between either the pT3 assay or t-tau (Figures 16A-16B). In both cases, the results accurately predicted CDR status for 9 out of 10 subjects. One outlier subject was determined by Innotest to have abnormally low tau. This may represent dementia not caused by tauopathy. Additionally, a correlation between the p217+ tau / t-tau ratio and MMSE was observed, which was consistent with the pT3 uptake. This suggests that cognition can be tracked by signals detected by the sei.

[0215] [Table 7] * CDR1 and Aβ were positive, but low titers were detected in Innotest and Simoa. having p-tau and p-tau

[0216] Cohort 5: "HV vs. MCI vs. AD cohort" Clinically and biochemically defined (Innotest AB42 > 600 pg / mL) LF CSF samples from HV (n=7) were analyzed by Precision Medicine (San Diego, CA). Clinically and biochemically AB42 < 600 pg / mL) due to defined MCI (n = 28) and AD (n = 12) The original LF CSF samples were obtained from the University of Antwerp. rpHPLC and Simoa p217+ tau assays were performed by Janssen Neurochem. The study was conducted at Science Biomarkers (La Jolla).

[0217] pT3-based assays (pT3xhT43 and pT3xpT82) and t-tau (hT 7xpT82) were used to prepare CSF samples either crude or after rpHPLC fractionation. Both pT3- and hT7-based assays showed significant differences in HV vs. MCI vs. AD groups. The signal gradually increased with increasing intensity (Fig. 17A-C), and they correlated well with each other. Cohort 1, as seen in Figure 9 (Figures 17D and 17E). Sei was also somewhat correlated with Innotest t tau and p tau 181 ( 17F and 17G), and there was no correlation with Innotest AB42 or the AB42 / 40 ratio. No significant difference was observed between the crude CSF measurements (Figs. 17A-17C) and rpHP (Figs. 17H and 17II). Similar results were obtained for diagnostic staging in LC-fractionated material (Figs. 17J–17T). As seen in cohort 3, the pT3-based assay was more effective than the t-tau assay. The separation of HV vs. MCI vs. AD was more pronounced (high statistical significance) when using the assay. ), thereby highlighting the pathological relevance of this pT3 assay measurement.

[0218] Cohort 6 ("Disease Severity and Progression Cohort") Samples from subjects with clinically defined AD (Clinical Dementia Scale 1+) (n=235) These samples were obtained from Janssen Research ELN115727301 / 302. Samples were baseline (pre-dose) samples from all subjects in the trial. , including CSF samples from placebo subjects (n=90) at 78 weeks of follow-up to assess disease progression Biomarkers were evaluated. Cognitive assessment (ADAS-COG, MMSE, NTB, and C DR.SOB), ApoE genotype, sex, and age were obtained from the clinical trial. AB42, Innotest AB40, Simoa neurofilament light(NFL), pT3xpT82, pT3xhT43, and hT7xpT82 The assay was funded by Janssen Neuroscience Biomarkers (La Subjects were randomly selected based on an AB42 / 40 ratio cutoff of 0.09. Subjects were confirmed as amyloid positive or negative (e.g., subjects with a ratio of <0.09 = amyloid Amyloid positive = AD, whereas subjects >0.09 = amyloid negative = cognitive impairment due to causes other than AD Of the 235 subjects, 27 were determined to be amyloid-negative, so each group was divided separately. analyzed.

[0219] Again, the signal from the crude CSF measurements was comparable between the two pT3 assays and the tTau assay. Although it correlates well with serotonin (Fig. 18A and 18B), it is a suspected marker of systemic neurodegeneration. It was found that the NFL, which is the target of pT3 adenosine triphosphate (NFL), was not correlated with the NFL (Fig. 18C). These results suggest that the assay may recognize specific forms or stages of neurodegeneration.

[0220] Again, the pT3-based assay showed higher IL-1 expression in amyloid-positive versus amyloid-negative subjects. A clear signal was revealed (Figs. 18D to 18E).

[0221] pT3-based assays revealed several cognitive scores (ADAS-COG, MMSE, A moderate correlation with NTB, CDR.SOB, Figures 18F-18M) was revealed, which is This corroborated the findings in Cohort 4 (Figure 16C). Say's baseline signal was similar to the change in cognitive scores over an 18-month follow-up period. was moderately correlated with , suggesting its ability to predict cognitive decline (Figure 18N- 18P).

