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WO2019213612A1 - Méthodes de diagnostic et de traitement basées sur la phosphorylation de tau spécifique à un site - Google Patents

Méthodes de diagnostic et de traitement basées sur la phosphorylation de tau spécifique à un site Download PDF

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Publication number
WO2019213612A1
WO2019213612A1 PCT/US2019/030725 US2019030725W WO2019213612A1 WO 2019213612 A1 WO2019213612 A1 WO 2019213612A1 US 2019030725 W US2019030725 W US 2019030725W WO 2019213612 A1 WO2019213612 A1 WO 2019213612A1
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Prior art keywords
tau
phosphorylation
ratio
total
tau phosphorylation
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Nicolas BARTHELEMY
Randall Bateman
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University of Washington
Washington University in St Louis WUSTL
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University of Washington
Washington University in St Louis WUSTL
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Priority to CA3097667A priority Critical patent/CA3097667A1/fr
Priority to BR112020021782-4A priority patent/BR112020021782A2/pt
Priority to EP19797038.7A priority patent/EP3788062A4/fr
Priority to MX2020011458A priority patent/MX2020011458A/es
Priority to KR1020207029980A priority patent/KR102787585B1/ko
Priority to JP2020561809A priority patent/JP7301394B2/ja
Priority to CN201980030025.7A priority patent/CN112166117A/zh
Priority to SG11202010094TA priority patent/SG11202010094TA/en
Priority to KR1020257009592A priority patent/KR20250048591A/ko
Priority to AU2019262220A priority patent/AU2019262220B2/en
Application filed by University of Washington, Washington University in St Louis WUSTL filed Critical University of Washington
Publication of WO2019213612A1 publication Critical patent/WO2019213612A1/fr
Priority to US17/015,985 priority patent/US11085935B2/en
Priority to ZA2020/06348A priority patent/ZA202006348B/en
Priority to IL278266A priority patent/IL278266B2/en
Anticipated expiration legal-status Critical
Priority to US17/368,403 priority patent/US11402392B2/en
Priority to US17/843,470 priority patent/US11635440B2/en
Priority to US18/305,586 priority patent/US20230341421A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the microtubule-associated protein tau plays an essential role in the morphology and physiology of neurons.
  • Tau has six different isoforms of the full-length protein and undergoes a number of possible post-translational modifications including acetylation, glycosylation and phosphorylation.
  • Phosphorylation is important for regulating the normal function of tau in axonal stabilization and can occur at over 80 different residues.
  • excessive phosphorylation of tau appears to increase the probability of tau aggregating into intracellular insoluble paired helical filaments (PHF) and neurofibrillary tangles (NFT), which are primarily composed of hyperphosphorylated tau.
  • PHF insoluble paired helical filaments
  • NFT neurofibrillary tangles
  • Intracellular neurofibrillary tangles in the cerebral cortex are a defining pathological feature of Alzheimer disease (AD) and correlate with the onset of clinical symptoms long after the appearance of extracellular amyloid-b (Ab) plaques, which begin to develop up two decades before symptom onset.
  • AD soluble p-tau and unphosphorylated tau are increased by two-fold in the cerebrospinal fluid (CSF). It has been proposed that these changes reflect the effects of neuronal death
  • tau comprises a hallmark AD pathology and can be measured in aggregated or soluble forms
  • important gaps remain in our understanding of how the post-translational modifications of this critical neuronal protein lead to the development of NFT and neurodegeneration in humans.
  • the relationship of tau to amyloid-b plaques is unknown.
  • tau can be used to stage subjects prior to the onset of symptoms associated with AD and guide treatment decisions.
  • the present disclosure encompasses a method to diagnose a subject as having an increased risk for conversion to mild cognitive impairment (MCI) due to Alzheimer’s disease (AD).
  • the method comprises (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 ,
  • T205 and T217 and optionally measuring total tau and (b) diagnosing the subject as having an increased risk for conversion to MCI due to AD when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p-T181 and p- T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p- T205 and total tau, or p-T217 and total tau. Mathematical operations other than a ratio may also be used.
  • the present disclosure encompasses a method to stage a subject prior to the onset of mild cognitive impairment (MCI) due to
  • AD Alzheimer’s disease
  • the method comprises (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 , T205 and T217 and optionally measuring total tau; and (b) diagnosing the subject as being a certain number of years from onset of MCI due to AD when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • phosphorylation level(s) may be a ratio between p-T181 and p-T205, p-T217 and p- T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. Mathematical operations other than a ratio may also be used.
  • the present disclosure encompasses a method to stage a subject after onset of Alzheimer’s disease (AD) symptoms.
  • the method comprises (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 , T205 and T217 and optionally measuring total tau; and (b) diagnosing the subject as being a certain number of years after onset of MCI due to AD when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • AD Alzheimer’s disease
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau. Mathematical operations other than a ratio may also be used.
  • the present disclosure encompasses a method for treating a subject in need thereof.
  • the method comprises (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 , T205 and T217 and optionally measuring total tau; and (b) administering a pharmaceutical composition to the subject when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p-T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau.
  • present disclosure encompasses a method for enrolling a subject into a clinical trial.
  • the method comprises (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 , T205 and T217 and optionally measuring total tau; and (b) enrolling the subject into a clinical trial when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p-T181 and p- T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p- T205 and total tau, or p-T217 and total tau. Mathematical operations other than a ratio may also be used.
  • FIG. 1 is a schematic of the longest human tau isoform (2N4R) and epitopes of tau antibodies.
  • the N-terminus, mid domain, MTBR, and C-terminus are identified for this isoform and will vary in a predictable way for other tau isoforms (e.g., 2N3R, 1 NR4, 1 N3R, 0N4R, and 0N3R).
  • FIG. 2 is a schematic showing the principle of the Parallel Reaction Monitoring experiment.
  • FIG. 3 shows data from a PRM screening of the mono- phosphorylated tau sequence at 103-126 (ON isoform).
  • FIG. 4 shows data from a PRM screening of mono-phosphorylated tau sequence 68-126 (1 N isoform) containing six potential phosphorylation sites. Three phosphorylation sites are shared by peptides containing residues 103-126 as described in FIG. 3 (d-f). Six LC-MS patterns were identified. The Y28 fragment carrying
  • phosphate shared by pS68 (a) or pT69 (b), is found in the two LC-MS patterns 4 and 5. This demonstrates the existence of the two phosphorylated peptides but corresponding LC-MS patterns cannot be strictly assigned without the detection of ion fragment y29 to differentiate pS68 and pT69. Specific fragments corresponding to pT71 (c) and pT 111 (d) are found in LC-MS patterns 6 and 1 , respectively. Specific fragments for pS113 (e) and pT123 (f) (y15 with phosphate) are found in the LC-MS pattern 2.
  • FIG. 5 shows data from a PRM screening of mono-phosphorylated tau sequence 45-67 (1 N and 2N isoforms).
  • a strong signal from a conformer was identified on the front of a non-phosphorylated peptide LC-MS pattern.
  • PRM scan interpretation led to the detection of pT50 (b) as the main phosphorylation site on this sequence (pattern 2).
  • Pattern 1 with a similar fragmentation fingerprint, was attributed to a conformer of pT50.
  • pS46 (a) able to differentiate signals from pT50 (b), was not detected, suggesting this
  • FIG. 6 shows data from a PRM screening of mono-phosphorylated tau sequence 88-126 (2N isoform). 6 potential phosphorylation sites are located in this sequence and 6 LC-MS patterns were identified. Fragments found in pattern 1 and 2 were consistent with phosphorylated peptides at residues T111 (d) and S113 (e), respectively. No specific fragment from the phosphorylated peptide at residue T123 (f) was found. Patterns 4 and 6 contained a low signal of the y29 fragment matching with phosphorylation on residue T101 (b) or T102 (c), but no specific fragment able to differentiate them was detected.
  • Patterns 3 and 5 shared fragments found in patterns 4 and 6 but in lower abundance, locating the phosphorylated residue at the N-terminus on residue G109. This could indicate the presence of an additional phosphorylated peptide, likely at residue T95 (a) or abundant conformers from peptides found in patterns 4 and 6.
  • FIG. 7 shows a PRM scan of mono-phosphorylation tau sequence 68-87, containing 4 potential phosphorylation sites. 3 LC-MS patterns were detected. Patterns 2 and 3 were consistent with phosphorylation at residues S68 (a) or T69 (b). Pattern 1 contained both fragments compatible with the presence of two co-eluted phosphorylated peptides at T71 (c) and T76 (d). Comparison of y14 XIC with and without phosphate in pattern 1 indicates pT71 (c) is more abundant than pT76 (d).
  • FIG. 8H, FIG. 8I, FIG. 8J, FIG. 8K, and FIG. 8L show detection of phosphorylation sites in the mid-domain and C-terminus of brain p-tau protein.
  • FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 89E, and FIG. 9F show phosphorylated peptide profiles from tau sequences 195-209 (SEQ ID NO: 38) and 212- 221 (SEQ ID NO: 64) are variable between the soluble brain fraction, normal CSF, and AD CSF tau protein. Brain soluble tau extracts are diluted as indicated to approximately match corresponding CSF tau level. Phosphorylated peptides on 195-209: in brain lysate, one signal corresponding to the co-elution of two phosphorylated peptides pS199 and pS202 is observed. In CSF, two additional signals are observed.
  • Fragment analysis allowed the assignment of the signal on left to pT205. In AD CSF, the two signals are increased allowing the identification of specific fragments assigning the signal on the right to pS208.
  • Phosphorylated peptides on 212-221 two signals with similar MS intensities corresponding to pT217 and pS214 are identified in brain lysate.
  • the signal corresponding to pT217 is the most intense while pS214 is close to the limit of detection, indicating a dramatic change in their relative abundance in comparison to the brain extract.
  • pT217 is significantly increased due to specific hyperphosphorylation.
  • FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E, FIG. 10F, FIG. 10G, FIG. 10H, and FIG. 101 show pT153, pT175 and pT231 phosphorylated peptides identified in CSF.
  • AQUA internal standard signals are shown for pT175 and pT231. Fragmentation pattern of pT153 is similar to unmodified.
  • FIG. 11 shows phosphorylation abundance on T111 is higher in the CSF than the brain relative to S113 phosphorylation.
  • the MS/MS fragment y18 common to all mono-phosphorylated peptides on tau sequence 103-126 is detected.
  • the relative abundance of the y15 fragment from pS113 (b) is significantly lower in CSF in comparison to brain extract.
  • the y15 fragment from pT 111 (a) is abundant in CSF and not detectable in brain soluble extract diluted to match the AD CSF tau level.
  • FIG. 12 shows relative abundance of tau phosphorylation depends on the biological extract and varies across the protein sequence. Comparison of tau phosphorylation abundance measured by MS in normal brain lysate, normal CSF and AD CSF extracted by immuno capture using H J8.5 and Tau1. Circle area is proportional to site phosphorylation abundance. Red and green colors indicate an increase or decrease, respectively, in comparison to the brain soluble profile taken as reference (blue). Tau is c-terminally truncated in CSF, which explains the absence of detection of the C-terminal cluster of phosphorylation sites. Phosphorylation on T205 and S208 is specific to CSF (red X on Brain Soluble - top).
  • FIG. 13 shows tau phosphorylation sites are differentially modified in brain, normal CSF and AD CSF. Measurements are the relative abundances of the phosphorylated signal compared to the corresponding non phosphorylated site
  • FIG. 14A, FIG. 14B, FIG. 14C, and FIG. 14D show antibody effect on phosphorylation ratio measurement by IP-MS.
  • Radar plots of phosphorylation ratios measured on main CSF sites are shown in brain lysate (FIG. 14A, left panel), non-AD CSF (FIG. 14A, middle panel) and AD CSF (FIG. 14A, right panel).
  • FIG. 14A Tau phosphorylation ratio
  • FIG. 14B Low recovery of pS199 by Tau1 or Tau1 +HJ8.5 IP underestimate pS199/S199 ratio measurements compared to other antibodies tested.
