WO2024107745A1 - Methods to detect csf mtbr-tau and uses thereof - Google Patents

Methods to detect csf mtbr-tau and uses thereof Download PDF

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WO2024107745A1
WO2024107745A1 PCT/US2023/079668 US2023079668W WO2024107745A1 WO 2024107745 A1 WO2024107745 A1 WO 2024107745A1 US 2023079668 W US2023079668 W US 2023079668W WO 2024107745 A1 WO2024107745 A1 WO 2024107745A1
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tau
mtbr
tau275
sample
tau282
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PCT/US2023/079668
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French (fr)
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Kanta HORIE
Chihiro Sato
Randall Bateman
Nicolas BARTHELEMY
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Washington University
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    • 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
    • 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
    • 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
    • G01N33/6875—Nucleoproteins
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number

Definitions

  • the present disclosure relates to detection of microtubule-binding region (MTBR)-tau peptides in cerebral spinal fluid (CSF), and use of tau peptides for detecting pathological features or clinical symptoms of a primary tauopathy, which may be used to diagnose, stage, or choose treatments appropriate for the primary tauopathy.
  • MTBR microtubule-binding region
  • CSF cerebral spinal fluid
  • tau BACKGROUND Accumulation of tau protein in the brain as insoluble aggregates is a hallmark of neurodegenerative diseases classified as tauopathies, such as Alzheimer’s disease.
  • Tau pathology appears to propagate and spread across brain regions by cell-to-cell transmission of certain pathological tau species in a prion-like manner, although the spreading process and nature of these species (e.g., monomeric, oligomeric, and fibril species) are uncertain (Frost et al., 2009; Goedert et al., 2010, 2017; Sanders et al., 2014; Wu et al., 2016; Mirbaha et al., 2018; Lasagna-Reeves et al., 2012).
  • Tau has six different isoforms of the full-length protein, and more than one hundred potential post-translational modification sites, including phosphorylation sites and multiple truncation sites (Meredith et al., 2013; Sato et al., 2018; Barthélemy et al., 2019; Cicognola et al., 2019; Blennow et al., 2020).
  • MS Mass spectrometry
  • Cryogenic electron microscopy (Cryo-EM) demonstrates that the core structure of tau aggregates contains a sub-segment of MTBR-tau and the particular conformation depends on the tauopathy (Fitzpatrick et al., 2017; Falcon et al., 2018, 2019; Zhang et al., 2020). These findings strongly suggest that MTBR-tau is critical for tau aggregation. However, these studies used postmortem brain tissue. Little is known about the pathophysiology of corresponding extracellular MTBR-tau species in biological samples such as CSF, which may serve as a surrogate biomarker of brain tau aggregates in living humans. [004] CSF is routinely obtained from study participants via lumbar puncture during clinical visits.
  • CSF cerebrospinal fluid
  • a method of detecting tau in a cerebrospinal fluid (CSF) sample may comprise (a) providing a CSF sample; and (b) detecting and measuring the amount of one or more of Microtubule Binding Region (MTBR)-tau275 peptides and MTBR-tau282 peptides.
  • MTBR Microtubule Binding Region
  • a method of detecting 4R tau aggregates in a subject which may comprise one or more of (a) purifying endogenously cleaved fragments of tau from a CSF sample from the subject, without contacting the endogenously cleaved fragments of tau in vitro with a protease; (b) contacting the purified endogenously cleaved fragments of tau with an endopeptidase to obtain one or more of proteolytic MTBR-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and proteolytic MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptid
  • a solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptides may be desalted before step (c), optionally wherein desalting is by solid phase extraction.
  • the purifying in step (a) may comprise contacting the CSF sample with one or more anti-tau epitope binding agents.
  • the epitope binding agent may be an anti-tau antibody or antigen-binding fragment thereof.
  • the purifying in step (a) may comprise immunoprecipitation, such as immunoprecipitation of N-terminal to mid-domain tau to deplete N-terminal to mid-domain tau from the CSF sample.
  • the one or more anti-tau epitope binding agents may specifically bind to an epitope within amino acids 1-243 of the sequence set forth in SEQ ID NO:1.
  • the epitope binding agent may be an anti- tau antibody or antigen fragment thereof, optionally selected from anti-tau antibody Tau1, HJ8.5, or HJ8.7.
  • the purifying in step (a) may comprise immunoprecipitation of one or more MTBR-tau species from the biological sample to capture the one or more MTBR-tau species from the biological sample.
  • the one or more captured MTBR-tau species may be a MTBR-tau275 peptide, MTBR-tau282 peptide, or combination thereof.
  • the immunoprecipitation of one or more MTBR-tau species may comprise contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to an 2 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web epitope within amino acids 244-368 relative to SEQ ID NO:1, or within amino acids 316-335 of the sequence set forth in SEQ ID NO:1.
  • the epitope binding agent may be anti-tau antibody 77G7 or an antigen-binding fragment thereof.
  • the endopeptidase may be trypsin.
  • the CSF sample may comprise an internal standard.
  • the method may further comprise one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and an ApoE isoform, in the CSF sample.
  • the method may quantify the amount of mid-domain tau, optionally wherein the mid-domain tau comprises tau212-221 relative to SEQ ID NO:1 (t-tau).
  • the one or more of MTBR-tau275 peptides and MTBR-tau282 peptides may be normalized to the amount of t-tau, and the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau is indicative of 4R tau aggregates in the subject.
  • a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value may be indicative of 4R tau aggregates in the subject.
  • a method of detecting 4R tau aggregates in a subject may comprise (a) performing affinity depletion on a CSF sample from the subject by contacting the biological sample with one or more affinity depletion agents comprising one or more epitope binding agents that each binds to one of N-terminal tau, mid-domain tau, or C-terminal tau, but not to an antigen within MTBR-tau, wherein the CSF sample comprises endogenously cleaved fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, mid-domain tau, and/or C-terminal tau, and wherein the enriched sample is enriched for endogenously cleaved fragments of tau comprising one or more
  • the one or more of proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides may be desalted before step (d), optionally wherein desalting is by solid phase extraction.
  • the one or more affinity depletion agents may comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino 3 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web acids 1-243 of the sequence set forth in SEQ ID NO:1.
  • the epitope binding agent may be an anti-tau antibody or antigen fragment thereof, optionally selected from anti-tau antibody Tau1, HJ8.5, or HJ8.7.
  • the one or more immunoprecipitation agents may comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244-368 of the sequence set forth in SEQ ID NO:1, or within amino acids 316-335 of the sequence set forth in SEQ ID NO:1.
  • the one or more immunoprecipitation agents may comprise the anti-tau antibody 77G7 or an antigen-binding fragment thereof.
  • the endopeptidase may be trypsin.
  • the method may further comprise one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and an ApoE isoform, in the CSF sample.
  • the method may quantify the amount of mid-domain tau, optionally wherein the mid-domain tau comprises tau212-221 relative to SEQ ID NO: 1 (t-tau).
  • the one or more of MTBR-tau275 peptides and MTBR-tau282 peptides may be normalized to the amount of t-tau, and wherein the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t- tau is indicative of 4R tau aggregates in the subject.