[0222] The ratio of pT3-based signal to t-tau signal (p217_tau / t-tau) Similar results were obtained, but the data are not shown.

[0223] The correlation between cognition and cognitive change was observed in both the amyloid-positive and -negative groups, but the latter This group was a small sample set. If confirmed, this may be a sign of a relationship between p217+ vs. cognition. This suggests that the association may not be AD-specific.

[0224] Example 9. Quantification of p217+ tau bound to antibody versus no antibody The assay described in Example 3 was carried out as follows.

[0225] Assay 1: Immunocapture / depletion followed by rpHPLC to identify free peptides in biological fluids Quantification of bound p217+ tau The assay was tested by spiking the antibody into pooled CSF samples. AD CSF was treated with 10 μg of pT3 mAb, humanized pT3 mAb, msIgG, or The cells were spiked with an equal volume of PBS (mock) and incubated at 4°C for 24 hours. The samples, as well as the parent CSF that was not subjected to immunocapture, were fractionated by rpHPLC. Each fraction was measured using the pT3xhT43 assay to determine total and bound p217+ Tau levels were assessed in parental samples (total p217 + tau) and in mice treated with either pT3 mAb or humanized p T3 mAb immunocapture (bound p217+tau) showed similar results to those seen in Example 7 and Figure 7. Similarly, substantial signal was observed in one major peak, but not in mock or Ig This was not observed with G immunocapture (Fig. 19).

[0226] Pooled AD CSF was spiked with appropriately titrated humanized pT3 mAb and incubated at 22°C. The mixture was incubated for 2 hours at RT, followed by immunocapture, rpHPLC, and analysis of the pT3xhT43 antigen. Assays were performed to assess bound p217+ tau (Figure 20A). IgG-depleted supernatants were also fractionated. , and free p217+ tau was assessed (Fig. 20B). The amount of bound p217+ tau measured was increased in a dose-dependent manner, while the amount of free p21 The amount of 7+ tau was reduced.

[0227] Taken together, these results demonstrate that this method, a direct measure of target association, is effective in treating p217+ tau epithelial cell death. The results showed that the antibody-targeting effect was specific to the target antibody (Figure 19) and was dose-dependent. (Figure 20).

[0228] Assay 2: Free versus bound p217+ tau in biological fluids via selective denaturation of antibodies Quantitation of A biological sample (e.g., CSF) is heated to near boiling point for 4 minutes and then cooled on ice. and then measured in the pT3xhT43 and / or pT3xpT82 assays. The exact time of the process cannot interfere with the assay, but rather with the p217+ tau signal itself. The antibody in the sample is irreversibly damaged so as not to affect the This is due to the lack of a specific tertiary structure in the tau protein. This is thought to be due to the fact that it can be made particularly stable at high temperatures. The samples were designated as total p217+ tau, while the parallel samples that were not subjected to heat treatment were designated as total p217+ tau. The total concentration was subtracted from the free to obtain the bound p217+ tau. Tau measurements were obtained.

[0229] The effect of heat on the assay was determined as follows.

[0230] Effect of heat on CSF / humanized pT3 mAb mixtures: Analysis of pooled AD CSF Spike the recoat with humanized pT3 mAb to 1 μg / mL and incubate for 2 hours at 22°C. The solution was incubated, heated at 95°C for 0-20 minutes, cooled to 4°C, and then diluted 1:10 with pT p217+ tau signals were measured using the 3xpT82 assay (Figure 21A). The 2-minute heat treatment reduced the levels, which then returned to the levels seen in unspiked CSF. The solution returned to the original state and was stable through heating for approximately 10 minutes before the addition.

[0231] Heat effects on naive CSF: Aliquots of pooled CSF were heated to 95°C for 0–20 min. After heating and cooling to 4°C, use the pT3xpT82 assay at a 1:10 dilution. The p217+ tau signal was measured after heating for approximately 10 minutes before the instillation (Fig. 21B). It was stable.