  • Tau13, HJ8.5, and HJ8.7 antibodies indicate no significant changes of pS199 phosphorylation ratio between brain and CSF.
  • pS202/S202 CSF hypophosphorylation (FIG. 14C) and pT217/pT217 hyperphosphorylation (FIG. 14D) are evidenced independently of the antibody used for IP-MS.
  • the legend for FIG. 14B-D is the same and shown in FIG. 14C - Brain (blue), nonAD CSF (green), and AD CSF (red).
  • FIG. 15 shows CSF incubation does not impact phosphorylation rate measurement on tau.
  • FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG. 16E, FIG. 16F, and FIG. 16G show amyloid plaques are strongly correlated with tau hyperphosphorylation but differ by site of phosphorylation.
  • FIG. 17A, FIG. 17B, FIG. 17C, FIG. 17D, FIG. 17E, and FIG. 17F show longitudinal changes of different phosphorylated-tau sites are stage of disease specific and change in opposite directions as AD progresses. Individual, z-transformed, longitudinal changes in the ratio of phosphorylation of (FIG. 17A) p-T217, (FIG. 17B) p- T181 (FIG. 17C) total tau, (FIG. 17D) p-T205, and (FIG.
  • FIG. 18A and FIG. 18B show phosphorylated-tau sites are differentially related to brain hypometabolism and atrophy.
  • FIG. 19A, FIG. 19B, FIG. 19C, and FIG. 19D show decreasing phosphorylation at p-T217, p-T181 and p-T205 is associated with dementia and cognitive decline.
  • FIG. 20 shows tau PET increases near symptom onset in DIAD mutation carriers.
  • NFT neurofibrillary tangle
  • FIG. 21 A, FIG. 21 B, FIG. 21C, FIG. 21 D, and FIG. 21 E show longitudinal change in tau and tau phosphorylation sites are differentially related to neurofibrillary tau (tau-PET) in dominantly inherited AD.
  • tau-PET neurofibrillary tau
  • the vertical line is an SUVR of 1.22 and represents a conservative estimate of the point when NFT tau-PET (a composite of multiple cortical and limbic regions) is considered elevated compared to non-carriers.
  • FIG. 22 is an illustration showing tau pathology evolves through distinct phases in Alzheimer Disease. Measuring four different soluble tau species and insoluble tau in a group of participants with deterministic Alzheimer disease mutations we show over the course of 35 years (x-axis) tau related changes unfold (y-axis) and differ based on the stage of disease and other measurable biomarkers.
  • FIG. 23A, FIG. 23B, and FIG. 23C show quantitation of
  • FIG. 23A T181 monitoring using a microLC system.
  • FIG. 23B S199, S202, T205 (coeluted in framed signal) and T217 monitoring using a nanoLC system. Endogenous signals (full blue line), 15N labeled peptides (red dotted line), AQUA peptides (green dotted line).
  • FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D and FIG. 24E show CSF tau phosphorylation on T217 is associated to amyloidosis status.
  • FIG. 24A T217 phosphorylation significantly increases in participants having amyloidosis (PiB-PET and CSF Ab42/40 ratio positive) compared to amyloid-negative controls with no or mild cognitive decline.
  • FIG. 24B ROC curves for the diagnosis of amyloid-positive from amyloid-negative participants using phosphorylation rate of T217, T181 by MS and T181 by ELISA.
  • FIG. 24C pT217/T217 ratio comparison demonstrates the specific
  • FIG. 24D-E Comparison of T217 phosphorylation with CSF Ab42/40 changes measured by MS and to amyloid plaque deposition measured by PiB-PET.
  • FIG. 24D The extent of T217
  • FIG. 24E PiB-PET loading (FBP Total Cortical Mean) is correlated with T217 phosphorylation state in amyloid-positive participants. Cut-off value differentiating amyloid positive from amyloid negative by PiB is 0.18.
  • FIG. 25 shows CSF tau phosphorylation on T217 is independent from cognitive status and is significantly modified in preclinical AD.
  • Left Panel No correlation exists between T217 phosphorylation and the cognitive profile measured by the clinical dementia rating sum of boxes (CDR-SB).
  • Tau protein aggregation into neurofibrillary tangles in the central nervous system contributes to the etiology of certain neurodegenerative disorders, including Alzheimer’s disease (AD). Though the mechanism of tau destabilization is not fully understood yet, tau protein has been found to be hyperphosphorylated in tau aggregates. Applicants have discovered that certain methods to quantify tau
  • FIG. 22 illustrates the dynamic pattern of tau phosphorylation measurable at T181 , T205 and T217 created by the applicant’s method in relation to years from onset of MCI due to AD and to the development of certain pathophysiological changes.
  • the present disclosure encompasses use of the methods to quantify tau phosphorylation at specific amino acid residues to predict time to onset of mild cognitive impairment due to Alzheimer’s disease, guide treatment decisions, select subjects for clinical trials, and evaluate the clinical efficacy of certain therapeutic interventions. Other aspects and iterations of the invention are described more thoroughly below.
  • the term“about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like.
  • the term“about” also encompasses these variations, which can be up to ⁇ 5%, but can also be ⁇ 4%, 3%,
  • An antibody may be a complete antibody as understood in the art, i.e. , consisting of two heavy chains and two light chains, or may be any antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab’, Fab, F(ab’)2, single domain antibodies, Fv, and single chain Fv.
  • the term antibody also refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody and a humanized antibody.
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; herein incorporated by reference in its entirety).
  • aptamer refers to a polynucleotide, generally a RNA or DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes.
  • an aptamer has a molecular activity such as binging to a target molecule at a specific epitope (region). It is generally accepted that an aptamer, which is specific in it binding to a polypeptide, may be synthesized and/or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art. See, for instance US 7,939,313, herein incorporated by reference in its entirety.
  • Ab refers to peptides derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the gene encoding APP is located on chromosome 21.
  • Ab peptides are typically 37-43 amino acid sequences long, though they can have truncations and modifications changing their overall size. They can be found in soluble and insoluble compartments, in monomeric, oligomeric and aggregated forms, intracellularly or extracellularly, and may be complexed with other proteins or molecules.
  • the adverse or toxic effects of Ab may be attributable to any or all of the above noted forms, as well as to others not described specifically.
  • Ab typically refers to a plurality of Ab species without discrimination among individual Ab species. Specific Ab species are identified by the size of the peptide, e.g., Ab42, Ab40, Ab38 etc.
  • the term“Ab42/ Ab40 value” means the ratio of the concentration of Ab42 in a sample obtained from a subject compared to the
  • Ab amyloidosis is clinically defined as evidence of Ab deposition in the brain.
  • a subject that is clinically determined to have Ab amyloidosis is referred to herein as“amyloid positive,” while a subject that is clinically determined to not have Ab amyloidosis is referred to herein as“amyloid negative.”
  • Ab amyloidosis likely exists before it is detectable by current techniques. Nonetheless, there are accepted indicators of Ab amyloidosis in the art.
  • Ab amyloidosis is typically identified by amyloid imaging (e.g., PiB PET, fluorbetapir, or other imaging methods known in the art) or by decreased cerebrospinal fluid (CSF) Ab42 or a decreased CSF Ab42/40 ratio.
  • amyloid imaging e.g., PiB PET, fluorbetapir, or other imaging methods known in the art
  • CSF cerebrospinal fluid
  • [ 11 C]PIB-PET imaging with mean cortical binding potential (MCBP) score > 0.18 is an indicator of Ab amyloidosis, as is cerebral spinal fluid (CSF) Ab42
  • IP/MS immunoprecipitation and mass spectrometry
  • amyloidosis may or may not be symptomatic, and symptomatic subjects may or may not satisfy the clinical criteria for a disease associated with Ab amyloidosis.
  • symptoms associated with Ab amyloidosis may include impaired cognitive function, altered behavior, abnormal language function, emotional dysregulation, seizures, dementia, and impaired nervous system structure or function.
  • Diseases associated with Ab amyloidosis include, but are not limited to, Alzheimer's Disease (AD), cerebral amyloid angiopathy, Lewy body dementia, and inclusion body myositis.
  • Subjects with Ab amyloidosis are at an increased risk of developing a disease
  • A“clinical sign of Ab amyloidosis” refers to a measure of Ab deposition known in the art.
  • Clinical signs of Ab amyloidosis may include, but are not limited to, Ab deposition identified by amyloid imaging (e.g. PiB PET, fluorbetapir, or other imaging methods known in the art) or by decreased cerebrospinal fluid (CSF)
  • Ab42 or Ab42/40 ratio See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, and Fagan AM et al. Ann Neurol 59(3) 2006, each hereby incorporated by reference in its entirety.
  • Clinical signs of Ab amyloidosis may also include measurements of the metabolism of Ab, in particular measurements of Ab42 metabolism alone or in comparison to measurements of the metabolism of other Ab variants (e.g. Ab37, Ab38, Ab39, Ab40, and/or total Ab), as described in U.S. Patent Serial Nos. 14/366,831 , 14/523,148 and 14/747,453, each hereby incorporated by reference in its entirety.
  • a subject with clinical signs of Ab amyloidosis may or may not have symptoms associated with Ab deposition. Yet subjects with clinical signs of Ab amyloidosis are at an increased risk of developing a disease associated with Ab amyloidosis.
  • A“candidate for amyloid imaging” refers to a subject that has been identified by a clinician as in individual for whom amyloid imaging may be clinically warranted.
  • a candidate for amyloid imaging may be a subject with one or more clinical signs of Ab amyloidosis, one or more Ab plaque associated symptoms, on one or more CAA associated symptoms, or combinations thereof.
  • a clinician may recommend amyloid imaging for such a subject to direct his or her clinical care.
  • a candidate for amyloid imaging may be a potential participant in a clinical trial for a disease associated with Ab amyloidosis (either a control subject or a test subject).
  • An“Ab plaque associated symptom” or a“CAA associated symptom” refers to any symptom caused by or associated with the formation of amyloid plaques or CAA, respectively, being composed of regularly ordered fibrillar aggregates called amyloid fibrils.
  • Exemplary Ab plaque associated symptoms may include, but are not limited to, neuronal degeneration, impaired cognitive function, impaired memory, altered behavior, emotional dysregulation, seizures, impaired nervous system structure or function, and an increased risk of development or worsening of Alzheimer's disease or CAA.
  • Neuronal degeneration may include a change in structure of a neuron
  • Impaired cognitive function may include but is not limited to difficulties with memory, attention, concentration, language, abstract thought, creativity, executive function, planning, and organization.
  • Altered behavior may include, but is not limited to, physical or verbal aggression, impulsivity, decreased inhibition, apathy, decreased initiation, changes in personality, abuse of alcohol, tobacco or drugs, and other addiction-related behaviors.
  • Emotional dysregulation may include, but is not limited to, depression, anxiety, mania, irritability, and emotional incontinence.
  • Seizures may include but are not limited to generalized tonic-clonic seizures, complex partial seizures, and non-epileptic, psychogenic seizures.
  • Impaired nervous system structure or function may include, but is not limited to, hydrocephalus, Parkinsonism, sleep disorders, psychosis, impairment of balance and coordination. This may include motor impairments such as monoparesis, hemiparesis, tetraparesis, ataxia, ballismus and tremor. This also may include sensory loss or dysfunction including olfactory, tactile, gustatory, visual and auditory sensation. Furthermore, this may include autonomic nervous system impairments such as bowel and bladder dysfunction, sexual
  • this may include hormonal impairments attributable to dysfunction of the hypothalamus and pituitary gland such as deficiencies and dysregulation of growth hormone, thyroid stimulating hormone, lutenizing hormone, follicle stimulating hormone, gonadotropin releasing hormone, prolactin, and numerous other hormones and modulators.
  • the term“subject” refers to a mammal, preferably a human.
  • the mammals include, but are not limited to, humans, primates, livestock, rodents, and pets.
  • a subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.
  • the term“healthy control group,”“normal group” or a sample from a“healthy” subject means a subject, or group subjects, who is/are diagnosed by a physician as not suffering from Ab amyloidosis, or a clinical disease associated with Ab amyloidosis (including but not limited to Alzheimer's disease) based on qualitative or quantitative test results.