  • a decrease in the amount of one or more of MTBR- tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value may be indicative of 4R tau aggregates in the subject.
  • a method for detecting a primary tauopathy in a subject may comprise providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR- tau282/t-tau from a CSF sample from a subject.
  • One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove.
  • the primary tauopathy may be selected from the group consisting of frontotemporal lobar degeneration (FTLD) (e.g., FTLD-MAPT) and corticobasal degeneration (CBD).
  • FTLD frontotemporal lobar degeneration
  • CBD corticobasal degeneration
  • a decrease in the amount of one or more of MTBR-tau275/t-tau and MTBR- tau282/t-tau relative to a threshold value may be indicative of FTLD (e.g., FTLD-MAPT) or CBD).
  • FTLD e.g., FTLD-MAPT
  • CBD MTBR-tau
  • the 4 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web FTLD-MAPT may be P301L, R406W, or S305I.
  • the FTLD-MAPT may be symptomatic or asymptomatic.
  • a method of diagnosing FTLD e.g., FTLD-MAPT
  • FTLD FTLD-MAPT
  • a method of diagnosing FTLD may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR-tau282/t-tau from a CSF sample from the subject; and (b) diagnosing FTLD-MAPT when the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau detected is decreased relative to a threshold value.
  • the FTLD-MAPT may be P301L, R406W, or S305I.
  • the FTLD-MAPT may be symptomatic or asymptomatic.
  • a method of measuring FTLD (e.g., FTLD-MAPT) progression in a subject may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR-tau282/t-tau from a CSF sample from the subject; and (b) calculating a difference between amounts of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample and the first sample, wherein a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample as compared to the first sample indicates progression of the subject’s FTLD (e.g., FTLD-MAPT).
  • FTLD e.g., FTLD-MAPT
  • One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove.
  • the FTLD-MAPT may be P301L, R406W, or S305I.
  • the FTLD-MAPT may be symptomatic or asymptomatic.
  • a method of diagnosing CBD in a subject may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR-tau282/t- tau from a CSF sample from the subject; and (b) diagnosing CBD when the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau detected is decreased relative to a threshold value.
  • One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove.
  • a method of measuring CBD progression in a subject may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR- tau282/t-tau from a CSF sample from the subject; and (b) calculating a difference between amounts of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample and the first sample, wherein a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample as compared to the first sample indicates progression of the subject’s CBD.
  • One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. 5 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [014]
  • a method of treating a primary tauopathy in a subject in need thereof may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR- tau282, and MTBR-tau282/t-tau from a CSF sample from the subject; and (b) administering to the subject a treatment that alters tau pathology if the amount of the one or more of MTBR-tau275/t-tau and MTBR- tau282/t-tau is decreased relative to the thresh-old value.
  • One or more of MTBR-tau275 and MTBR- tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove.
  • a method of treating a primary tauopathy in a subject in need thereof comprising administering to the subject a treatment that alters tau pathology, wherein the subject has been identified as having a decreased amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau, as measured according to a method as described hereinabove, relative to the threshold value.
  • the treatment may alter or stabilize the amount of the detected one or more of MTBR-tau275 peptides and MTBR-tau282 peptides.
  • the treatment may be selected from the group consisting of lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, Bepranemab, Dasatinib + Quercetin, E2814, Epothilone D, Gosuranemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, Semorinemab, TPI 287, Tideglusib, Tila-vonemab, Zagotenemab, an anti-tau monoclonal antibody, an anti-tau anti-sense oligonucleotide, an anti-tau small interfering RNA, an tau production inhibitor, and a tau active vaccine.
  • the treatment may be selected from the group consisting of anti-A ⁇ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, gamma-secretase inhibitors, beta-secretase inhibitors, a kinase inhibitor, a phosphatase activator, a vaccine, and a tau protein aggregation inhibitor.
  • the kinase inhibitor may be an inhibitor of a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3 ⁇ , MARK, CDK5, or Fyn.
  • the phosphatase activator may increase the activity of protein phosphatase 2A.
  • the vaccine may be CAD106 or AF20513.
  • the anti-A ⁇ antibody may be aducanumab or another anti-amyloid anti-body that removes plaques.
  • FIG 1 shows demographics and brain MTBR-tau measures of participants in primary tauopathy cohort.
  • FTLD-TDP frontotemporal lobar degeneration with TAR DNA-binding protein
  • PiD Pick's disease
  • PSP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • AD Alzheimer’s disease
  • FTLD - MAPT frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16)
  • HS 6 92044717.7 PATENT-PRO Atty.
  • FIG.2 shows demographics and CSF MTBR-tau measures of participants in pathologically- confirmed primary tauopathy cohort.
  • FTLD-TDP frontotemporal lobar degeneration with TAR DNA-binding protein
  • PiD Pick's disease
  • AGD argyrophilic grain disease
  • PSP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • AD Alzheimer’s disease
  • FTLD-MAPT frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16)
  • HS hippocampal sclerosis
  • MND motor neuron disease
  • LBD Lewy body dementia.
  • CDR plus NACC FTLD-SB Clinical Dementia Rating plus National Alzheimer's Coordinating Center FTLD sum of boxes.
  • ALS amyotrophic lateral sclerosis
  • MCI mild cognitive impairment
  • bvFTD behavioral variant of frontotemporal dementia
  • CBS corticobasal syndrome
  • DLB dementia with Lewy bodies
  • nfvPPA nonfluent variant primary progressive aphasia
  • PPS pallidopyramidal syndrome
  • svPPA semantic variant of primary progressive aphasia
  • PAGF pure akinesia with gait freezing
  • PSP-RS Progressive supranuclear palsy with Richardson's Syndrome
  • TES traumatic encephalopathy syndrome
  • EOAD early-onset Alzheimer's disease
  • lvPPA logopenic variant primary progressive aphasia
  • PCA posterior cortical atrophy
  • CN cognitively normal.
  • FIG.3 shows diagnostic accuracies of 4R specific CSF MTBR-tau to distinguish CBD from FTLD-tau and control.
  • Abbreviations include CBD: corticobasal degeneration, NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, FTLD-tau: frontotemporal lobar degeneration with TAR DNA-binding protein, PSP: progressive supranuclear palsy, AGD: argyrophilic grain disease, PiD: Pick's disease, AUC: area under the curve, CI: confidence interval.
  • FIGS.4A-4C show 4R specific insoluble brain MTBR-tau is enriched in CBD, FTLD-MAPT, AD and PSP.
  • FIG.4A depicts a schematic of the quantified peptides of total-tau (t-tau, 181-190) and 4R isoform specific microtubule binding region of tau (MTBR-tau) in R2 region (grey bars, MTBR-tau275 and MTBR-tau282). The relative abundance of each MTBR-tau was normalized to the t-tau peptide.
  • Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar 7 92044717.7 PATENT-PRO Atty.
  • FIGS.5A-5C show 4R specific CSF MTBR-tau decreases in CBD, FTLD-MAPT, and AD.
  • FIG.5A depicts a schematic of the quantified peptides of total-tau (t-tau, 212-221), truncation, and 4R isoform specific microtubule binding region of tau (MTBR-tau) in R2 region (grey bars, MTBR-tau275 and MTBR-tau282).