[0232] The effect of heat on the ability of humanized pT3 mAb to inhibit the pT3xpT82 assay: P An aliquot of 10 μg / mL humanized pT3 mAb in BS was heated at 95°C for 0–20 min. These samples were then mixed with pooled AD CSF and cooled to 4°C. (The final concentration of humanized pT3 mAb was 1 μg / mL.) Mix and incubate at 22°C for 2 hours. After incubation, the results were measured using the pT3xpT82 assay at a 1:10 dilution (Figure 21). C). p217+ tau signal is reduced by ~2 min of JNJ heat treatment, followed by return to levels seen in unspiked CSF (see Figure 21B) and It was stable through heating for 0 minutes.

[0233] Parallel aliquots of pooled AD CSF were titrated with humanized pT3 mAb and The mixture was incubated at 4°C for 2 hours, and then subjected to a heat denaturation process (heat treatment for 4 minutes) (Figure 122 pT3xpT82 after either immunocapture / rpHPLC (Figure 22A) or immunocapture / rpHPLC (Figure 22B). Both methods demonstrated that humanized pT3 mAb was able to bind in a dose-dependent manner. The results showed that the free and total p217+ tau signal was unchanged. In addition, the more laborious immunocapture / rpHPLC method of Assay 1 was used by heat denaturation. The humanized pT3 mAb dose-dependence and relative free vs. bound vs. total activity were comparable to those obtained with p217+ measurements were obtained (Figure 22C). Therefore, standard sample analysis requires the use of thermal The method is recommended.

[0234] Example 10. p217+ tau signaling in preclinical animal models Naive samples from a variety of common laboratory animals are available to support preclinical studies. , assessed using pT3-based assays, and / or sequence alignment to identify cross-reactivity. The responsiveness was predicted.

[0235] Cynomolgus Macaque CSF from two cynomolgus monkeys was analyzed using pT3- and hT7-based assays. For comparison, the same detection antibody was used as two capture antibodies. In some cases, two individual CSFs were tested separately (cytoplasmic CSF). (Cynomolgus monkey 1 or cynomolgus monkey 2); in other cases, CSF samples were pooled to conserve volume. Regardless of the detection antibody, all assays using hT7 as the capture antibody showed Although a substantial signal (AEB) was observed, the assay used pT3 as the capture antibody No signal was detected in either case. The assay showed that despite the large signal in AD human brains, the homozygous signal in cynomolgus monkey brains was Even in the presence of ribosomal nucleotides, little or no pT3-based signal was observed. This suggests that despite high levels of tau, the pT3 epitope was not present. This suggests that the protein is not conserved in this species. Analysis of the protein sequence revealed a single amino acid sequence in the pT3 core epitope between humans and cynomolgus monkeys. The structure of the humanized pT3 mAb with tau suggests that the amino acids are different. Modeling suggests that this change may result in a loss of pT3 binding (data not shown). (Data not shown).

[0236] Common marmoset CSF from common marmosets was used in pT3- and hT7-based assays. CSF from three common marmosets was tested using the pT3xhT43 , pT3xpT82, and hT7xpT82 assays at various dilutions. For comparison, pooled cynomolgus monkey CSF (negative control) and pooled AD human CSF were used. F (positive control) was tested simultaneously. pT3xpT82 (Figure 24B) and hT7xpT82 (Fig. 20C) Marmoset CSF using the assay showed a substantial signal (AEB). was seen in the pT3xhT43 assay but not in the pT3xhT43 assay (Fig. 24A).

[0237] This suggests that the hT43 epitope is missing in this species, and indeed Furthermore, protein sequence alignment revealed common markers with humans in the hT43 epitope. pT3, hT7, and pT82 epitopes differ by one amino acid between the two mosets. indicates that the α-glucan is preserved. Therefore, there is substantial signal in the pT3xpT82 and hT7xpT82 assays. However, the pT3xhT43 assay confirmed that it was very small (data not shown). Therefore, analysis of p217+ tau signals in marmosets reveals that pT3xpT8 This was achieved using two assays.

[0238] Mice, rats, dogs, pigs Mouse, rat, dog, or pig (NCBI accession number NP_00 1033698.1, NP_058908.2, NP_001104271.1, and A Alignment of predicted tau protein sequences with human sequences in (GJ26517.1) suggests that pT3 is 100% conserved in these species. The mouse, rat, dog, and pig hT43 and pT82 sequences are identical to the human sequences. Therefore, the samples derived from these were pT3xhT43 and pT3xpT82 It needs to be evaluated using an assay.