  • A“normal” subject is usually about the same age as the individual to be evaluated, including, but not limited, subjects of the same age and subjects within a range of 5 to 10 years.
  • blood sample refers to a biological sample derived from blood, preferably peripheral (or circulating) blood.
  • the blood sample can be whole blood, plasma or serum, although plasma is typically preferred.
  • the term“isoform”, as used herein, refers to any of several different forms of the same protein variants, arising due alternative splicing of mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein, genetic variations and somatic recombination.
  • the terms“isoform” and“variant” are used interchangeably.
  • tau protein or “tau” encompasses all tau isoforms, whether full-length, truncated, or post-translationally modified.
  • tau is encoded by the gene MAPT.
  • isoforms of tau that are generated by alternative splicing of exons 2, 3, and 10 of MAPT. These isoforms range in length from 352 to 441 amino acids.
  • Exons 2 and 3 encode 29-amino acid inserts each in the N- terminus (called N), and full-length human tau isoforms may have both inserts (2N), 1 one insert (1 N), or no inserts. All full-length human tau isoforms also have three repeats of the microtubule binding domain (called R). Inclusion of exon 10 at the C-terminus leads to inclusion of a fourth microtubule binding domain encoded by exon 10. Hence, full-length human tau isoforms may be comprised of four repeats of the microtubule binding domain (exon 10 included) or three repeats of the microtubule binding domain (exon 10 excluded).
  • Human tau may or may not be post-translationally modified.
  • tau may be phosphorylated, ubiquinated, glycosylated, and glycated.
  • the term“human tau” encompasses the (2N, 3R), (2N, 4R), (1 N, 3R), (1 N, 4R), (ON, 3R), and (ON, 4R) isoforms, isoforms that are N- and/or C-terminally truncated species thereof, and all post-translationally modified isoforms.
  • Alternative splicing of the gene encoding tau similarly occurs in other animals. In animals where the gene is not identified as MAPT, a homolog may be identified by methods well known in the art.
  • a disease associated with tau deposition in the brain may be referred to as a "tauopathy”.
  • Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy, dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-Bodig disease, Parkinson-dementia complex of Guam, tangle- predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration, argyrophilic grain disease (AGD), Frontotemporal lobar degeneration, Alzheimer's Disease, and frontotemporal dementia.
  • APD argyrophilic grain disease
  • “Significantly deviate from the mean” refers to values that are at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations or even more preferably at least 2 standard deviations, above or below the mean.
  • Ab and tau therapies collectively refers to any imaging agent or therapeutic agent contemplated for, or used with, subjects at risk of developing Ab amyloidosis or AD, subjects diagnosed as having Ab amyloidosis, subjects diagnosed as having a tauopathy, or subjects diagnosed as having AD.
  • Methods of the present disclose comprise providing an isolated tau sample obtained from a subject and measuring tau phosphorylation at one or more amino acid residue and optionally total tau.
  • An isolated tau sample refers to a composition comprising tau, wherein tau has been purified from blood or cerebrospinal fluid (CSF) obtained from a subject.
  • CSF cerebrospinal fluid
  • a subject is a mammal, preferably a human.
  • CSF may be obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple blood or CSF samples contemporaneously collected from the subject may be pooled. Blood may be collected by veni-puncture with or without an intravenous catheter, or by a finger stick (or the equivalent thereof).
  • blood or CSF samples may be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cellular debris, use of additives designed to stabilize and preserve the specimen prior to analytical testing, etc.). Blood or CSF samples may be used
  • tau has been either partially or completely purified from blood or CSF.
  • Methods for purifying tau from blood or CSF include, but are not limited to, selective
  • Suitable methods concentrate both phosphorylated tau and unphosphorylated tau from blood or CSF.
  • isolated tau samples of the present disclosure comprise tau that has been purified from blood or CSF by affinity purification.
  • Affinity purification refers to methods that purify a protein of interest by virtue of its specific binding properties to an immobilized ligand.
  • an immobilized ligand is a ligand attached to a solid support, such as a bead, resin, tissue culture plate, etc.
  • Suitable ligands specifically bind both phosphorylated and unphosphoryated tau.
  • a suitable ligand may bind an epitope within the mid domain of tau.
  • a suitable ligand may bind an epitope within the N-terminus of tau, preferably within amino acids 1 to 35 of tau.
  • a suitable ligand may bind an epitope within the MTBR of tau. In another example, a suitable ligand may bind an epitope within the C-terminus of tau. In still further embodiments, tau may be affinity purified from blood or CSF using two or more immobilized ligands. In one example, an immobilized ligand binds an epitope within the N-terminus of tau and another
  • immobilized ligand binds an epitope within the mid domain of tau.
  • an immobilized ligand binds an epitope within the MTBR of tau and another immobilized ligand binds an epitope within the mid domain of tau.
  • an immobilized ligand binds an epitope within the MTBR of tau and another immobilized ligand binds an epitope within the mid domain of tau.
  • immobilized ligand binds an epitope within the C-terminus of tau and another
  • immobilized ligand binds an epitope within the mid domain of tau.
  • an immobilized ligand binds an epitope within the C-terminus of tau and another immobilized ligand binds an epitope within the N-terminus of tau.
  • an immobilized ligand binds an epitope within the MTBR of tau and another immobilized ligand binds an epitope within the N-terminus of tau.
  • an immobilized ligand binds an epitope within the MTBR of tau and another immobilized ligand binds an epitope within the N-terminus of tau.
  • the immobilized ligand binds an epitope within the MTBR of tau and another immobilized ligand binds an epitope within the C-terminus of tau.
  • the ligand may be an antibody or an aptamer. Non-liming examples of suitable antibodies are shown in FIG. 1.
  • An isolated tau sample may be used immediately or may be stored indefinitely by methods known in the art.
  • Phosphorylation of specific amino acids (i.e.“sites”) in tau results in phosphorylated tau (p-tau) isoforms.
  • Methods of the present disclosure provide means to measure the stoichiometry of phosphorylation at one or more specific amino acids of tau.
  • methods herein comprise measuring tau phosphorylation at one or more residue chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231.
  • methods herein comprise measuring tau phosphorylation at one or more residue chosen from T111 , T181 , T205, S208, S214, T217, and T231.
  • methods herein comprise measuring tau phosphorylation at one or more residue chosen from T181 , S214, and T217. In other embodiments, methods herein comprise measuring tau phosphorylation at one or more residue chosen from T181 , T205, and T217. In other embodiments, methods herein comprise measuring tau phosphorylation at one or more residue that includes S199. In other embodiments, methods herein comprise measuring tau phosphorylation at one or more residue that includes S202. In other embodiments, methods herein comprise measuring tau phosphorylation at one or more residue that includes S199. In other embodiments, methods herein comprise measuring tau phosphorylation at one or more residue that includes T181.
  • methods herein comprise measuring tau phosphorylation at one or more residue that includes T205. In other embodiments, methods herein comprise measuring tau phosphorylation at one or more residue that includes T217. In other embodiments, methods herein comprise measuring tau phosphorylation at two or more residues that include T153 and T175. In other embodiments, methods herein comprise measuring tau phosphorylation at two or more residue chosen from T 181 , T205, and T217. In other embodiments, methods herein comprise measuring tau phosphorylation at three or more residues that include T181 , T205, and T217.
  • MS mass spectrometry
  • PRM parallel reaction monitoring
  • the present disclosure is not limited to any one particular method to quantitatively assess site-specific phosphorylation of tau. Suitable methods should discriminate tau isoforms that differ only in the phosphorylation status of a single amino acid, discriminate p-tau isoforms that are phosphorylated at different amino acids, and quantify changes in phosphorylation occurring at specific sites independently from the global change in total tau.
  • phosphorylated peptide is normalized with any absolute quantitation value obtained for any peptide from the tau protein. All three approaches use internal normalization for comparing relative phosphorylation changes for each site. Other methods known in the art may also be used.
  • site-specific phosphorylation of tau is measured by high-resolution mass spectrometry.
  • mass spectrometers are known in the art. These include, but are not limited to, quadrupole, time-of-flight, ion trap and Orbitrap, as well as hybrid mass spectrometers that combine different types of mass analyzers into one architecture (e.g., Orbitrap FusionTM TribridTM Mass
  • tau may be proteolytically digested. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C, and Arg-N.
  • affinity purification is used to produce an isolated tau sample, digestion may occur after eluting tau from the immobilized ligand or while tau is bound.
  • digested tau peptides may be separated by a liquid chromatography system interfaced with a high-resolution mass spectrometer. The chromatography system may be optimized by routine experimentation to produce a desired LC-MS pattern. A wide array of LC-MS techniques may be used to
  • Total tau refers to all tau isoforms in a given sample. Tau can be found in soluble and insoluble compartments, in monomeric and aggregated forms, in ordered or disordered structures, intracellularly and extracellularly, and may be complexed with other proteins or molecules. Accordingly, the source of the biological sample (e.g., brain tissue, CSF, blood, etc.) and any downstream processing of the biological sample will affect the totality of tau isoforms in a given sample.
  • the source of the biological sample e.g., brain tissue, CSF, blood, etc.
  • Total tau may be measured by monitoring abundance of unmodified tau peptides. For each phosphorylated tau site, a tau peptide sharing the common amino acid sequence with the phosphorylated peptide of interest may preferentially be used to measure total tau level, but any peptide from the tau sequence can be used.
  • Tau peptides measurement can be performed by mass spectrometry and accuracy of the measurement can be improved by using labeled internal standards as reference. Alternatively, total tau can be measured by immunoassays or other method quantifying tau concentration.
  • One aspect of the present disclosure encompasses methods to diagnose subjects as having a high risk of conversion to mild cognitive impairment due to Alzheimer’s disease, and to optionally stage or classify the subject in terms of the number of years of onset to MCI due to AD.
  • Mild cognitive impairment (MCI) due to Alzheimer’s disease (AD) refers to the symptomatic predementia phase of AD. This degree of cognitive impairment is not normal for age and, thus, constructs such as age- associated memory impairment and age-associated cognitive decline do not apply.
  • MCI due to AD is a clinical diagnosis, and clinical criteria for the diagnosis of MCI due to AD are known in the art. See, for instance, Albert et al. Alzheimer’s & Dementia, 2011 , 7(3): 270-279.
  • Cognitive testing is optimal for objectively assessing the degree of cognitive impairment for a subject. Scores on cognitive tests for subjects with MCI are typically 1 to 1.5 standard deviations below the mean for their age and education matched peers on culturally appropriate normative data (i.e. , for the impaired domain(s), when available). The designation of MCI is often supported by a global rating of 0.5 on the Clinical Dementia Rating (CDR) scale. The CDR is a numeric scale used to quantify the severity of symptoms of dementia. Other suitable cognitive tests are known in the art. While suitable tests exist to assess the severity of cognitive impairment, there is a need in the art for a test that identifies subjects with a high degree of confidence years before the onset of MCI due to AD.
  • CDR Clinical Dementia Rating
  • a method to diagnose a subject as having a high risk of conversion to MCI due to AD may comprise (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; and (b) diagnosing the subject as having a high risk of conversion to MCI due to AD when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a method to diagnose a subject as having a high risk of conversion to MCI due to AD may comprise (a) providing a first and a second isolated tau sample obtained from a subject and measuring, in each isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) diagnosing the subject as having a high risk of conversion to MCI due to AD when the calculated change(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.“Significantly deviate from the mean” refers to values that are at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5
  • o is the standard deviation defined by the normal distribution measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF).
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used to diagnose a subject.
  • An isolated tau sample can be obtained from a subject that may or may not be asymptomatic.
  • An“asymptomatic subject” refers to a subject that does not show any signs or symptoms of AD.
  • a subject may however exhibit signs or symptoms of AD (e.g., memory loss, misplacing things, changes in mood or behavior, etc.,) but not show sufficient cognitive or functional impairment for a clinical diagnosis of mild cognitive impairment.
  • a subject may carry one of the gene mutations known to cause dominantly inherited Alzheimer’s disease.