  • FTLD-TDP frontotemporal lobar degeneration with TAR DNA-binding protein
  • PiD Pick's disease
  • AGD argyrophilic grain disease
  • PSP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • AD Alzheimer’s disease
  • FTLD- MAPT frontotemporal lobar degeneration with MAPT mutations.
  • the box plots show the minimum, 25 percentile, median, 75 percentile, and maximum. Differences in biomarker values were assessed with one-way ANOVAs.
  • FIGS.6A-6F show CSF soluble MTBR-tau correlates with brain insoluble MTBR-tau aggregates.
  • Gray shadow represents 95% confidential intervals for linear regression.
  • Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR 8 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations.
  • FIG.7 shows 4R CSF MTBR-tau assay is reproducible and stable in repeated lumbar punctures.
  • CSF MTBR-tau275/t-tau was measured in 25 participants (#01-#25) who had 3 to 5 repeated lumbar punctures within 4 months.
  • CBS corticobasal syndrome
  • PSP- RS progressive supranuclear palsy-Richardson’s syndrome
  • FTLD-MAPT frontotemporal lobar degeneration with MAPT mutations.
  • FIG.8 shows CSF MTBR-tau275/t-tau decreases in clinically diagnosed CBS-PSP continuum and FTLD-MAPT.
  • Differences in biomarker values were assessed with one-way ANOVAs.
  • a two-sided p ⁇ 0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%. *P ⁇ 0.05.
  • FDR Benjamini-Hochberg false discovery rate
  • FIG.9 shows demographic and CSF MTBR-tau measures of participants in repeated lumbar puncture cohort.
  • FIG.10 shows CSF MTBR-tau275/t-tau biomarker positivity based on clinical syndromes in pathologically-confirmed cohort
  • FIG.11 shows CSF MTBR-tau282/t-tau biomarker positivity based on clinical syndromes in pathologically-confirmed cohort.
  • FIGS.12A-12C show that CSF MTBR-tau and t-tau concentrations do not reflect CBD and FTLD-MAPT pathologies.
  • FIGS.13A-13B show CSF MTBR-tau/tau correlates with disease duration in CBD.
  • FIGS.14A-14C show CSF pT217/T217 identifies AD from other tauopathies.
  • FDR Benjamini-Hochberg false discovery rate
  • FIGS.15A-15E show CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau do not correlate with amyloid pathology in primary tauopathies.
  • FIGS.16A-16L ROC curves for 4R specific CSF MTBR-tau to distinguish CBD from control and other tauopathies.
  • CSF MTBR-tau275/t-tau (FIG.16A, FIG.16C, FIG.16E, FIG.16G, FIG. 16I, and FIG.16K) and MTBR-tau282/t-tau (FIG.16B, FIG.16D, FIG.16F, FIG. 16H, FIG.16J, and FIG.16L) can distinguish CBD from NC (FIG.16A and FIG.16B), FTLD-TDP (FIG.16C and FIG.16D), FTLD-tau (i.e.
  • AUC improves when AD co-pathology cases are excluded from CBD and PSP (FIG.16K and FIG.16L).
  • Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick’s disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation.
  • FIGS.17A-17E show retrospective clinical syndromes and CSF MTBR-tau markers in pathologically-confirmed cohort.
  • the box plots show the minimum, 25 percentile, median, 75 percentile, and maximum.
  • FIG.16E depicts a schematic of relationship between clinical syndrome and pathological diagnoses in FTLD. Red and black lines show CSF MTBR-tau biomarkers positive and negative, respectively. Biomarker positivity was determined by the median of each disease group.
  • Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I), bvFTD: behavioral variant of frontotemporal dementia, CBS: corticobasal syndrome, nfvPPA: nonfluent variant primary progressive aphasia, PAGF: pure akinesia with gait freezing, PSP-RS: Progressive supranuclear palsy with Richardson's Syndrome, CN: cognitively normal.
  • a binding molecule e.g., an antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain, and that the binding entails some recognition between the antigen-binding domain and the epitope.
  • a binding 12 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web molecule is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specifically binds,” as used herein with regards to epitope binding agents, means that an epitope binding agent does not cross react to a significant extent with other epitopes on the protein of interest (e.g., Tau), or on other proteins in general.
  • the term “antibody”, as used herein, is used in the broadest sense and encompasses various antibody and antibody-like structures, including but not limited to full-length monoclonal, polyclonal, and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as heavy chain antibodies and antibody fragments provided they exhibit the desired antigen-binding activity.
  • variable region The domain(s) of an antibody that is involved in binding an antigen is referred to as a “variable region” or “variable domain,” and is described in further detail below.
  • a single variable domain may be sufficient to confer antigen-binding specificity.
  • antibodies useful in the discovery are produced recombinantly.
  • Antibodies may or may not be glycosylated, though glycosylated antibodies may be preferred.
  • An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by methods known in the art.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • Non-limiting examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; single- chain forms of antibodies and higher or-der variants thereof; single-domain antibodies, and multi-specific antibodies formed from antibody fragments.
  • Single-chain forms of antibodies may include, but are not limited to, single-domain antibodies, single chain variant fragments (scFvs), divalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra-scFvs), diabodies, and triabodies and tetrabodies.
  • ScFv’s are comprised of heavy and light chain variable regions connected by a linker. In most instances, but not all, the linker may be a peptide.
  • a linker peptide is preferably from about 5 to 30 amino acids in length, or from about 10 to 25 amino acids in length.
  • the linker allows for stabilization of the variable domains without interfering with the proper folding and creation of an active binding site.
  • a linker peptide is rich in glycine, as well as serine or threonine.
  • ScFvs can be used to facilitate phage display or can be used for flow cytometry, immunohistochemistry, or as targeting domains. Methods of making and using scFvs are known in the art.
  • ScFvs may also be conjugated to a human constant domain (e.g., a heavy constant domain is derived from an IgG do-main, such as lgG1, lgG2, lgG3, or lgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE).
  • a human constant domain e.g., a heavy constant domain is derived from an IgG do-main, such as lgG1, lgG2, lgG3, or lgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE.
  • Diabodies, 13 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web triabodies, and tetrabodies and higher order variants are typically created by varying the length of the linker peptide from zero to several amino acids.
  • multivalent binding antibody variants can be generated using self-assembling units linked to the variable domain.
  • a “single-domain antibody” refers to an antibody fragment consisting of a single, monomeric variable antibody domain.
  • Multi-specific antibodies include bi-specific antibodies, tri-specific, or anti-bodies of four or more specificities. Multi-specific antibodies may be created by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains can be covalently linked.
  • An antibody of the disclosure may be a Dual-affinity Re-targeting Antibody (DART).
  • the DART format is based on the diabody format that separates cognate variable domains of heavy and light chains of the two antigen-binding specificities on two separate polypeptide chains. Whereas the 2 polypeptide chains associate noncovalently in the diabody format, the DART format provides additional stabilization through a C-terminal disulfide bridge. DARTs can be produced in high quantity and quality and reveal exceptional stability in both formulation buffer and human serum.
  • "Monoclonal antibody” refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone. "Monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
  • Monoclonal antibodies can be produced using hybridoma techniques well known in the art, as well as recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies and other technologies readily known in the art. Furthermore, the monoclonal antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.