[0239] Taken together, the data presented herein demonstrate the utility of the Simoa platform for CSF measurement. The pT3xhT43 and pT3xpT82 assays developed on this form were highly sensitive. It has femtogram sensitivity, high precision, accuracy, dilution linearity, and analyte stability. The assay correlated well with classical AD biomarkers and dementia scores. and may be superior to measurements in identifying and staging AD subjects. There is a gender.

[0240] The assay was used to measure levels of total p217+ tau in CSF, or rpHPL To assess the fragment profile of p217+ in C-fractionated CSF. The assay also allows for antibody-free detection of endogenous or exogenous p217+ tau. To measure the level of p217+ tau bound to the administered antibody, preanalytical procedures and Thus, the assay can be performed on individuals with high levels of p217+ tau targets. To identify subjects suitable for anti-p217+ tau antibody therapy by identifying the subjects. It can be used as a predictive biomarker. Total, free, and therapeutic antibody-bound The assay can be used as a pharmacodynamic marker by measuring the levels of p217+ tau in the target cells. It can also be used.

[0241] Example 11. p217+ tau signal in blood Measurement of tau in CSF has great value in diagnosing and staging neurodegenerative disorders However, collection of CSF is subject to limitations (e.g., patient burden, clinical facility experience, collection time, etc.). Therefore, blood products (e.g., serum, plasma) have limitations in terms of volume and frequency. There is considerable interest in adapting tau measurements for use in reported that tau measurement in crude serum or plasma does not show ideal diagnostic performance, with limitations on sensitivity and matrix interference. However, pT3-based assays may suffer from the hurdle of may represent a new opportunity due to its high sensitivity and specificity.

[0242] Sera from clinically defined AD and HV subjects (n=4 each) were analyzed by D'Abra As in Mo et al. 2016, crude samples of various dilutions ("crude", Figure 25A- 25D), acid (NaOAc, pH 5) treated and denatured samples ("boiled", Figure 26A ~26B) using the pT3xpT82 and hT7xpT82 assays This was used to remove most matrix interference and immunoprecipitated (IP) with pT3 beads. The eluate was then heat denatured ("pT3 IP", Figure 27).

[0243] Measurements in crude serum showed that most samples were below the limit of quantitation (LOQ). However, much higher levels were reported in a few outlier samples. However, the signal does not survive moderate dilution and is therefore an interference artifact. The highest dilutions tested (Figs. 25B and 25D) and therefore the interference The lowest impact assessment was achieved with the pT3xpT82 assay, which showed slightly more activity in the AD samples. Although it is possible to detect signals, all are below the LOQ, and therefore, accurate and / or high It was suggested that the accuracy may not be sufficient.

[0244] Acid treatment (to dissociate protein-protein interactions) and heat (to remove most non- Measurements in serum after denaturing tau protein revealed that all pT3xpT82 and hT7xpT82 signals were reduced to near or below the LOQ (Figures 26A-26 B). Again, the pT3xpT82 assay showed slightly more cytotoxicity in the AD samples. Signals can be detected, but all are near the LOQ, so accuracy and / or precision are not possible. It may not be a degree.

[0245] pT3-IP and denaturation to remove most interfering substances and enrich for p217+ tau Subsequent serum measurements showed that levels were significantly higher in AD samples than in HV samples. The P217+ level was higher than that measured by crude or boiling method (Fig. 27). Because it was about 4 times higher, the HV sample was the LOQ here, and the AD sample was the LOQ here. All were within the linear range.

[0246] These results demonstrate that the pT3-based assay described herein is particularly useful for enrichment of cells, such as IP. When paired with a cytochrome P450 antibody-binding strategy, it may be useful as a blood-based measure of pathological tau. did.

[0247] Although the present invention has been described in detail and with reference to specific embodiments thereof, those skilled in the art will appreciate that Various changes and modifications may be made to the present invention without departing from the spirit and scope of the invention. It will be clear.