  • a subject may not carry a gene mutation known to cause dominantly inherited Alzheimer’s disease.
  • Alzheimer’s disease Alzheimer’s disease that has no specific family link is referred to as sporadic Alzheimer’s disease.
  • a“stage of AD” may be defined as an amount of time (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 months, etc.) that has elapsed since the onset of MCI due to AD.
  • a method to diagnose a subject’s stage of AD may comprise (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; and (b) diagnosing the stage of the subject’s AD when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a method to diagnose a subject prior to the onset of MCI due to AD may comprise (a) providing a first and a second isolated tau sample obtained from a subject and measuring, in each isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) diagnosing the subject as being a certain number of years from onset of MCI due to AD when the calculated change(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.“Significantly deviate from the mean” includes values that are at least 1 standard deviation, preferably at least 1.3 standard deviations or more preferably at least 1.5 standard deviation
  • o is the standard deviation defined by the normal distribution measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF).
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used to diagnose a subject.
  • An isolated tau sample can be obtained from a subject that may or may not have a clinical diagnosis of MCI due to AD, dementia, or AD.
  • a subject may carry one of the gene mutations known to cause dominantly inherited Alzheimer’s disease.
  • a subject may not carry a gene mutation known to cause dominantly inherited Alzheimer’s disease.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used. Both approaches are detailed in the examples. Mathematical operations other than a ratio may also be used. For instance, the examples use site-specific tau phosphorylation values in various statistical models (e.g., linear regressions, LME curves, LOESS curves, etc.) in conjunction with other known biomarkers (e.g. APOE e4 status, age, sex, cognitive test scores, functional test scores, etc.).
  • biomarkers e.g. APOE e4 status, age, sex, cognitive test scores, functional test scores, etc.
  • diagnostic accuracy may be evaluated by area under the ROC curve and in some embodiments, an ROC AUC value of 0.7 or greater is set as a threshold (e.g., 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.).
  • Brain amyloid plaques in humans are routinely measured by amyloid-positron emission tomography (PET).
  • PET amyloid-positron emission tomography
  • 11 C-Pittsburgh compound B (PiB) PET imaging of cortical Ab-plaques is commonly used to detect Ab-plaque pathology.
  • the standard uptake value ratio (SUVR) of cortical PiB-PET reliably identifies significant cortical Ab-plaques and is used to classify subjects as PIB positive (SUVR > 1 .25) or negative (SUVR ⁇ 1 .25).
  • a control population without brain amyloid plaques as measured by PET imaging may refer to a population of subjects that have a cortical PiB-PET SUVR ⁇ 1 .25.
  • Other values of PiB binding e.g., mean cortical binding potential
  • analyses of regions of interest other than the cortical region may also be used to classify subjects as PIB positive or negative.
  • Other PET imaging agents may also be used.
  • a control population without brain amyloid plaques as measured by Ab42/40 measurement in CSF may refer to a population of subjects that has an
  • a method to diagnose a subject as having a high risk of conversion to MCI due to AD or a subject’s stage of AD may comprise (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 , T205 and T217 and optionally measuring total tau; and (b) diagnosing the subject as having a high risk of conversion to MCI due to AD, or as being a certain number of years from onset of MCI due to AD, or staging the subject’s AD when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • FIG. 22 illustrates the dynamic pattern of tau phosphorylation measurable at T181 , T205 and T217 in an isolated tau sample in relation to years from onset of MCI due to AD.
  • Phosphorylation levels at T217 that significantly deviate from the mean first occur about 21 years from onset of MCI due to AD;
  • phosphorylation levels at T181 that significantly deviate from the mean first occur about 19 years from onset of MCI due to AD; an increase in total tau that significantly deviates from the mean first occurs about 17 years from onset of MCI due to AD;
  • phosphorylation levels at T205 that significantly deviate from the mean first occur about 13 years from onset of MCI due to AD.
  • symptom onset e.g., MCI due to AD
  • phosphorylation levels at T217 and T181 plateau and then decrease.
  • additional mathematical operations may be performed with the measurements of phosphorylation at T181 , T205 and/or T217, including but not limited to ratio between the measured phosphorylation level(s) and ratio between the measured phosphorylation level(s) and total tau.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p-T181 and p- T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p- T205 and total tau, or p-T217 and total tau.
  • a method of the present disclosure comprises (a) providing an isolated tau sample obtained from a subject and measuring tau
  • tau phosphorylation at T217 and/or tau phosphorylation at T181 may be about 1 3o, about 1 35o, about 1 4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2 o, or above 2o. In other embodiments, tau phosphorylation at T217 and/or tau phosphorylation at T181 may be about 1.85o, about 1.9o, about 1.95o, about 2o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • tau phosphorylation at T205 may be about 1.3o, about 1.35o, about 1.4o, about 1.45o, about 1.5o, about 1.51 o, about 1.55o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2.0o, or below 2.0o.
  • tau phosphorylation at T205 may be about 2.0o, about 2.05o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o, or below 2.5o.
  • tau phosphorylation at T217 and/or tau phosphorylation at T181 about 2o or above and tau phosphorylation at T205 may be about 2o or less.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used to diagnose a subject.
  • measured levels of tau phosphorylation at T205 and at T181 and/or T217 may be used in various mathematical operations to improve the predictive power compared to each by itself.
  • ratio(s) may be calculated from the measured phosphorylation levels.
  • Mathematical operations other than a ratio may also be used.
  • a method of the present disclosure comprises (a) providing an isolated tau sample obtained from a subject and measuring total tau and tau phosphorylation at (i) T217 and T205, (ii) T 181 and T205, or (iii) T181 , T205 and T217; and (b) diagnosing the subject as being about 10 to about 25 years, or about 10 to about 20 years from the onset of MCI due to AD when the ratio of tau
  • phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau is about 1 5o or above and the ratio of tau phosphorylation at T205 to total tau is about 1.5o or below, where o is the standard deviation defined by the normal distribution of total tau and tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 1 3o, about 1 35o, about 1 4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2 o, or above 2o. In other embodiments, the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 1 85o, about 1 9o, about 1 95o, about 2o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • the ratio of tau phosphorylation at T205 to total tau may be about 1 3o, about 1 35o, about 1 4o, about 1 45o, about 1 50o, about 1 55o, about 1 6o, about 1 7o, about 1 8o, about 1 9o, about 2.0 o, or below 2o.
  • the ratio of tau phosphorylation at T205 to total tau may be about 2.0 o, about 2.05 o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o, or below 2.5o.
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 2o or above and the ratio of tau phosphorylation at T205 to total tau may about 2o or less.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used to diagnose a subject.
  • a method of the present disclosure comprises (a) providing an isolated tau sample obtained from a subject and measuring tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; and (b) diagnosing the subject as being about 15 years or less, or about 10 years or less, from the onset of MCI due to AD when tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) is about 1 5o or above, where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 1 3o, about 1 35o, about 1 4o, about 1 45o, about 1 5o, about 1 6o, about 1.la, about 1 8o, about 1 9o, about 2a, or above 2o.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 1 85o, about 1.9o, about 1.95o, about 2o, about 2.1o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 2o or above.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • a method of the present disclosure comprises (a) providing an isolated tau sample obtained from a subject and measuring total tau and tau phosphorylation at (i) T 181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; and (b) diagnosing the subject as being about 15 years or less, or about 10 years or less, from the onset of MCI due to AD when the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau is about 1 5o or above, where o is the standard deviation defined by the normal distribution of total tau and tau phosphorylation at T217 and T205, T 181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 1.3o, about 1.35o, about 1.4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1 8o, about 1 9o, about 2 o, or above 2o.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 1.85o, about 1.9o, about 1.95o, about 2o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 2o or above.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used to diagnose a subject.
  • a method of the present disclosure comprises (a) providing a first and a second isolated tau sample obtained from a subject, wherein “first” and“second” refer to the order in which the samples were collected, and measuring tau phosphorylation at (i) T 181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) diagnosing the stage of a subject’s AD when the phosphorylation level at T181 and/or T217 decreases or stays the same and the phosphorylation level at T205 and optionally total tau increases.
  • the first and the second isolated tau samples may be collected days, weeks, or months apart.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) will also be about 1 5o or above for both samples and , where o is the standard deviation defined by the normal distribution tau phosphorylation at T217 and T205,
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • a method of the present disclosure comprises (a) providing a first and a second isolated tau sample obtained from a subject, wherein “first” and“second” refer to the order in which the samples were collected, and measuring total tau and tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T 181 , T217 and T205; (b) calculating the change in the site-specific phosphorylation at each residue measured and the change in total tau; and (c) diagnosing the stage of a subject’s AD when the phosphorylation level at T181 and/or T217 decreases or stays the same, the phosphorylation level at T205 decreases or stays the same, and total tau increases.
  • the first and the second isolated tau samples may be collected days, weeks, or months apart. Typically, tau phosphorylation at the specific sites recited in (a)(i),
  • (a)(ii) or (a)(iii) will also be about 1 5o or above for both samples and , where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • tau phosphorylation at T181 , T205 and T217 indicated as percentage of ptau/tau ratio is 21.7 ⁇ 2.3, 0.34 ⁇ 0.13, and 1.2 ⁇ 0.66, respectively, in a control population without brain amyloid plaques as measured by PET imaging, as measured in an isolated tau sample that was purified from CSF (see Table 3, mutation non-carriers column). Accordingly, twice the standard deviation above the mean found for the mutation non-carrier population (i.e.
  • p-T 181 /T181 , p- T205/T205 and p-T217/T217 is 43.4, 0.68, and 2.4, respectively.
  • the absolute value may vary depending upon the protocol and the source/specifications of internal standards used for absolute quantitation.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification and tau phosphorylation is measured by mass spectrometry.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification using a ligand that specifically binds an epitope within the mid domain of tau, and optionally with a second ligand that specifically binds an epitope within the N-terminus of tau, and tau phosphorylation is measured by high resolution mass spectrometry.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification using a ligand that specifically binds an epitope within the mid domain of tau, and optionally with a second ligand that specifically binds an epitope within the MTBR or the C-terminus of tau, and tau phosphorylation is measured by high resolution mass spectrometry.
  • a mass spectrometry protocol outlined in the Examples is used. IV.
  • Another aspect of the present disclosure is a method for treating a subject in need thereof.
  • the terms“treat,” “treating,” or “treatment” as used herein, refers to the provision of medical care by a trained and licensed professional to a subject in need thereof.
  • the medical care may be a diagnostic test, a therapeutic treatment, and/or a prophylactic or preventative measure.
  • the object of therapeutic and prophylactic treatments is to prevent or slow down (lessen) an undesired physiological change or disease/disorder.
  • Beneficial or desired clinical results of therapeutic or prophylactic treatments include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented.
  • a subject receiving treatment is asymptomatic.
  • An“asymptomatic subject,” as used herein, refers to a subject that does not show any signs or symptoms of AD.
  • a subject may exhibit signs or symptoms of AD (e.g., memory loss, misplacing things, changes in mood or behavior, etc.,) but not show sufficient cognitive or functional impairment for a clinical diagnosis of mild cognitive impairment due to Alzheimer’s disease.
  • a symptomatic or an asymptomatic subject may have Ab amyloidosis; however, prior knowledge of Ab amyloidosis is not a requisite for treatment.
  • a subject may be diagnosed as having AD.
  • a subject may carry one of the gene mutations known to cause dominantly inherited Alzheimer’s disease.
  • a subject may not carry a gene mutation known to cause dominantly inherited Alzheimer’s disease.
  • a method for treating a subject as described above may comprise (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; and (b) administering a pharmaceutical composition to the subject when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a method for treating a subject as described above may comprise (a) providing a first and a second isolated tau sample obtained from a subject and measuring, in each isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) administering a pharmaceutical composition to the subject when the calculated change(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.“Significantly deviate from the mean” refers to values that are at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations or even more preferably at least 2 standard deviations, above or below
  • the extent of change above or below the mean may be used as criteria for treating a subject.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p- T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau.
  • Mathematical operations other than a ratio may also be used.