  • a heterologous compound e.g., an enzyme or toxin
  • a “heavy chain antibody” refers to an antibody that consists of two heavy chains. A heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from a cartiliaginous fish.
  • a “humanized antibody” refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response in humans following administration but retains similar binding specificity and affinity as the starting non-human antibody.
  • a humanized antibody binds 14 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web to the same or similar epitope as the non-human antibody.
  • the term “humanized antibody” includes an antibody that is composed partially or fully of amino acid sequences derived from a human antibody germline by altering the sequence of an antibody having non-human hypervariable regions (“HVR”).
  • variable region of the antibody is also humanized by techniques that are by now well known in the art.
  • the framework regions of a variable region can be substituted by the corresponding human framework regions, while retaining one, several, or all six non-human HVRs.
  • Some framework residues can be substituted with corresponding residues from a non-human VL domain or VH domain (e.g., the non-human antibody from which the HVR residues are derived), e.g., to restore or improve specificity or affinity of the humanized antibody.
  • Substantially human framework regions have at least about 75% homology with a known human framework sequence (i.e., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity).
  • HVRs may also be randomly mutated such that binding activity and affinity for the antigen is maintained or enhanced in the context of fully human germline framework regions or framework regions that are substantially human.
  • the term "humanized antibody” refers to an antibody comprising a substantially human framework region, at least one HVR from a nonhuman antibody, and in which any constant region present is substantially human.
  • Substantially human constant regions have at least about 90% with a known human constant sequence (i.e., about 90%, about 95%, or about 99% sequence identity). Hence, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences.
  • a ⁇ refers to peptides derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of A ⁇ that may have toxic effects: A ⁇ peptides are typically 37-43 amino acid sequences long, though they can have truncations and modifications changing their overall size.
  • a ⁇ typically refers to 15 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web a plurality of A ⁇ species without discrimination among individual A ⁇ species.
  • a ⁇ 42/A ⁇ 40 value or “A ⁇ 42/A ⁇ 40 ratio” means the ratio of the amount of A ⁇ 42 in a sample obtained from a subject compared to the amount of A ⁇ 40 in the same sample.
  • 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.
  • control population refers to a subject, or group of subjects, who are clinically determined to not have a tauopathy (including but not limited to Alzheimer’s disease, FTLD (e.g., FTLD-MAPT), and CBD), based on qualitative or quantitative test results.
  • FTLD FTLD-MAPT
  • CBD CBD
  • tau refers to a plurality of isoforms encoded by the gene MAPT (or homolog thereof), as well as species thereof that are C-terminally truncated in vivo, N-terminally truncated in vivo, post- translationally modified in vivo, or any combination thereof.
  • the terms “tau” and “tau protein” and “tau species” may be used interchangeably.
  • tau is encoded by the gene MAPT.
  • a homolog may be identified by methods well known in the art.
  • 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), one insert (1N), or no inserts (0N). 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.
  • full-length human tau isoforms may be comprised of four repeats of the microtubule binding domain (exon 10 included: R1, R2, 16 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web R3, and R4) or three repeats of the microtubule binding domain (exon 10 excluded: R1, R3, and R4).
  • Human tau may or may not be post-translationally modified. For example, it is known in the art that tau may be phosphorylated, ubiquinated, glycosylated, and glycated.
  • Human tau also may or may not be proteolytically processed in vivo at the C-terminus, at the N-terminus, or at the C-terminus and the N- terminus. Accordingly, the term “human tau” encompasses the 2N3R, 2N4R, 1N3R, 1N4R, 0N3R, and 0N4R isoforms, as well as species thereof that are C-terminally truncated in vivo, N-terminally truncated in vivo, post-translationally modified in vivo, or any combination thereof. Alternative splicing of the gene encoding tau similarly occurs in other animals.
  • tau-441 or “full length tau” as used herein, refers to the longest human tau isoform (2N4R), which is 441 amino acids in length.
  • the amino acid sequence of tau-441 is provided as: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSET SDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM VSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSG DRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCG SLGNIHHKPGGGQVEVKSEKLDFK
  • N term tau The N-terminus (N term) tau, mid-domain tau, MTBR-tau, and C-terminus (C term) tau are described hereinbelow for this isoform. These regions will vary in a predictable way for other tau isoforms (e.g., 2N3R, 1 NR4, 1 N3R, 0N4R, and 0N3R). Accordingly, when amino acid positions are identified relative to tau-441, a skilled artisan will be able to determine the corresponding amino acid position for the other isoforms.
  • N-terminal tau refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the N-terminus of tau (e.g., amino acids 1-103 of tau-441).
  • mid-domain tau refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the mid-domain of tau (e.g., amino acids 104- 243 of tau-441).
  • MTBR tau or “MTBR-tau” as used herein, refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the microtubule binding region (MTBR) of tau (e.g., amino acids 244-368 of tau-441). 17 92044717.7 PATENT-PRO Atty.
  • MTBR microtubule binding region
  • C-terminal tau refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the C-terminus of tau (e.g., amino acids 369- 441 of tau-441, etc.).
  • tauopathy A disease associated with tau deposition in the brain is referred to herein as a “tauopathy.”
  • tau deposition is inclusive of all forms pathological tau deposits including but not limited to neurofibrillary tangles, neuropil threads, and tau aggregates in dystrophic neurites.
  • Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy (PSP), 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 (CBD), argyrophilic grain disease (AGD), Frontotemporal lobar degeneration (FTLD), Alzheimer’s disease (AD), and frontotemporal dementia (FTD).
  • PPP progressive supranuclear palsy
  • AD frontotemporal dementia
  • FTD frontotemporal dementia
  • Tauopathies are classified by the predominance of tau isoforms found in the pathological tau deposits. Those tauopathies with tau deposits predominantly composed of tau with three MTBRs are referred to as “3R-tauopathies.” Pick’s disease is a non-limiting example of a 3R-tauopathy.
  • pathological tau deposits of some 3R-tauopathies may be a mix of 3R and 4R tau isoforms with 3R isoforms predominant.
  • Intracellular neurofibrillary tangles i.e., tau deposits
  • brains of subjects with Alzheimer’s disease are generally thought to contain both approximately equal amounts of 3R and 4R isoforms.
  • a clinical sign of a tauopathy may be aggregates of tau in the brain, including but not limited to neurofibrillary tangles.
  • tau PET using tau-specific ligands such as [ 18 F]THK5317, [ 18 F]THK5351 , [ 18 F]AV1451 , [ 11 C]PBB3, [ 18 F]MK-6240, [ 18 F]RO-948, [ 18 F]P I-2620, [ 18 F]GTP1 , [ 18 F]PM-PBB3, and [ 18 F]JNJ64349311 , [ 18 F]JNJ-067), etc.
  • 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 18 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 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. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of disease.
  • tau therapy collectively refers to any imaging agent, therapeutic treatment, and/or a prophylactic or preventative measure contemplated for, or used with, subjects at risk of developing a tauopathy, or subjects clinically diagnosed as having a tauopathy.
  • imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.).
  • functional imaging agents e.g., fluorodeoxyglucose, etc.