[0248] References Abhinandan and Martin,Mol Immunol.45:38 32-9,2008 Almagro,Mol Recognit.17:132-43,2004 Barthelemy et al.,J Alzheimers Dis.51(4 ):1033-43,2016 Butner and Kirschner,J Cell Biol.115(3) :717-30,1991 Chothia and Lesk, J Mol Biol. 196:901-17, 1987 Chothia et al.,J.Mol.Biol.227:799-817,1 992 Clavaguera et al., Nat Cell Biol. 11:909- 13,2009 D'Abramo et al Neurobiol Aging.37:58-65 ,2016 Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996) Fishwild et al., Nat Biotechnol.14:845-5 1,1996 Frost et al., J Biol Chem.284:12845-52.2 009 Hanger et al. J Biol Chem. 282(32):23645- 54,2007 Hanger et al., Trends Mol Med.15:112-9.2 009 Iqbal et al.,Curr Alzheimer Res.7(8):65 6-664,2010 Knappik et al., J Mol Biol.296:57-86,200 0 Kohler and Milstein, Nature.256:495-7.19 75 Krebs et al., J Immunol Methods.254:67-8 4,2001 Lefranc et al.,Dev Comp Immunol.27:55-7 7,2003 Lonberg et al., Nature.368:856-9, 1994 Mendez et al., Nat Genet.15:146-56,1997 Meredith et al. PLoS One.8(10):e76523,20 13 Morris et al., Neuron, 70:410-26, 2011 Russell et al.,J Alzheimers Dis.55(1):3 03-313,2017 Shi et al., J Mol Biol.397:385-96,2010 Tramontano et al.,J.Mol.Biol.215:175-18 2,1990 Wu and Kabat, J Exp Med.132:211-50,1970

Claims

1. 1. A method for measuring p217+ tau peptide in a sample, comprising: (i) contacting the sample with a capture antibody against the p217+ tau epitope to obtain a pre- capturing the p217+ tau peptide in the sample; (ii) cleaving the captured p217+tau peptide by cleaving the captured p217+tau peptide at amino acid residue 1 of the tau protein; A first detection antibody against an epitope including amino acid residues 19-126 of tau protein contacting the antibody with at least one of the second detection antibodies directed against an epitope containing groups 7-20; and determining the amount of the p217+tau peptide and the amount of the long p217+tau peptide, respectively. and measuring at least one of the amounts of The method, wherein the amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO:

1.

2. The captured p217+tau peptide was subjected to the first detection antibody and the second detection antibody. and determining the amount of the p217+tau peptide and the amount of the long p217+tau peptide, respectively. measuring the amount of said long p217+tau peptide; and optionally determining the amount of said long p217+tau peptide. and determining a ratio of the amount of p217+tau peptide to the amount of p217+tau peptide. method.

3. (i) subtracting the amount of the long p217+tau peptide from the amount of the p217+tau peptide; determining the amount of short p217+ tau peptides via subtraction; (ii) optionally, determining the amount of said short p217+tau peptide; the ratio of the amount of said long p217+tau peptide to the amount of said short p217+tau peptide and determining a ratio of the amount of 217+ tau peptide to the amount of 217+ tau peptide. Law.

4. 1. A method for measuring p217+ tau peptide in a sample, comprising: (i) contacting the sample with a capture antibody against the p217+ tau epitope to obtain a pre- The p217+tau peptide in the sample is captured, and the sample is subjected to analysis of the tau protein. Epitope of amino acids 150-250, preferably amino acids 159-16 of tau protein 3, and contacting the sample with a phosphorylation-independent capture antibody against an epitope containing capturing total tau peptides; (ii) a. The captured p217+tau peptide is ligated to amino acid residue 11 of the tau protein. contacting the p217+ taupeptide with a first detection antibody directed against an epitope including 9-126; and measuring the amount of total tau peptide captured, contacting the captured total tau peptide with the first detection antibody to determine the total tau peptide. The amount of tau peptide is measured, and the amount of p217+tau peptide is the total amount of tau peptide. Finding the ratio to b. The captured p217+tau peptide is purified by cleaving the captured p217+tau peptide from amino acid residues 7 to 10 of the tau protein. 20, and contacting the long p217+ taupeptide with a second detection antibody directed against an epitope containing and measuring the amount of total tau peptide captured, contacting the captured total tau peptide with the second detection antibody. measuring the amount of total long tau peptides, and determining the amount of the long p217+ tau peptides. determining the ratio to the amount of long tau peptide; and performing at least one of: The method, wherein the amino acid numbering refers to the amino acid sequence set forth in SEQ ID NO:

1.