  • the examples use site-specific tau phosphorylation values in various statistical models (e.g., linear regressions, LME curves, LOESS curves, etc.) in conjunction with other known biomarkers (e.g. APOE e4 status, age, sex, cognitive test scores, functional test scores, etc.).
  • Ab targeting therapies are generally designed to decrease Ab production, antagonize Ab aggregation or increase brain Ab clearance
  • tau targeting therapies are generally designed to alter tau phosphorylation patterns, antagonize tau aggregation, or increase NFT clearance
  • a variety of therapies are designed to reduce CMS inflammation or brain insulin resistance; etc.
  • the efficacy of these various agents can be improved by administering the agents to subjects that have certain tau
  • the efficacy of imaging agents and therapeutic agents contemplated for, or used with, subjects at risk of developing Ab amyloidosis or AD, subjects diagnosed as having Ab amyloidosis, subjects diagnosed as having a tauopathy, or subjects diagnosed as having AD can be improved by administering the Ab or tau therapy to subjects that have certain tau phosphorylation levels at T181 , T205 and/or T217, as measured by methods disclosed herein and illustrated, for example, in FIG.
  • preferred therapeutic agents may include those designed to prevent a subject from becoming amyloid positive (e.g., amyloid targeting therapies designed to decrease Ab production, antagonize Ab aggregation, etc ).
  • preferred therapeutic agents may include those designed to prevent amyloid deposition from increasing or reduce a subject’s existing plaque load.
  • preferred therapeutic agents may include those designed to prevent amyloid deposition from increasing, reduce a subject’s existing plaque load, prevent tau aggregation, or target NFTs.
  • preferred therapeutic agents may include those designed to prevent amyloid deposition from increasing, reduce a subject’s existing plaque load, prevent tau aggregation, or target NFTs, as well as those specific for subjects with AD.
  • the details disclosed herein can similarly be used to administer therapeutic agents designed for other targets (e.g., CNS
  • the present disclosure provides a method for treating a subject having an increased risk of conversion to MCI due to AD, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring tau phosphorylation at (i) T217 and T205, (ii) T 181 and T205, or (iii) T181 , T205 and T217; and (b) administering a pharmaceutical composition to the subject when tau phosphorylation at T217 and/or T181 is about 1 5o or above and tau phosphorylation at T205 is about 1 5o or below, where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T 181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • tau phosphorylation at T217 and/or tau phosphorylation at T181 may be about 1 3o, about 1.35o, about 1.4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1.8o, about 1 9o, about 2 o, or above 2o. In other embodiments, tau phosphorylation at T217 and/or tau phosphorylation at T181 may be about 1 85o, about 1 9o, about 1 95o, about 2a, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • tau phosphorylation at T205 may be about 1 3o, about 1 35o, about 1.4o, about 1.45o, about 1.5o, about 1.51 o, about 1.55o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2.0 o, or below 2.0 o.
  • tau phosphorylation at T205 may be about 2.0 o, about 2.05 o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o, or below 2.5o.
  • tau phosphorylation at T217 and/or tau phosphorylation at T181 about 2o or above and tau phosphorylation at T205 may be about 2o or less.
  • measured levels of tau phosphorylation at T205 and at T181 and/or T217 may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • the present disclosure provides a method for treating a subject having an increased risk of conversion to MCI due to AD, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring total tau and tau phosphorylation at (i) T217 and T205, (ii) T 181 and T205, or (iii) T181 , T205 and T217; and (b) administering a pharmaceutical composition to the subject when the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau is about 1 5o or above and the ratio of tau phosphorylation at T205 to total tau is about 1 5o or below, where o is the standard deviation defined by the normal distribution of total tau and tau phosphorylation at T217 and T205, T 181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 1.3o, about 1.35o, about 1.4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1 8o, about 1 9o, about 2a, or above 2o.
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 1 85o, about 1 9o, about 1 95o, about 2 o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o. In each of the above
  • the ratio of tau phosphorylation at T205 to total tau may be about 1 3o, about 1.35o, about 1.4o, about 1.45o, about 1.50o, about 1.55o, about 1.6o, about 1 7o, about 1 8o, about 1 9o, about 2.0o, or below 2o.
  • the ratio of tau phosphorylation at T205 to total tau may be about 2.0o, about 2.05o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o, or below 2.5o.
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 2o or above and the ratio of tau phosphorylation at T205 to total tau may about 2o or less.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used as criteria to treat a subject.
  • the present disclosure provides a method for treating a subject having an increased risk of conversion to MCI due to AD, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring tau phosphorylation at (i) T 181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; and (b) administering a pharmaceutical composition to the subject when tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) is about 1 5o or above, where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T 181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 1 3o, about 1 35o, about 1.4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2 o, or above 2o.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 1 85o, about 1 9o, about 1 95o, about 2 o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 2o or above.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • the present disclosure provides a method for treating a subject having an increased risk of conversion to MCI due to AD, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring total tau and tau phosphorylation at (i) T 181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; and (b) administering a pharmaceutical composition to the subject when the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau is about 1 5o or above, where o is the standard deviation defined by the normal distribution of total tau and tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 1 3o, about 1 35o, about 1 4o, about 1 45o, about 1 5o, about 1 6o, about 1 7o, about 1 8o, about 1 9o, about 2 o, or above 2o.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 1 85o, about 1 9o, about 1 95o, about 2 o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 2o or above.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used as criteria to treat a subject.
  • the present disclosure provides a method for treating a subject with symptoms of AD, the method comprising (a) providing a first and a second isolated tau sample obtained from a subject, wherein“first” and“second” refer to the order in which the samples were collected, and measuring tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) administering a pharmaceutical composition to the subject when the phosphorylation level at T181 and/or T217 decreases or stays the same and the phosphorylation level at T205 and optionally total tau increases.
  • the first and the second isolated tau samples may be collected days, weeks, or months apart.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) will also be about 1 5o or above for both samples and , where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • the present disclosure provides a method for treating a subject with symptoms of AD, the method comprising (a) providing a first and a second isolated tau sample obtained from a subject, wherein“first” and“second” refer to the order in which the samples were collected, and measuring total tau and tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205;
  • the first and the second isolated tau samples may be collected days, weeks, or months apart. Typically, tau phosphorylation at the specific sites recited in (a)(i),
  • phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • a pharmaceutical composition may comprise an imaging agent.
  • imaging agents include functional imaging agents (e.g. fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radionuclide- labeled antibodies, etc.)
  • a pharmaceutical composition may comprise an active pharmaceutical ingredient.
  • active pharmaceutical ingredients include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-Ab antibodies (including antigen-binding fragments, variants, or derivatives thereof), anti- tau antibodies (including antigen- binding fragments, variants, or derivatives thereof), stem cells, dietary supplements (e.g.
  • TRx0237 methylthionimium chloride, etc.
  • therapies to improve blood sugar control e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc
  • a pharmaceutical composition may comprise a kinase inhibitor.
  • Suitable kinase inhibitors may inhibit a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3P, MARK, CDK5, Fyn, 5' adenosine monophosphate- activated protein kinase (AMPK), Calcium-calmodulin kinase II, Cyclin-dependent kinase-5 (cdk5), Casein kinase 1 (CK1 ), Casein kinase 2 (CK2), Cyclic AMP-dependent protein kinase (PKA), Dual-specificity tyrosine-phosphorylation regulated kinase 1A (DYRK1A), Glycogen synthase kinase-3 (GSK-3), JNK, LRRK2, Microtubule affinity- regulating kinase (MARK), MSK1 , p35/41 , p42/p44 mitose (TA
  • PSK1/TAOK2 Prostate-derived sterile 20-like kinase 2
  • PSK2/TAOK1 Prostate-derived sterile 20-like kinase 2
  • SAPK Stress- activated protein kinase 1 gamma
  • SAPK2a Prostate-derived sterile 20-like kinase 2
  • SAPK3 Prostate-derived sterile 20-like kinase 2
  • SAPK Stress- activated protein kinase 1 gamma
  • SAPK2a SAPK2b
  • Tau-tubulin kinase 1/2 Tau-tubulin kinase 1/2
  • a pharmaceutical composition may comprise a phosphatase activator.
  • a phosphatase activator may increase the activity of protein phosphatase 1 , 2A, 2B, or 5.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification and tau phosphorylation is measured by mass spectrometry.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification using a ligand that specifically binds an epitope within the mid domain of tau, and optionally with a second ligand that specifically binds an epitope within the N-terminus of tau, and tau phosphorylation is measured by high resolution mass spectrometry.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification using a ligand that specifically binds an epitope within the mid domain of tau, and optionally with a second ligand that specifically binds an epitope within the MTBR or the C-terminus of tau, and tau phosphorylation is measured by high resolution mass spectrometry.
  • a mass spectrometry protocol outlined in the Examples is used.
  • a method for enrolling a subject into a clinical trial may comprise (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; and (b) enrolling the subject into a clinical trial when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a method for a method for enrolling a subject into a clinical trial may comprise (a) providing a first and a second isolated tau sample obtained from a subject and measuring, in each isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T111 , S113, T181 , S199, S202, S208, T153, T175, T205, S214, T217, and T231 , and optionally measuring total tau; (b) calculating the change in the site-specific
  • a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF is defined in Section IV.“Significantly deviate from the mean” refers to values that are at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations or even more preferably at least 2 standard deviations, above or below the mean (i.e.
  • o is the standard deviation defined by the normal distribution measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF).
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used as criteria for enrolling a subject.
  • a ratio calculated from the measured phosphorylation level(s), or a ratio calculated from the measured phosphorylation level(s) and total tau may be used.
  • a ratio calculated from the measured phosphorylation level(s) may be a ratio between p- T181 and p-T205, p-T217 and p-T205, or p-T181 and p-T217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between p-T181 and total tau, p-T205 and total tau, or p-T217 and total tau.
  • Mathematical operations other than a ratio may also be used.
  • the examples use site-specific tau phosphorylation values in various statistical models (e.g., linear regressions, LME curves, LOESS curves, etc.) in conjunction with other known biomarkers (e.g. APOE e4 status, age, sex, cognitive test scores, functional test scores, etc.).
  • Section V the efficacy of these various agents can be improved by ad inistering the agents to subjects that have certain site-specific tau phosphorylation levels, as measured by methods disclosed herein and illustrated.
  • clinical trials enrolling subjects with symptoms of AD e.g., after the onset of MCI due to AD
  • measuring tau phosphorylation levels as described herein prior to enrolling a subject in a clinical trial, in particular into a treatment arm of a clinical trial may result in smaller trials and/or improved outcomes.
  • methods described herein may be developed and used as a companion diagnostic for a therapeutic agent.
  • a method for a method for enrolling a subject into a clinical trial may comprise (a) providing an isolated tau sample obtained from a subject and measuring, in the isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from chosen from T181 , T205, and T217, and optionally measuring total tau; and (b) enrolling the subject into a clinical trial when the measured phosphorylation level(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a method for a method for enrolling a subject into a clinical trial may comprise (a) providing a first and a second isolated tau sample obtained from a subject and measuring, in each isolated tau sample, tau phosphorylation at one or more amino acid residue chosen from T181 , T205, and T217, and optionally measuring total tau; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) enrolling the subject into a clinical trial when the calculated change(s) significantly deviate from the mean in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF is defined in Section IV.“Significantly deviate from the mean” refers to values that are at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations or even more preferably at least 2 standard deviations, above or below the mean (i.e. , 1o, 1 3o, 1 5o, or 1 5o, respectively, where o is the standard deviation defined by the normal distribution measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF).
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used as criteria for enrolling a subject.
  • the present disclosure provides a method for enrolling a subject into a clinical trial, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring tau phosphorylation at (i) T217 and T205, (ii) T181 and T205, or (iii) T181 , T205 and T217; and (b) administering a pharmaceutical composition to the subject when tau phosphorylation at T217 and/or T181 is about 1 5o or above and tau phosphorylation at T205 is about 1 5o or below, where o is the standard deviation defined by the normal distribution of tau
  • tau phosphorylation at T217 and/or tau phosphorylation at T181 may be about 1 3o, about 1 35o, about 1 4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2 o, or above 2o.