  • molecular imaging agents e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.
  • Non-limiting examples of therapeutic agents 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
  • EFS Web receptor agonists Via EFS Web receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.
  • AD Alzheimer’s disease
  • FTLD-tau frontotemporal lobar degeneration with tau
  • CBD corticobasal degeneration
  • Biomarkers in cerebrospinal fluid (CSF) and blood provide the accurate performance for identifying AD.
  • CSF cerebrospinal fluid
  • some FTLD-tau such as CBD brains accumulate tau species from 4-repeat (4R) tau- specific domain in the microtubule-binding region (MTBR)
  • MTBR microtubule-binding region
  • the present disclosure examined if CSF levels of these MTBR-tau species altered in the FTLD-tau CSF as a reflection of accumulating tau aggregates in brain.
  • a method quantifying various tau species in CSF was designed and applied for two discovery studies to analyze CSF from individuals with AD and frontotemporal dementia (FTD) syndrome including genetically-confirmed as well as pathologically-unconfirmed cases.
  • FTD frontotemporal dementia
  • the specific MTBR-tau species level against total tau (t-tau) specifically decreased in CSF from individuals with symptomatic AD and FTD syndrome compared to the cases with asymptomatic AD and healthy control.
  • pathogenic FTLD-tau mutants i.e., P301L and R406W
  • P301L and R406W pathogenic FTLD-tau mutants
  • the MTBR-tau species in all pathologically- or genetically-confirmed CSF were analyzed as a validation study.
  • P301L and R406W FTLD-tau
  • P301L and R406W FTLD-tau
  • FTLD-tau FTLD unrelated to tau
  • the present disclosure provides methods for measuring MTBR-tau peptides in a biological sample (e.g., by mass spectrometry or immunoassay).
  • the present disclosure further contemplates in each of the methods described herein, determining the presence/absence of one or more protein in the biological sample and/or measuring the concentration of one or more additional protein in the biological sample.
  • the one or more protein may be a protein depleted from the biological sample prior to purification of tau.
  • N-terminal tau and/or mid-domain tau species may be identified and/or quantified separately from tau species (e.g., MTBR-tau, C-terminal tau) quantified by the methods disclosed herein. 20 92044717.7 PATENT-PRO Atty.
  • post-translation modifications such as phosphorylation of tau at specific residues can be measure and quantified in addition to the endogenously cleaved fragment of tau.
  • a ⁇ e.g. A ⁇ 42/A ⁇ 40
  • ApoE or any other protein of interest may be identified and/or quantified either by processing a portion of the biological sample in parallel, by depleting the protein of interest from the biological sample prior to utilization in the methods disclosed herein, or by depleting the protein of interest from the biological sample during the sample processing steps disclosed herein.
  • the biological sample, suitable internal standards, and the steps of, purifying tau, optionally depleting one or more proteins, optionally cleaving purified tau with a protease, and detecting and measuring the endogenously cleaved peptide fragment of tau are described in more detail below.
  • Methods of Detecting Fragments of Tau [070] The present disclosure provides methods for detecting tau (e.g., peptide fragments of tau) in a cerebrospinal fluid (CSF) sample. The detection may comprise confirming the presence of tau, such as for measuring the peptide fragment of tau or quantifying the amount of the peptide fragment of tau.
  • CSF cerebrospinal fluid
  • the presence, measurement, or quantified amount of a peptide fragment of tau may be indicative of aggregated insoluble tau or tangles (e.g., neurofibrillary tangles) associated with a tauopathy.
  • the detection and measuring may be performed by mass spectrometry or an immunoassay.
  • the biological sample may be obtained from a subject having or suspected of having a tauopathy.
  • a peptide fragment of tau (e.g., fragment of tau) as described herein may be endogenously cleaved (for example, cleaved in cells or tissues of a subject).
  • a peptide fragment of tau comprises amino acids of a 4R tau splicing isoform of tau.
  • a peptide fragment of tau comprises an R2 region fragment of tau. In some embodiments, a peptide fragment of tau comprises amino acids of a portion of the R2 region of tau. In some embodiments, a peptide fragment of tau comprises a MTBR-tau275 peptide, as described herein. In some embodiments, a peptide fragment of tau (e.g., an MTBR-tau275 peptide) comprises or consists of amino acids 275-280 of tau-441. In some embodiments, a peptide fragment of tau (e.g., an MTBR-tau275 peptide) comprises or consists of amino acids 275-280 as set forth SEQ ID NO:1.
  • a peptide fragment of tau comprises or consists of the amino acid sequence VQIINK (SEQ ID NO:2).
  • a peptide fragment of tau comprises a MTBR-tau282 peptide, as described herein.
  • a peptide fragment of tau (e.g., an MTBR-tau282 peptide) comprises or consists of amino acids 282-290 of tau-441.
  • a peptide fragment of tau (e.g., an MTBR-tau282 peptide) comprises or consists of amino 21 92044717.7 PATENT-PRO Atty.
  • a peptide fragment of tau comprises or consists of the amino acid sequence LDLSNVQSK (SEQ ID NO:3).
  • Detecting tau e.g., tau peptide fragments
  • the CSF sample comprises endogenously cleaved tau.
  • An endogenously cleaved fragment of tau may not be a tryptic tau peptide.
  • the purification may be performed without first exposing the sample to additional in-vitro proteolytic cleavage.
  • a pre-clearing step e.g., protein precipitation and/or immunodepleting
  • An endogenously cleaved fragment of tau may be further cleaved in vitro with a protease before or after purification.
  • the protease is trypsin.
  • a sample as described herein e.g., a CSF sample comprising endogenously cleaved fragments of tau
  • Each round of immunodepletion or immunoprecipitation may be performed using one or more anti-tau or anti-MTBR-tau antibodies described herein, or a combination thereof.
  • the purified tau peptides from one or more of the immunoprecipitated sample or the immunodepleted sample may be further cleaved with one or more proteases described herein.
  • LC/MS Liquid chromatography–mass spectrometry
  • an immunoassay with a sample comprising a purified peptide fragment of tau e.g., an endogenously cleaved fragment of tau
  • concentration relative or absolute
  • One or more fragments of tau may be used to detect and measure the amount of tau, such as the amount of insoluble tau aggregates, present in the biological sample.
  • the immunoassay may comprise contacting a sample comprising a purified peptide fragment of tau with an anti-MTBR-tau antibody disclosed herein.
  • detecting tau e.g., a tau peptide fragment
  • detecting tau in a CSF sample may comprise one or more of confirming the presence of a tau peptide, measuring a tau peptide, and/or quantifying a tau peptide (e.g., an amount of a tau peptide) in a CSF sample.
  • a method as described herein comprises detecting and measuring the amount of proteolytic MTBR-tau275 peptides, proteolytic MTBR-tau282 peptides, or both.
  • a method of detecting a tau peptide in a CSF sample comprising the steps of: (a) providing a CSF sample (e.g., a CSF sample comprising 22 92044717.7 PATENT-PRO Atty.
  • MTBR-tau peptides e.g., MTBR-tau275 peptides and MTBR-tau282 peptides.
  • the one or more of MTBR-tau peptides may be detected by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay of the sample to detect and/or measure the amount of the one or more of MTBR-tau peptides.