5. The amount of the long p217+tau peptide is subtracted from the amount of the p217+tau peptide. determining the amount of short p217+ tau peptides through the analysis of the total tau peptides; The total short tau peptides are obtained by subtracting the amount of the total long tau peptides from the amount of the total short tau peptides. and determining the amount of said short p217+tau peptide in relation to said total short tau peptide. and determining a ratio of the amount of the peptide.

6. the sample consists of blood, brain homogenate, or cerebrospinal fluid (CSF) from the subject. The method according to any one of claims 1 to 5, wherein the biological sample is derived from the subject selected from the group consisting of: How to post.

7. The method of claim 6 , wherein the biological sample is blood.

8. The method of claim 6 , wherein the biological sample is CSF.

9. The biological sample is analyzed using reversed-phase high performance liquid chromatography (rpHPLC).

7. The method of claim 6, wherein

10. a. determining whether the subject suffers from or is at risk of developing a tauopathy determining whether there is a problem; b. determining whether the subject is suitable for treatment with an anti-p217+tau antibody; and, c. Determining the effectiveness of treating a tauopathy in said subject; or d. Monitoring treatment with an anti-p217+ tau antibody in a subject; Further comprising: The determining or monitoring comprises determining or monitoring the amount of the p217+ tau peptide from the subject, the amount of long p217 + tau peptide, the amount of said short p217 + tau peptide, and comparing at least one of these ratios to a corresponding baseline value; The method of claim 6.

11. and monitoring treatment with an anti-p217+ tau antibody in the subject. Item 11. The method according to item 10, wherein the method comprises: (i) obtaining a biological sample from said subject; (ii) subjecting the biological sample to a p217+ tau antibody treatment without the anti-p217+ tau antibody. a first sample containing a peptide, and a p21 bound to the anti-p217+tau antibody; separating a second sample containing 7+ tau peptides; (iii) separating the second sample, preferably via rpHPLC, to obtain anti-p2 Obtaining a third sample containing p217+ tau peptides without p217+ tau antibodies and, (iv) subjecting each of the first sample and the third sample to p217+ tau and contacting the first sample and the third sample with a capture antibody against a target antigen. capturing p217+tau peptides in each; (v) (a) separating the captured p217+tau peptide from the amino acid residues of the tau protein; contacting said first subunit with a first detection antibody directed to an epitope comprising residues 119-126; The amount of p217+tau peptide in each of the first sample and the third sample is measured. and (b) transferring the captured p217+tau peptide to an amino acid sequence of a tau protein. and contacting the first sample with a second detection antibody directed against an epitope comprising acid residues 7-20. The amount of long p217+ tau peptide in each of the first and second samples was measured. and optionally, (c) determining the first sample and the previous sample. The amount of p217+tau peptide in each of the third samples is used to determine the long p The amount of short p217+ tau peptide was calculated by subtracting the amount of 217+ tau peptide. To ask for and to carry out (vi) the p217 in each of the first sample and the third sample + amount of tau peptide, said long p217 + amount of tau peptide, said short p217 the anti-p217 based on at least one of the amount of tau peptide, the amount of tau peptide, and the ratio thereof. and monitoring treatment with the tau antibody.

12. and monitoring treatment with an anti-p217+ tau antibody in the subject. Item 11. The method according to item 10, wherein the method comprises: (i) obtaining a biological sample from said subject; (ii) Obtaining a semi-denatured sample from a biological sample containing total p217+tau peptides wherein the semi-denatured sample is heated to denature the antibodies in the sample. and containing the p217+ tau peptide without the anti-p217+ tau antibody. obtaining a non-denatured sample from the biological sample; (iii) each of the semi-denatured sample and the non-denatured sample is subjected to p217+ tau p217+ in each of the samples by contacting with a capture antibody against the epitope. capturing tau peptides; (iv) (a) separating the captured p217+tau peptide from the amino acid sequence of tau protein; contacting the sample with a first detection antibody directed to an epitope comprising residues 119-126; (b) measuring the amount of p217+tau peptide in each of said capture tubes; and The resulting p217+tau peptide was then subjected to an epitope analysis containing amino acid residues 7-20 of the tau protein. and contacting each of the samples with a second detection antibody against the long p2 measuring the amount of 17+ tau peptide; and optionally, ) the p217+ taupe in each of the first sample and the third sample; and subtracting the amount of the long p217+ tau peptide from the amount of the short p217+ tau peptide. determining the amount of p217+ tau peptide; (v) the amount of the p217+tau peptide in each of the samples, the amount of p217+tau peptide, the amount of said short p217+tau peptide, and and monitoring treatment with the anti-p217+tau antibody based on at least one of the ratios. and

13. The capture antibody is conjugated to a bead and the detection antibody is biotinylated. The method according to any one of claims 1 to 12, wherein

14. the lower limit of quantitation of the method is about 40 fg / mL of the p217+ tau peptide; 1. The method of claim 1, wherein the lower limit of detection is about 2 fg / mL of said p217+tau peptide.