  • tau phosphorylation at T181 may be about 1.85o, about 1.9o, about 1.95o, about 2o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • tau phosphorylation at T205 may be about 1.3o, about 1.35o, about 1.4o, about 1.45o, about 1.5o, about 1.51 o, about 1.55o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2.0o, or below 2.0o.
  • tau phosphorylation at T205 may be about 2.0o, about 2.05o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o, or below 2.5o.
  • tau phosphorylation at T217 and/or tau phosphorylation at T181 about 2o or above and tau phosphorylation at T205 may be about 2o or less.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • measured levels of tau phosphorylation at T205 and at T181 and/or T217 may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • the present disclosure provides a method for enrolling a subject into a clinical trial, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring total tau and tau phosphorylation at (i) T217 and T205, (ii) T181 and T205, or (iii) T181 , T205 and T217; and (b)
  • o is the standard deviation defined by the normal distribution of total tau and tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 1 3o, about 1 35o, about 1 4o, about 1.45o, about 1.5o, about 1.6o, about 1.7o, about 1.8o, about 1.9o, about 2 o, or above 2o. In other embodiments, the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 1 85o, about 1 9o, about 1 95o, about 2o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • the ratio of tau phosphorylation at T205 to total tau may be about 1 3o, about 1 35o, about 1 4o, about 1 45o, about 1 50o, about 1 55o, about 1 6o, about 1 7o, about 1 8o, about 1 9o, about 2.0o, or below 2o.
  • the ratio of tau phosphorylation at T205 to total tau may be about 2.0o, about 2.05o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o, or below 2.5o.
  • the ratio of tau phosphorylation at T217 to total tau and/or the ratio of tau phosphorylation at T181 to total tau may be about 2o or above and the ratio of tau phosphorylation at T205 to total tau may about 2o or less.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used as criteria for enrolling a subject.
  • the present disclosure provides a method for enrolling a subject into a clinical trial, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; and (b) administering a pharmaceutical composition to the subject when tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) is about 1 5o or above, where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 1.3o, about 1.35o, about 1.4o, about 1.45o, about 1.5o, about 1 6o, about 1 7o, about 1 8o, about 1 9o, about 2 o, or above 2o.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 1.85o, about 1.9o, about 1.95o, about 2o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be about 2o or above.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • the present disclosure provides a method for enrolling a subject into a clinical trial, the method comprising (a) providing an isolated tau sample obtained from a subject and measuring total tau and tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; and (b)
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 1 3o, about 1 35o, about 1 4o, about 1 45o, about 1 5o, about 1 6o, about 1 7o, about 1 8o, about 1 9o, about 2 o, or above 2o.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 1 85o, about 1 9o, about 1 95o, about 2 o, about 2.1 o, about 2.2o, about 2.3o, about 2.4o, about 2.5o or above 2.5o.
  • the ratio of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) to total tau may be about 2o or above.
  • a threshold e.g. at least 1 standard deviation above or below the mean
  • the extent of change above or below the mean may be used as criteria for enrolling a subject.
  • the present disclosure provides a method for enrolling a subject into a clinical trial, the method comprising, (a) providing a first and a second isolated tau sample obtained from a subject, wherein“first” and“second” refer to the order in which the samples were collected, and measuring tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; (b) calculating the change in the site-specific phosphorylation at each residue measured and optionally the change in total tau; and (c) enrolling the subject into the clinical trial when the
  • tau phosphorylation level at T181 and/or T217 decreases or stays the same and the phosphorylation level at T205 and optionally total tau increases.
  • the first and the second isolated tau samples may be collected days, weeks, or months apart.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) will also be about 1 5o or above for both samples and , where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • the present disclosure provides a method for enrolling a subject into a clinical trial, the method comprising (a) providing a first and a second isolated tau sample obtained from a subject, wherein“first” and“second” refer to the order in which the samples were collected, and measuring total tau and tau phosphorylation at (i) T181 and T205, (ii) T217 and T205, or (iii) T181 , T217 and T205; (b) calculating the change in the site-specific phosphorylation at each residue measured and the change in total tau; and (c) enrolling the subject into the clinical trial when the phosphorylation level at T181 and/or T217 decreases or stays the same, the
  • phosphorylation level at T205 decreases or stays the same, and total tau increases.
  • the first and the second isolated tau samples may be collected days, weeks, or months apart.
  • tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) will also be about 1 5o or above for both samples and , where o is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205, or T181 , T205 and T217 measured in a control population without brain amyloid plaques as measured by PET imaging and/or Ab42/40 measurement in CSF.
  • measured levels of tau phosphorylation at the specific sites recited in (a)(i), (a)(ii) or (a)(iii) may be used in various mathematical operations to improve the predictive power compared to each by itself. For instance, ratio(s) may be calculated from the measured phosphorylation levels. Mathematical operations other than a ratio may also be used.
  • tau phosphorylation at T181 , T205 and T217 is 21 7 ⁇ 2.3, 0.34 ⁇ 0.13, and 1 2 ⁇ 0.66, respectively, in a control population without brain amyloid plaques as measured by PET imaging, as measured in an isolated tau sample that was purified from CSF (see Table 3, mutation non-carriers column).
  • twice the standard deviation above the mean found for the mutation non- carrier population (i.e. 2a) for p-T 181 , p-T205 and p-T217 is 43.4, 0.68, and 2.4, respectively.
  • the absolute value may vary depending upon the protocol.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification and tau phosphorylation is measured by mass spectrometry.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification using a ligand that specifically binds an epitope within the mid domain of tau, and optionally with a second ligand that specifically binds an epitope within the N-terminus of tau, and tau phosphorylation is measured by high resolution mass spectrometry.
  • an isolated tau sample comprises tau that has been purified from blood or CSF by affinity purification using a ligand that specifically binds an epitope within the mid domain of tau, and optionally with a second ligand that specifically binds an epitope within the MTBR or the C-terminus of tau, and tau phosphorylation is measured by high resolution mass spectrometry.
  • a mass spectrometry protocol outlined in the Examples is used.
  • a subject may be enrolled into a treatment arm of the clinical trial.
  • the "treatment” is defined in Section V.
  • Subjects enrolled in the treatment arm of a clinical trial may be administered a pharmaceutical composition.
  • a pharmaceutical composition may comprise an imaging agent.
  • imaging agents include functional imaging agents (e.g. fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.).
  • a pharmaceutical composition may comprise an active
  • Non-limiting examples of active pharmaceutical ingredients include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists,
  • antidepressants e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.
  • gamma-secretase inhibitors beta-secretase inhibitors
  • anti-Ab antibodies including antigen-binding fragments, variants, or derivatives thereof
  • anti- tau antibodies including antigen- binding fragments, variants, or derivatives thereof
  • stem cells e.g.
  • TRx0237 methylthionimium chloride, etc.
  • therapies to improve blood sugar control e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • anti-inflammatory agents e.g., insulin, exenatide, liraglutide pioglitazone, etc
  • a pharmaceutical composition may comprise a kinase inhibitor. Suitable kinase inhibitors may inhibit a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3p, MARK, CDK5, or Fyn.
  • a pharmaceutical composition may comprise a phosphatase activator. As a non-limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A.
  • a subject may or may not be symptomatic.
  • An“asymptomatic subject,” as used herein, refers to a subject that does not show any signs or symptoms of AD.
  • a subject may exhibit signs or symptoms of AD (e.g., memory loss, misplacing things, changes in mood or behavior, etc.,) but not show sufficient cognitive or functional impairment for a clinical diagnosis of mild cognitive impairment.
  • a symptomatic or an asymptomatic subject may have Ab amyloidosis; however, prior knowledge of Ab amyloidosis is not a requisite for treatment.
  • a subject may have AD. In any of the
  • a subject may carry one of the gene mutations known to cause dominantly inherited Alzheimer’s disease. In alternative embodiments, a subject may not carry a gene mutation known to cause dominantly inherited Alzheimer’s disease.
  • Example 1 Phosphorylation sites on tau protein in the normal non-AD human brain
  • CSF tau purification using tau-specific antibodies and digestion generated detectable peptides mainly from the mid-domain of the protein sequence (residues 150-221 ). Peptides were detectable to a lesser extent from the N-terminus, and almost no sequence was detectable from the microtubule binding repeat (MTBR) domain or the C-terminus of tau (Barthelemy et al., 2016; Sato et al., 2018). This difference in signal recovery may result from tau truncation during its release from neurons (Sato et al., 2018). The peptide recovery of this mid-domain was sufficient to monitor corresponding minor phosphorylated isoforms using the current PRM method. Conversely, a significant technological advance in MS method will be required to detect phosphorylated peptides in the MTBR and C-terminal domains.
  • MTBR microtubule binding repeat
  • PRM screening of tau phosphopeptides from normal control CSF identified several phosphorylation sites in common with brain soluble tau at T181 (not shown), S199, S202 and T217 (FIG. 9). A low signal corresponding to pS214 was also detected (FIG. 9). A specific fragmentation pattern corresponding to pT205 was identified in the CSF extract and was separated by chromatography from the
  • AD CSF pools from AD patients with mild to moderate dementia.
  • We expected AD CSF pools would contain increased concentrations of tau as well as increased levels of phosphorylated tau.
  • the same phosphorylated residues found in normal CSF (T181 , S199, S202, T217 and T231 ) were detected in AD CSF.
  • signals corresponding to pS113 and pT175 previously found in tau from the brain but not in the normal CSF were detected in AD CSF (FIG. 9, FIG. 10).
  • a LC- MS/MS pattern containing specific fragments and distinct retention times from
  • Brain and CSF tau have different truncation patterns: brain tau isoforms are mainly full length while CSF tau isoforms are truncated. Brain and CSF tau isoforms and corresponding peptides recovered after IP depend on the antibody used for the immunoprecipitation (Sato et al., 2018). Similarly, antibodies used to
  • Normalizing p-tau signal or level using non-phosphorylated tau allows for quantifying changes in phosphorylation stoichiometry (i.e. hyper- or hypo-phosphorylation compared to normal CSF or brain tau) occurring at specific sites independently from the global change in total tau.
  • phosphorylation stoichiometry i.e. hyper- or hypo-phosphorylation compared to normal CSF or brain tau
  • soluble tau production is increased in AD and correlates with amyloid plaques. Therefore, controlling for not just the amount, but the rate of phosphorylation is key to understand.
  • pT 181 , pT217, pT231 , pT205, pS208 and pS214 were hyperphosphorylated in AD CSF as was 0N-specific pT 111 (FIG. 13).
  • pT153 and pT175 were not detected in non-AD and likely increased slightly in AD.
  • pT181 , pS214, and pT217 showed approximately 1.2-fold, 1.6-fold, and 4-fold increases in phosphorylation respectively.
  • not all the monitored sites were hyperphosphorylated:
  • phosphorylation rate changes (hyper- vs hypo-phosphorylation) across the protein, in different compartments (intracellular brain vs extracellular CSF), and in different pathological conditions (AD vs non-AD).
  • This N-terminus projection domain has been recently assigned as a part of the dominant component of the repulsive barrier that prevents neighboring microtubules (associated to tau via the MTBR domain) from getting close to each other (Chung et al., 2016).
  • This sequence contains numerous acidic residues and an increase in phosphorylation may contribute to increased global acidity, enforcing the repulsive barrier. Together with a variable amount of N-terminal extension induced by alternative splicing on exon 2-3, tau phosphorylation could regulate tau/tau N-terminal interactions and microtubule intermolecular distance.
  • phosphorylation at S202, T205 and S208 is recognized by the anti-AT8 antibody (Malia et al., 2016) commonly used to characterize Braak stages of tau aggregation in brain (Braak and Braak, 1995). Indeed, pS208 was detected by MS in PFIF (Flanger et al. 1998). pT205 and pS208 were absent in normal soluble brain tau in our study, confirming the unlikeliness to detect immunoreactivity of AT8 in normal brain tau. Furthermore, a recent study implicates pT205 and pS208 as a combinational phosphorylation pattern that, together with pS202, leads to tau self-aggregation
  • Phosphorylation rate measurements from this study enabled for the first time the comparison of the degree and distribution of phosphorylation rate changes (hyper- vs hypo-phosphorylation) across the protein, in different compartments, (intracellular brain vs extracellular CSF) and in different pathological conditions (AD vs non-AD).