  • a method of the present disclosure further comprises purifying the one or more of MTBR-tau peptides from the CSF sample, wherein the one or more of MTBR-tau peptides is optionally further cleaved in vitro with one or more proteases before or after purification.
  • the one or more of MTBR-tau peptides are not cleaved in vitro (e.g., not endogenously) before purification.
  • a method of the present disclosure further comprises decreasing in the CSF sample by affinity depletion or immunoprecipitation of at least one peptide of tau, wherein the at least one peptide of tau that is affinity depleted or immunoprecipitated is not the one or more of MTBR-tau peptides for detection and/or measurement (e.g., not MTBR-tau275 or MTBR-tau282).
  • the affinity depletion or immunoprecipitation is of N-terminal tau, mid-domain tau, and optionally, C-terminal tau, or a combination thereof.
  • removing additional proteins from the CSF sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant can be performed before or after purification of the of one or more of MTBR-tau peptides.
  • a method of the present disclosure further enriching one or more of MTBR- tau peptides comprising purifying one or more of the MTBR-tau peptides from the affinity depleted sample.
  • the enriched one or more of MTBR-tau peptides may be enriched for MTBR-tau275 peptide and/or MTBR-tau282 peptide.
  • the MTBR-tau275 peptide and/or MTBR-tau282 peptide may be detected and/or measured using an immunoassay, which may comprise the use of anti-tau antibody 77G7, or an antigen-binding fragment thereof.
  • the sample comprising enriched MTBR-tau275 peptide and/or MTBR- tau282 peptide may be proteolytically cleaved with a protease.
  • the resultant cleavage may be desalted (e.g., by solid phase extraction) to obtain a sample comprising the one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and/or MTBR-tau282 peptide).
  • the method further comprises desalting (e.g., by solid phase extraction) to obtain a sample comprising proteolytic peptides of tau (e.g., endogenously cleaved tau) prior to purification.
  • the method comprises detecting and/or measuring the amount of 23 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web MTBR-tau275 peptides.
  • the method comprises detecting and/or measuring the amount of MTBR-tau282 peptides.
  • the method comprising one or more of the following steps: (a) purifying endogenously cleaved fragments of tau from a CSF sample from the subject, without contacting the endogenously cleaved fragments of tau in-vitro with a protease; (b) contacting the purified endogenously cleaved fragments of tau with an endopeptidase to obtain one or more of proteolytic MTBR-tau275 peptides comprising amino acids 275- 280 relative to SEQ ID NO:1 and proteolytic MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptides and/or proteo
  • the method further comprises desalting a solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR- tau282 peptides is before step (c).
  • the desalting is by solid phase extraction.
  • the purifying in step (a) comprises contacting the CSF sample with one or more anti- tau epitope binding agents.
  • the purifying in step (a) comprises immunoprecipitation.
  • the immunoprecipitation of the purification step (a) is for immunoprecipitation of N- terminal to mid-domain tau (e.g., to deplete N-terminal to mid-domain tau from the CSF sample).
  • the immunoprecipitation of N-terminal to mid-domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid- domain tau.
  • the immunoprecipitation of the purification step (a) is for immunoprecipitation of N-terminal tau (e.g., to deplete N-terminal tau from the CSF sample).
  • the immunoprecipitation of N-terminal tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal tau.
  • the immunoprecipitation of the purification step (a) is for immunoprecipitation of mid-domain tau (e.g., to deplete mid-domain tau from the CSF sample).
  • the immunoprecipitation of mid- domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to mid-domain tau.
  • the immunoprecipitation of purification step (a) is for immunoprecipitation of one or more MTBR-tau species from the CSF sample (e.g., to capture the one or more MTBR-tau species from the biological sample).
  • the 24 92044717.7 PATENT-PRO Atty is for immunoprecipitation of mid-domain tau (e.g., to deplete mid-domain tau from the CSF sample).
  • the immunoprecipitation of mid- domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to mid-domain tau.
  • Via EFS Web immunoprecipitation of one or more MTBR-tau species comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species.
  • a method of detecting 4R tau aggregates in a subject comprising one or more of the following steps: (a) performing affinity depletion on a cerebrospinal fluid (CSF sample from the subject by contacting the biological sample with one or more affinity depletion agents comprising one or more epitope binding agents that each binds to one of N- terminal tau, mid-domain tau, or C-terminal tau, but not to an antigen within MTBR-tau, wherein the CSF sample comprises endogenously cleaved fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, mid-domain tau, and/or C-terminal tau, and wherein the enriched sample is enriched for endogenously cleaved fragments of tau comprising one or more of endogenous MTBR-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and endogenous MT
  • the sample comprising one or more of proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides is desalted before step. In some embodiments, the desalting is by solid phase extraction.
  • the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau. In some embodiments, the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to N-terminal tau. In some embodiments, the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to mid-domain tau.
  • the one or more immunoprecipitation agents comprises one or more anti-tau epitope 25 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web binding agents that specifically bind to one or more MTBR-tau species (e.g., MTBR-tau275 and/or MTBR-tau282).
  • MTBR-tau species e.g., MTBR-tau275 and/or MTBR-tau282.
  • a biological sample comprising endogenously cleaved fragments of tau is contacted with one or more, or all of, HJ8.5, HJ8.7, and Tau1.
  • the biological sample is contacted with Tau1, HJ8.5, and HJ8.7.
  • a sample depleted of tau peptides using the aforementioned antibodies may be proteolytically cleaved in vitro using one or more proteases disclosed herein, and the resulting in vitro cleaved tau peptides (proteolytic fragments) may be contacted with an antibody that binds to the one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and/or MTBR-tau282 peptide, such as anti-tau.
  • the protease is trypsin.
  • the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau bind to an epitope within amino acids 1-243 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau bind to an epitope within amino acids 1-243 relative to SEQ ID NO:1.
  • the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau to mid-domain tau bind to an epitope within the amino acid sequence of: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSET SDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM VSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSG DRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRL (SEQ ID NO:4).
  • the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid- domain tau is selected from anti-tau antibodies HJ8.5, HJ8.7, or Tau1, or an antigen-binding fragment thereof.
  • the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within amino acids 1-103 of tau (e.g., of tau-441).
  • the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within amino acids 1-103 relative to SEQ ID NO:1.
  • the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within the amino acid sequence of: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSET SDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTA (SEQ ID NO:5). 26 92044717.7 PATENT-PRO Atty.
  • the one or more anti-tau epitope binding agents that specifically bind to mid-domain tau bind to an epitope within amino acids 104-243 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to mid-domain tau bind to an epitope within amino acids 104-243 relative to SEQ ID NO:1.
  • the one or more anti-tau epitope binding agents that specifically bind to mid-domain tau bind to an epitope within the amino acid sequence of: EEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQA NATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SPSSAKSRL (SEQ ID NO:6).
  • the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species specifically bind to an epitope within amino acids 244-368 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope within amino acids 244-368 relative to SEQ ID NO:1.
  • the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope of the amino acid sequence of: QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGN (SEQ ID NO:7).