3. The method according to any one of the preceding claims.

15. The tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, and 17th chromosome aberration syndrome. Frontotemporal dementia with somatic link and parkinsonism (FTDP-17), progressive supranuclear Paralysis, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle-predominant type Tangle-only dementia, diffuse neurofibrillary tangle disease with calcifications, argyrophilic granules Dementia, amyotrophic lateral sclerosis / Parkinson-dementia complex, Down syndrome, Gerstmann-Schwarz syndrome Reussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease Feldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein brain atrophy Myloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, neurofibrillary tangles Non-Guam motor neuron disease with encephalitis, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (boxer's disease), and preferably, the tauopathy The method according to any one of claims 7 to 14, wherein the disease is Alzheimer's disease.

16. The capture antibodies have the polypeptide sequences of SEQ ID NOs: 32, 33, and 34, respectively. Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3, and their respective SEQ ID NOs: Immunoglobulin light chain LCDR1, LC having polypeptide sequences 35, 36, and 37 DR2, and LCDR3, and preferably, the capture antibody comprises the polypeptide of SEQ ID NO:

28. a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region comprising the polypeptide sequence of SEQ ID NO: 30; The method according to any one of claims 1 to 15, comprising:

17. The first detection antibodies have the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively. Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3, and their respective SEQ ID NOs: Immunoglobulin light chain LCDR1, LCDR2 having polypeptide sequences 5, 6, and 7 and LCDR3, and preferably, the first detection antibody comprises the polypeptide of SEQ ID NO:

8. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10 The method of any one of claims 1 to 16, comprising:

18. The second detection antibody has the polypeptide sequences of SEQ ID NOs: 12, 13, and 14, respectively. Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the sequences Immunoglobulin light chain LCDR1 having the polypeptide sequences of sequence numbers 15, 16, and 17 , LCDR2, and LCDR3, and preferably, the detection antibody comprises the polypeptide of SEQ ID NO:

18. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 19 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 19 The method of any one of claims 1 to 17, comprising a variable region.

19. A kit comprising: a. a capture antibody against the p217+ tau epitope, optionally an amino acid sequence of the tau protein a phosphorylation-independent capture antibody against the tau epitope between amino acids 150 and 250; b. Tau protein containing amino acid residues 7 to 20 or 116 to 127 of tau protein and at least one detection antibody against the epitope.

20. The capture antibodies have the polypeptide sequences of SEQ ID NOs: 32, 33, and 34, respectively. Immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3, and their respective SEQ ID NOs: Immunoglobulin light chain LCDR1, LC having polypeptide sequences 35, 36, and 37 DR2, and LCDR3, and preferably, the capture antibody comprises the polypeptide of SEQ ID NO:

28. a heavy chain variable region comprising the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region comprising the polypeptide sequence of SEQ ID NO: 30; wherein the phosphorylation-independent capture antibody binds to amino acids 159 to 163 of tau protein.

20. The kit of claim 19, directed against a tau epitope comprising:

21. a. the first detection antibodies have the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively; immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3, and their respective sequences Immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having polypeptide sequences numbered 5, 6, and 7 R2, and LCDR3, and preferably, the first detection antibody is a polypeptide of SEQ ID NO:

8. a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 10 Including the area, b. the second detection antibody is a polypeptide sequence of SEQ ID NOs: 12, 13, and 14, respectively; immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having sequences immunoglobulin light chain LCD having the polypeptide sequences of SEQ ID NOs: 15, 16, and 17; R1, LCDR2, and LCDR3, and preferably the detection antibody is and a heavy chain variable region having the polypeptide sequence of SEQ ID NO:

19.

21. The kit of claim 19 or 20, comprising a light chain variable region.

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