  • This method could be used in the future to look at modifications of AD brain phosphorylations on numerous sites across brain regions and Braak stages as recently performed using immunochemistry (Neddens et al. 2018).
  • T111 , T205, S208 and T217 were more hyperphosphorylated than T 181 , which is the most commonly measured target used as a p-tau biomarker for AD (Fagan et al. 2009).
  • T153 was the most commonly measured target used as a p-tau biomarker for AD (Fagan et al. 2009).
  • AD pathology exacerbates cellular mechanisms contributing to specific p-tau isoforms enrichment during tau release into the CSF.
  • Brain soluble tau extraction Brain soluble tau extraction - Brain and CSF studies involving participants were approved by the Washington University Human Studies Committee and the General Clinical Research Center. Written informed consent was obtained from all participants prior to inclusion in the study. Brain soluble tau was extracted as described previously (Sato et al. , 2018). Briefly, frozen human brain tissues from controls without amyloid and tau pathologies as described before (Sato et al., 2018) were obtained from Knight Alzheimer’s Disease Research Center (ADRC) at
  • the supernatant was solubilized in 1 % Sarkosyl for 60min and centrifuged for 2hrs at 100,000xg.
  • Sarkosyl soluble fractions were pooled and 50uL fraction was diluted 10 times with 0.5% human plasma before immunoprecipitation.
  • brain lysate pool was diluted from 500 to 8000 times before immunopurification to match CSF tau levels.
  • Sepharose beads (GE Healthcare 17-0430-01 ) were crosslinked to antibodies Tau1 and HJ8.5, separately at a concentration of 3mg antibody per g of beads.
  • Samples are spiked with AQUA peptides (ThermoFisher Scientific) corresponding to 10 fmol phosphorylated and 100 fmol unphosphorylated tau for each sequence of interest per mL of sample. Tau and p-tau concentration is calculated using these internal standards. Soluble tau was immunoprecipitated in detergent (1 % NP-40), chaotropic reagent (5mM guanidine), and protease inhibitors (Roche Complete Protease Inhibitor Cocktail).
  • Anti- Tau1 and HJ8.5 antibodies conjugated to sepharose beads were diluted 10 and 5-fold, respectively, in inactivated sepharose beads, and 30uL of 50% slurry of the antibody beads were rotated with the solution for 90m in at room temperature.
  • the beads were washed three times in 25mM triethyl ammonium bicarbonate buffer (TEABC, Fluka 17902).
  • TABC triethyl ammonium bicarbonate buffer
  • the bound tau was digested on-beads with 400ng MS grade trypsin (Promega, V5111 ) for 16 hours at 37°C. Digests were loaded onto TopTip C18 (Glygen,
  • TT2C18.96 desalted, and eluted per manufacturer’s instructions.
  • the eluted peptides were dried by vacuum centrifugation (CentriVap Concentrator Labconco) and were resuspended in 25uL of a solution of 2% acetonitrile and 0.1 % formic acid in MS grade water.
  • solution A was composed of 0.1 % formic acid in MS grade water and solution B was composed of 0.1 % formic acid in acetonitrile.
  • Peptides were eluted from the column with a gradient of 2% to 20% of solution B in 28 minutes, then 20% to 40% solution B for another 13 minutes before ramping up to 85% solution B in another 3 minutes to clean the column.
  • the Orbitrap Fusion Lumos was equipped with a
  • Nanospray Flex electrospray ion source (Thermo Fisher Scientific, San Jose, CA).
  • Peptide ions sprayed from a 10um SilicaTip emitter (New Objective, Woburn, Ma) into the ion-source were targeted and isolated in the quadrupole. These were then fragmented by FICD and ion fragments were detected in the Orbitrap (resolution of 60,000, mass range 150-1200 m/z).
  • Monitoring of hydrophilic peptides (SSRcalc ⁇ 9, all without leucine) for peptide profiling was performed on a FISS T3 300um x 100um, 1.8mm column at a flow rate of 4ul/min with an elution occurring with a 2%to 12% solution B gradient and a spray operating on a 30mm SilicaTip emitter.
  • the quadrupole is set to select a mass window including the hypothesized precursor mass. After precursor collision, all generated fragments are simultaneously measured in the Orbitrap over the time of chromatographic elution. Potentially phosphorylated peptides can be searched in post analysis of the generated data.
  • Skyline software MacCoss Lab, University of Sun.
  • the hypothetical peptide being screened is detected as a LC-MS/MS fingerprint constituted by the strict co-elution of the extracted masses from predicted MS/MS fragments (FIG. 2).
  • the advantage of the PRM method over classic DDA or targeted-MS methods is its ability to use the MS instrument in the highest sensitive configuration possible and conserve discovery capability.
  • Sensitivity of the PRM measurement depends mainly on the number of ions from the target of interest transferred into the Orbitrap analyzer. This number was enhanced by increasing the fill time used to acquire one MS/HRMS scan.
  • the fill time corresponds to the time spent to accumulate the targeted precursor mass from the ion beam into the ion trap device. Accumulated precursors are then fragmented and product fragments are transferred into the Orbitrap to be analyzed.
  • fill time is set to a maximum limited by the need for a sufficient MS scan rate to acquire enough data points to describe the chromatogram signal (i.e. 8-15 scans per chromatographic peak).
  • Fill time for PRM screening on each investigated phosphorylated peptides was typically set to 1 second.
  • Each phosphorylated site ratio obtained can be compared across the protein sequence. This strategy may be biased by difference in fragmentation efficiency between the non-phosphorylated and the phosphorylated peptides. This method cannot be applied when the phosphorylated sites are part of a tryptic missed cleavage. 3) Absolute quantitation using internal synthetic labeled standards (e.g. AQUA) for each phosphorylated and non-phosphorylated peptide:
  • Signals from phosphorylated and non-phosphorylated standards are used to define an internal ratio. This strategy takes into account the fragmentation specificity of each compared peptide but requires peptides synthesis for each monitored species.
  • the second method was utilized to calculate phosphorylation rates.
  • AQUA measurement brain extracts were diluted 500 to 8000 times to be comparable to CSF tau levels. This dilution minimized the matrix effect that may results from significantly different ratio of AQUA internal standard to tau peptide levels in the brain versus CSF.
  • the first method was used for initial interpretation of the complex LC-MS/MS patterns from p-tau sequence containing numerous phosphorylated residues.
  • Lysine methylation is an endogenous post-translational modification of tau protein in human brain and a modulator of aggregation propensity. Biochem J 462, 77-88. doi: 10.1042/B J20140372.
  • Biopsy-derived adult brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau. Neuron 13, 989-1002.
  • Phosphorylated Tau Peptides in Cerebrospinal Fluid by Mass Spectrometry Provides New Biomarker Candidates. Journal of Alzheimer’s Disease 55, 303-313.
  • Example 5 Cerebral amyloid pathology is associated with site-specific
  • the standard uptake value ratio (SUVR) of cortical PiB-PET reliably identifies significant cortical Ab-plaques and is used to classify subjects as PIB positive (Amyloid +, SUVR > 1.25) or negative (Amyloid -, SUVR ⁇ 1.25).
  • PIB positive Amyloid +, SUVR > 1.25
  • Amyloid -, SUVR ⁇ 1.25 To explore the relationship of amyloid plaques and soluble tau species, we compared the SUVR of cortical PiB-PET with CSF total tau and with phosphorylation of CSF tau at multiple sites (i.e. ratio of phosphorylated to unphosphoryated sites of tau).
  • disease progression in DIAD can also be estimated using neuroimaging measures that track various components of disease progression, e.g. brain atrophy and metabolic decline. These measures have been shown to change at different periods of time before symptom onset, with declining cerebral metabolism (measured by [F18] fluorodeoxyglucose [FDG]-PET) occurring up to 18 years and brain atrophy (determined by MRI) occurring up to 13 years before symptom onset 28,31 33 . This raises the question of whether these biomarkers are likewise correlated with tau phosphorylation at specific sites.
  • FDG fluorodeoxyglucose
  • MRI - Flyperphosphorylation was inversely associated with cortical thickness in asymptomatic MCs: p-T205 was most strongly associated with a decrease in cortical and subcortical thickness throughout the brain, FIG. 18A, while p-T217 and total tau levels showed fewer regional associations and weaker correlations.
  • FDG PET In addition to cortical atrophy, a decline in glucose metabolism in neurons and glia is associated with disease progression in AD.
  • Example 8 Cognitive decline is specifically and differentially associated with site-specific differences in tau hyperphosphorylation
  • AD dementia is more closely related to neocortical NFT pathology than neocortical Ab pathology 35 , yet the relationship between soluble tau and cognition remains uncertain. Therefore, we assessed the longitudinal change in the soluble tau phosphorylation ratio and total tau levels over time in comparison to clinical outcomes 36 .
  • Non-phosphorylated tau monotonically increased with worsening cognition and phosphorylation at T217 and T181 decreased with worsening cognition, while pT205 demonstrated no change relative to cognitive decline.
  • Example 9 Increases in unphosphorylated tau are correlated with cortical NFTs by tau PET, but canonical phospho-tau species are not
  • tau comprises a hallmark AD pathology and can be measured in aggregated or soluble forms, important gaps remain in our understanding of how the post-translational modifications of this critical neuronal protein leads to the development of NFT 2 and neurodegeneration in humans.
  • tau comprises a hallmark AD pathology and can be measured in aggregated or soluble forms
  • AD soluble tau and p-tau
  • biomarkers representing AD specific and non-specific pathologies (e.g. Ab, p-tau and tau) 14 .
  • soluble p-tau and unphosphorylated tau are often presumed to be passively released from degenerating neurons, with p-tau associated with aggregated NFTs and
  • AD tauopathy could be defined as a modification of soluble tau
  • EYO Estimated Years to Symptom Onset
  • EYO 29 When a specific mutation average age of onset was unknown, the parental or proxy age of onset was used to define EYO 29 . For participants who were symptomatic at baseline, as assessed by a CDR >0, the reported age of actual symptom onset was subtracted from age at each clinical assessment to define EYO.
  • Clinical Assessments Standardized clinical evaluations, including the use of a study partner, were performed for each participant.
  • a comprehensive neuropsychological battery assessing general cognitive function, memory, attention, executive function, visuospatial function, and language was performed at each visit 46 . From these tests we developed a cognitive composite that reliably detects decline across the range of EYO and CDR 47 . The composite represents the average of the z scores from tests including episodic memory, complex attention, and processing speed and a general cognitive screen (Mini Mental State Examination).
  • CSF Tau Analyses - CSF was collected via standard lumbar puncture procedures (L4/L5) using an atraumatic Sprotte spinal needle (22Ga) into two 13ml polypropylene tubes. CSF was flash-frozen upright on dry ice. Samples collected in the United States were shipped overnight on dry ice to the DIAN biomarker core laboratory at Washington University, St. Louis, MO, USA, whereas samples collected at international sites were stored at -80°C and shipped quarterly on dry ice.
  • each sample was subsequently thawed, combined into a single polypropylene tube, and aliquoted (500pl each) into polypropylene microcentrifuge tubes (#05-538-69C, Corning Life Science, Corning, NY, USA), after which they were re-flash frozen on dry-ice and stored at -80°C.
  • Each thawed CSF sample was mixed with 25mI of a solution containing 15N-441 tau internal standard (2.5ng per sample), 50mM Guanidine, 10% NP-40 and 10X protease inhibitor cocktail (Roche).