  • the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species specifically bind to an epitope within amino acids 316-335 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope within amino acids 316-335 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope of the amino acid sequence of: SKVTSKCGSLGNIHHKPGGG (SEQ ID NO:8).
  • the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species is anti-tau antibody 77G7, or an antigen-binding fragment thereof.
  • the immunoprecipitation of one or more MTBR-tau species is for immunoprecipitation of MTBR-tau275 peptides and/or MTBR-tau282 peptides.
  • the one or more captured MTBR-tau species comprises MTBR-tau275 peptides and/or MTBR-tau282 peptides.
  • a CSF sample comprising tau is contacted with one or more, or all of: anti-tau antibody HJ8.5 or an antigen-binding fragment thereof; anti-tau antibody 27 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web HJ8.7 or an antigen-binding fragment thereof; and/or anti-tau antibody Tau1 or an antigen-binding fragment thereof.
  • a CSF sample is contacted with anti-tau antibodies Tau1, HJ8.5, and HJ8.7, or antigen-binding fragments thereof.
  • a sample depleted of tau peptides using the aforementioned antibodies may be contacted with an antibody that binds to MTBR-tau275 and MTBR- tau282, such as anti-tau antibody 77G7 or an antigen-binding fragment thereof.
  • Suitable biological samples include a cerebrospinal fluid (CSF) sample obtained from a subject.
  • the subject is a human.
  • a human subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.
  • a human subject may be a healthy subject, a subject at risk of developing a neurodegenerative disease, a subject with signs and/or symptoms of a neurodegenerative disease, or a subject diagnosed with a neurodegenerative disease.
  • the neurodegenerative disease may be a tauopathy, which may be a primary tauopathy.
  • the tauopathy may be frontotemporal lobar degeneration (FTLD-tau) (e.g., FTLD-MAPT), including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilc grain disease (AGD), globular glial tauopathy (GGT), Chronic Traumatic Encephalopathy (CTE), Pick’s disease (PiD), or Alzheimer’s disease.
  • FTLD-tau frontotemporal lobar degeneration
  • CBD corticobasal degeneration
  • PSP progressive supranuclear palsy
  • ATD argyrophilc grain disease
  • GTT globular glial tauopathy
  • CTE Chronic Traumatic Encephalopathy
  • Pick’s disease PiD
  • Alzheimer’s disease Alzheimer’s disease
  • the primary tauopathy is FTLD-tau or CBD.
  • the FTLD-tau is FTLD-MAPT.
  • the FTLD-MAPT is selected from FTLD- MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, and FTLD-MAPT IVS10+16.
  • the subject is a laboratory animal.
  • the subject is a laboratory animal genetically engineered to express human tau and optionally one or more additional human protein (e.g., human A ⁇ , human ApoE, etc.).
  • CSF may have been obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple CSF samples contemporaneously collected from the subject may be pooled.
  • CSF samples may have been 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.). CSF samples may be used immediately or may be frozen and stored indefinitely. Prior to use in the methods disclosed herein, the CSF sample may also have been modified, if needed or desired, to include protease inhibitors, isotope labeled internal standards, detergent(s) and chaotropic agent(s), and/or to optionally deplete other analytes (e.g., proteins peptides, metabolites). 28 92044717.7 PATENT-PRO Atty.
  • CSF samples volumes may be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In a specific example, the size of the sample may be about 0.05 mL to about 1 mL CSF.
  • a single sample is obtained from a subject. Alternatively, samples may be obtained from a subject over time. As such, more than one sample may be collected from a subject over time.
  • samples may be collected from a subject over time.
  • samples may be collected every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days.
  • a sample is collected a month apart, 3 months apart, 6 months apart, 1 year apart, 2 years apart, 5 years apart, 10 years apart, 20 years apart or more.
  • Purifying tau e.g., a peptide fragment of tau
  • Another step of the methods disclosed herein comprises purifying tau (e.g., a peptide fragment of tau), in particular, endogenously cleaved and/or proteolytically cleaved fragments of tau.
  • a method of the present disclosure comprises purifying tau peptides (e.g., an endogenously cleaved tau fragment) by affinity purification.
  • Affinity purification refers to methods that enrich for a protein of interest by virtue of its specific binding properties to a molecule.
  • the molecule is a ligand attached to a solid support, such as a bead, resin, tissue culture plate, etc. (referred to as an immobilized ligand).
  • Immobilization of a ligand to a solid support may also occur after the ligand- protein inter-action occurs.
  • Suitable ligands include antibodies, aptamers, and other epitope-binding agents.
  • Purifying a tau peptide by affinity purification comprises contacting a sample comprising an endogenously cleaved fragment tau with a suitable immobilized ligand, one or more wash steps, and elution of the cleaved fragment tau from the immobilized ligand.
  • a method of the present disclosure comprises purifying tau peptides (e.g., an endogenously cleaved tau fragment) tau by affinity purification using at least one epitope-binding agent that specifically binds to an epitope within amino acids 225-258 of tau-441, inclusive, or within amino acids 235-258 of tau-441, inclusive, or within amino acids 235-242, inclusive, (or within similarly defined regions for other full-length isoforms).
  • one, two, three or more epitope- 29 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web binding agents may be used.
  • a method of the present disclosure comprises purifying tau peptides by solid phase extraction. Purifying by solid phase extraction comprises contacting a sample comprising tau with a solid phase comprising a sorbent that adsorbs tau, one or more wash steps, and elution of tau from the sorbent. Suitable sorbents include reversed-phase sorbents.
  • Suitable re-versed phase sorbents are known in the art and include, but are not limited to alkyl-bonded silicas, aryl-bonded silicas, styrene/divinylbenzene materials, N- vinylpyrrolidone/divinylbenzene materials.
  • the reversed phase material is a polymer comprising N-vinylpyrrolidone and divinylbenzene or a polymer comprising styrene and divinylbenzene.
  • a sorbent is Oasis HLB (Waters).
  • a wash step may comprise the use of a liquid phase comprising about 0.05% v/v trifluoroacetic acid (TFA) to about 1% v/v TFA, or an equivalent thereof.
  • TFA trifluoroacetic acid
  • the wash may be with a liquid phase comprising about 0.05% v/v to about 0.5% v/v TFA or about 0.05% v/v to about 0.1% v/v TFA. In some examples, the wash may be with a liquid phase comprising about 0.1% v/v to about 1.0% v/v TFA or about 0.1% v/v to about 0.5% v/v TFA. Bound tau is then eluted with a liquid phase comprising about 20% v/v to about 50% v/v acetonitrile (ACN), or an equivalent thereof.
  • ACN v/v acetonitrile
  • tau is may be eluted with a liquid phase comprising about 20% v/v to about 40% v/v ACN, or about 20% v/v to about 30% v/v ACN. In some examples, tau is may be eluted with a liquid phase comprising about 30% v/v to about 50% v/v ACN, or about 30% v/v to about 40% v/v ACN.
  • the eluate may be dried by methods known in the art (e.g., vacuum drying (e.g., speed-vac), lyophilization, evaporation un-der a nitrogen stream, etc.). 2.5.
  • Methods of the present disclosure may comprise a step wherein one or more protein is depleted from a sample.