  • Tau was extracted by immune capture using incubation under rotation at room temperature during 2 hours with 20mI of sepharose beads cross-linked to Tau1 (tau epitope 192-199) and HJ8.5 (tau epitope 27- 35) antibodies. Beads were spun by centrifugation, then rinsed three times with 1 ml of 25mM TEABC. Samples were digested overnight at 37°C with 400 ng of trypsin Gold (Promega, Madison, Wl). AQUA peptides (Life Technologies, Carlsbad, CA) were spiked to obtain an amount of 5fmol per labeled phosphorylated peptide and 50fmol per labeled unmodified peptide in each sample.
  • the peptide mixture was loaded on TopTip C18 tips, washed with 0.1 % Formic Acid (FA) solution and eluted with 60% ACN 0.1 % FA solution. Eluates were dried using a Speedvac and dried samples were stored at - 80°C prior to analysis. Samples were resuspended in 25mI of 2% ACN 0.1 % FA.
  • FA Formic Acid
  • Extracts were analyzed by nanoLC-MS/HRMS using Parallel Reaction Monitoring using HCD fragmentation.
  • NanoLC-MS/MS experiments were performed using a nanoAcquity UPLC system (Waters, Mildford, Massachusetts) coupled to a Fusion Tribrid mass spectrometer (Thermo Scientific, San Jose, California). 5mI was injected for each sample. Peptide separation was achieved at 60°C in 24 minutes on a Waters HSS T3 column (75pm x 100mm, 1 8pm).
  • Mobile phases were (A) 0.1 % formic acid in water and (B) 0.1 % formic acid in acetonitrile.
  • phosphorylation on T181 , S202, T205 and T217 were measured using the ratio of the MS/HRMS transitions from phosphorylated peptides and corresponding unphosphorylated peptides. Each phosphorylated/unphosphorylated peptide endogenous ratio was normalized using the ratio measured on the MS/HRMS
  • the 18 F-FDG-PET scan started thirty minutes after a ⁇ 5 mCi bolus injection and lasted thirty minutes.
  • the 18 F-AV-1451 data was acquired from the 80-100 minute window after bolus injection and were converted to SUVRs.
  • the SUVRs were processed with grey cerebellum as reference regions and ROI data were corrected for partial volume effects using a regional point spread function (RSF) 48 in geometric transfer matrix framework.
  • RSF regional point spread function
  • Sex, years of education, and APOE e4 status were considered as covariates, but only those effects that were significant were retained in the models.
  • Random effects included in the models are the random intercepts for family clusters, individual random intercept and random slope with unstructured covariance matrix to account for the within-subject correlation due to repeated measures.
  • the adjusted difference in the mean level at baseline and difference in the rates of change between MC and NC were then tested using the approximate f-test derived from the models to determine the first EYO point where the difference became significant.
  • microtubule-associated protein tau in Alzheimer cytoskeletal pathology.
  • Jack, C. R., Jr. et al. A/T/N An unbiased descriptive classification scheme for Alzheimer disease biomarkers. Neurology 87, 539-547,
  • Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proceedings of the National Academy of Sciences 108, 5819-5824 (2011 ).
  • Alzheimer disease a systematic review and meta-analysis. Neurology 83, 253-260, doi: 10.1212/WNL.0000000000000596 (2014).
  • Alzheimer's disease a serial MRI study. Lancet Neurol 5, 828-834, doi: 10.1016/s1474- 4422(06)70550-6 (2006).
  • Alzheimer's disease Neurology 42, 631 -631 , doi:10.1212/wnl.42.3.631 (1992).
  • Alzheimer research The Dominantly Inherited Alzheimer Network. Clinical investigation 2, 975-984, doi: 10.4155/cli.12.93 (2012).
  • AD Alzheimer disease
  • CSF cerebrospinal fluid
  • PET brain Positron-Emission Tomography
  • phosphorylated tau isoforms in CSF we used an innovative targeted high-resolution MS (FIRMS) method targeting tau phosphorylated peptides in the mid-domain of the protein sequence, which is the most abundant domain in CSF and is phosphorylated on numerous sites in brain tau and AD tau aggregates.
  • FRMS targeted high-resolution MS
  • CSF tau purification using immunoprecipitation - 800 pi of CSF supernatant obtained after Ab immuno-precipitation and storage at -80C was used for tau analysis.
  • Thawed supernatants were spiked with 15N tau internal standard (5 ng per sample) and extracted using Tau1 immunoprecipitation.
  • 5mM Guanidine, 1 % NP-40 and protease inhibitor cocktail were added to the sample, then samples were mixed 3 hours at room temperature with 20 mI of sepharose beads cross-linked to Tau1 antibody. Beads were precipitated then rinsed with 0.5 M Guanidine and TEABC 25mM. Samples were digested with 400 ng of trypsin.
  • AQUA peptides (Life Technologies, Carlsbad, California) were spiked to achieve individual quantity of 10 fmol per labeled phosphopeptide and 100 fmol per labeled peptide per sample.
  • AQUA TPSLPpTPPTR (pT217) substituted the missed cleavage version used in the discovery cohort. Tryptic peptides were loaded on TopTip C18 tips, washed with 0.1 % FA solution and eluted with 60%ACN 0.1 %FA solution. Eluates were dried on speedvac. Samples were stored at -80C. Before LC-MS analysis, samples were resuspended in 25 mI 2%ACN 0.1 % FA. Extracts were analyzed by nanoLC-MS/HRMS.
  • T205 phosphorylation mainly in moderate AD CSF suggests an important role of this site in pathological mechanisms underlying amyloid- related tauopathy.
  • Modification in S202/T205 phosphorylation was not detected in the second cohort composed of preclinical and mild AD participant (data not shown), suggesting a dynamic process of tau phosphorylation on specific sites during the course of the disease.

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Abstract

La présente invention concerne des méthodes permettant de quantifier la phosphorylation de tau au niveau de résidus d'acides aminés spécifiques pour prédire le temps jusqu'à l'apparition d'une déficience cognitive légère due à la maladie d'Alzheimer, un stade de la maladie d'Alzheimer, des décisions de guidage de traitement, des sujets sélectionnés pour des essais cliniques, et pour évaluer l'efficacité clinique de certaines interventions thérapeutiques.
PCT/US2019/030725 2018-05-03 2019-05-03 Méthodes de diagnostic et de traitement basées sur la phosphorylation de tau spécifique à un site Ceased WO2019213612A1 (fr)

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KR1020207029980A KR102787585B1 (ko) 2018-05-03 2019-05-03 부위-특이적 타우 인산화에 기반한 진단 및 치료 방법
JP2020561809A JP7301394B2 (ja) 2018-05-03 2019-05-03 部位特異的タウリン酸化に基づく診断法及び治療方法
CN201980030025.7A CN112166117A (zh) 2018-05-03 2019-05-03 基于位点特异性tau磷酸化的诊断和治疗方法
SG11202010094TA SG11202010094TA (en) 2018-05-03 2019-05-03 Methods of diagnosing and treating based on site-specific tau phosphorylation
AU2019262220A AU2019262220B2 (en) 2018-05-03 2019-05-03 Methods of diagnosing and treating based on site-specific tau phosphorylation
KR1020257009592A KR20250048591A (ko) 2018-05-03 2019-05-03 부위-특이적 타우 인산화에 기반한 진단 및 치료 방법
CA3097667A CA3097667A1 (fr) 2018-05-03 2019-05-03 Methodes de diagnostic et de traitement basees sur la phosphorylation de tau specifique a un site
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ZA2020/06348A ZA202006348B (en) 2018-05-03 2020-10-13 Methods of diagnosing and treating based on site-specific tau phosphorylation
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US17/368,403 US11402392B2 (en) 2018-05-03 2021-07-06 Methods of treating based on site-specific tau phosphorylation
US17/843,470 US11635440B2 (en) 2018-05-03 2022-06-17 Methods of treating based on site-specific tau phosphorylation
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WO2021202777A1 (fr) * 2020-04-01 2021-10-07 Genentech, Inc. Utilisations de phosphosites de tau en tant que biomarqueurs pour la maladie d'alzheimer
WO2022115705A2 (fr) 2020-11-30 2022-06-02 Enigma Biointelligence, Inc. Évaluation non invasive de la maladie d'alzheimer
WO2022121040A1 (fr) * 2020-12-07 2022-06-16 佛山汉腾生物科技有限公司 Procédé de cartographie peptidique
WO2022212756A1 (fr) * 2021-03-31 2022-10-06 Washington University Profils de protéines tau et de bêta-amyloïde phosphorylés de csf utilisés en tant que biomarqueurs de tauopathies
WO2023288336A1 (fr) * 2021-07-16 2023-01-19 Washington University Procédés de détection des pentasaccharides et traitement et suivi de la maladie de stockage lysosomale
RU2794040C1 (ru) * 2021-12-23 2023-04-11 Автономная некоммерческая образовательная организация высшего образования "Сколковский институт науки и технологий" (Сколковский институт науки и технологий) Способ оценки риска развития болезни Альцгеймера с использованием панели белков крови
JP2024507328A (ja) * 2021-01-21 2024-02-19 ワシントン・ユニバーシティ アルツハイマー病のステージおよび進行に伴うｃｓｆタウ種を検出する方法、およびその使用
EP4013772A4 (fr) * 2019-08-13 2024-06-05 Washington University Procédés de détection d'isoformes tau de mtbr et leur utilisation
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KR102778075B1 (ko) * 2022-06-30 2025-03-12 광주과학기술원 타우 단백질 유래 인산화 펩타이드를 이용한 알츠하이머병 조기 진단, 단계 구분 및 뇌의 아밀로이드 베타 축적 판별 방법

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Publication number Priority date Publication date Assignee Title
US10830775B2 (en) 2014-09-30 2020-11-10 Washington University Tau kinetic measurements
EP4013772A4 (fr) * 2019-08-13 2024-06-05 Washington University Procédés de détection d'isoformes tau de mtbr et leur utilisation
WO2021050733A1 (fr) * 2019-09-10 2021-03-18 Washington University Analyse de sang pour diagnostic et traitement basés sur la phosphorylation de tau site-spécifique
US12493043B2 (en) 2019-09-10 2025-12-09 Washington University Blood-based assay for diagnosing and treating based on site-specific tau phosphorylation
WO2021202777A1 (fr) * 2020-04-01 2021-10-07 Genentech, Inc. Utilisations de phosphosites de tau en tant que biomarqueurs pour la maladie d'alzheimer
WO2022115705A2 (fr) 2020-11-30 2022-06-02 Enigma Biointelligence, Inc. Évaluation non invasive de la maladie d'alzheimer
WO2022121040A1 (fr) * 2020-12-07 2022-06-16 佛山汉腾生物科技有限公司 Procédé de cartographie peptidique
JP2024507328A (ja) * 2021-01-21 2024-02-19 ワシントン・ユニバーシティ アルツハイマー病のステージおよび進行に伴うｃｓｆタウ種を検出する方法、およびその使用
EP4281780A4 (fr) * 2021-01-21 2025-03-12 Washington University Méthodes de détection d'espèces de tau dans le lcr comprenant le stade et la progression de la maladie d'alzheimer et leur utilisation
US20230280357A1 (en) * 2021-03-31 2023-09-07 Washington University Csf phosphorylated tau and amyloid beta profiles as biomarkers of tauopathies
EP4314841A4 (fr) * 2021-03-31 2025-10-08 Washington University St Louis Profils de protéines tau et de bêta-amyloïde phosphorylés de csf utilisés en tant que biomarqueurs de tauopathies
WO2022212756A1 (fr) * 2021-03-31 2022-10-06 Washington University Profils de protéines tau et de bêta-amyloïde phosphorylés de csf utilisés en tant que biomarqueurs de tauopathies
WO2023288336A1 (fr) * 2021-07-16 2023-01-19 Washington University Procédés de détection des pentasaccharides et traitement et suivi de la maladie de stockage lysosomale
RU2794040C1 (ru) * 2021-12-23 2023-04-11 Автономная некоммерческая образовательная организация высшего образования "Сколковский институт науки и технологий" (Сколковский институт науки и технологий) Способ оценки риска развития болезни Альцгеймера с использованием панели белков крови
WO2024235859A1 (fr) * 2023-05-12 2024-11-21 EM Scientific Limited Dosage de preotéine tau

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