  • the term “deplete” means to diminish in quantity or number. Accordingly, a sample 30 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web depleted of a protein may have any amount of the protein that is measurably less than the amount in the original sample, including no amount of the protein.
  • Protein(s) may be depleted from a sample by a method that specifically targets one or more protein, for example by affinity depletion, solid phase extraction, or other method known in the art.
  • Targeted depletion of a protein, or multiple proteins may be used in situations where down-stream analysis of that protein is desired (e.g., identification, quantification, analysis of post-translation modifications, etc.).
  • a ⁇ peptides may be identified and quantified by methods known in the art following affinity depletion of A ⁇ with a suitable epitope-binding agent.
  • apolipoprotein E (ApoE) status may be determined by methods known in the art following affinity depletion of ApoE and identification of the ApoE isoform.
  • Targeted depletion may also be used to isolate other proteins for subsequent analysis including, but not limited to, apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, an interleukin, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, NFL, prion protein, pNFH, and DJ-1.
  • Targeted depletion of certain tau peptide fragments is also used herein to enrich for other tau peptides and/or eliminate proteins that cofound the mass spectrometry analysis.
  • N-terminal tau, mid-domain tau, and/or C-terminal MTBR-tau are depleted from a sample prior to further sample processing for analysis. Downstream analysis of the depleted tau proteins may or may not occur, but both options are contemplated by the methods of the present disclosure.
  • targeted depletion may occur by affinity depletion.
  • Affinity depletion refers to methods that deplete a protein of interest from a sample by virtue of its specific binding properties to a molecule.
  • the molecule is a ligand attached to a solid support, such as a bead, resin, tissue culture plate, etc. (referred to as an immobilized ligand).
  • Immobilization of a ligand to a solid support may also occur after the ligand-protein interaction occurs.
  • Suitable ligands include antibodies, aptamers, and other epitope-binding agents.
  • the molecule may also be a polymer or other material that selectively absorbs a protein of interest.
  • polyhydroxymethylene substituted by fat oxethylized alcohol e.g., PHM-L LIPOSORB, Sigma Aldrich
  • PHM-L LIPOSORB e.g., Sigma Aldrich
  • Two or more affinity depletion agents may be combined to sequentially or simultaneously deplete multiple proteins.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using at least one epitope-binding agent that specifically binds to an epitope within amino acids 1 to 243 of tau-441, inclusive (or within a similarly defined region for 0N or 1N 31 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web isoforms).
  • one, two, three or more epitope-binding agents may be used. When two or more epitope-binding agents are used, they may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within the N-terminus of tau (e.g., amino acids 1 to 103 of tau-441, inclusive), and an epitope-binding agent that specifically binds to an epitope within the mid-domain of tau (e.g., amino acids 104 to 243 of tau-441, inclusive).
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 of tau-441, inclusive, and an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive (or within similarly defined regions for 0N or 1N isoforms).
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 103 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive(or within a similarly defined region for 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta.
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta.
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 103 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); 32 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web and an epitope-binding agent that specifically binds to an epitope of amyloid be-ta.
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); and an epitope-binding agent that specifically binds to an epitope of amyloid be-ta.
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta.
  • the epitope-binding agents may be used sequentially or simultaneously.
  • a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 260 to 441 of tau-441, inclusive.
  • the epitope binding agent may comprise an antibody or an aptamer.
  • the epitope-binding agent that specifically binds to that specifically binds to an epitope within amino acids 1 to 103 of tau-441, inclusive is HJ8.5, or is an epitope-binding agent that binds the same epitope as HJ8.5 and/or competitively inhibits HJ8.5.
  • the epitope-binding agent that specifically binds to that specifically binds to an epitope within amino acids 104 to 221 of tau-441, inclusive is Tau1, or is an epitope-binding agent that binds the same epitope as Tau1 and/or competitively inhibits Tau1.
  • the epitope-binding agent that specifically binds to mid-domain tau is selected from HJ34.8, 77G7, RD3, RD4, UCB1017, PT76, E2815, or 7G6, or is an epitope-binding agent that binds the same epitope as HJ34.8, 77G7, RD3, RD4, UCB1017, PT76, E2815, or 7G6, and/or competitively inhibits HJ34.8, 77G7, RD3, RD4, UCB1017, or PT76 (e.g., as described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, and antibodies E2814 and 7G6 described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, as well as other epitope-binding agents that specifically bind the same epitopes as those antibodies.
  • protein(s) may be depleted from a sample by a more general method, for example by ultrafiltration or protein precipitation with an acid, an organic solvent or a salt. Generally speaking, these methods are used to reliably reduce high abundance and high molecular weight proteins, which in turn enriches for low molecular weight and/or low abundance proteins and peptides (e.g., tau, A ⁇ , etc.).
  • proteins may be depleted from a sample by precipitation.
  • precipitation comprises adding a precipitating agent to a sample and thoroughly mixing, incubating the sample with precipitating agent to precipitate proteins, and separating the precipitated proteins by centrifugation or filtration. The resulting supernatant may then be used in downstream applications.
  • the amount of the reagent needed may be experimentally determined by methods known in the art.
  • Suitable precipitating agents include perchloric acid, trichloroacetic acid, acetonitrile, methanol, and the like.
  • proteins are depleted from a sample by acid precipitation.
  • proteins are depleted from a sample by acid precipitation using perchloric acid.
  • proteins may be depleted from a sample by acid precipitation using perchloric acid.
  • perchloric acid refers to 70% perchloric acid unless otherwise indicated. In some embodiments, perchloric acid is added to a final concentration of about 1% v/v to about 15% v/v. In other embodiments, perchloric acid is added to a final concentration of about 1% v/v to about 10% v/v. In other embodiments, perchloric acid is added to a final concentration of about 1% v/v to about 5% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3% v/v to about 15% v/v.
  • perchloric acid is added to a final concentration of about 3% v/v to about 10% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3% v/v to about 5% v/v. In other embodiments, perchloric acid is added to a final concentration of 3.5% v/v to about 15% v/v, 3.5% v/v to about 10% v/v, or 3.5% v/v to about 5% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3.5% v/v.
  • samples are mixed well (e.g., by a vortex mixer) and held at a cold temperature, typically for about 10 minutes or longer, to facilitate precipitation.
  • samples may be held for about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes.
  • samples may be held for about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes.
  • samples may be held for about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes. In other examples, samples are held for about 30 minutes.
  • a cold temperature refers to a temperature of 10°C or less.
  • a cold temperature 34 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C.
  • a narrower temperature range may be preferred, for ex-ample, about 3°C to about 5°C, or even about 4°C.
  • a cold temperature may be achieved by placing a sample on ice.
  • Digesting purified tau with a protease comprises contacting a sample comprising purified tau with a protease under conditions suitable to digest tau. When affinity purification is used, digestion may occur after eluting tau from the immobilized ligand or while tau is bound. Suitable proteases include but are not limited to trypsin, Lys-N, Lys-C, Asp-C, Arg-N, and Arg-C. For detecting MTBR-tau275 of MTBR-tau282, the proteases comprise trypsin.
  • the resultant digestion product e.g., cleavage product
  • the resultant cleavage product comprises tryptic peptides of tau.
  • the resultant digestion product e.g., cleavage product
  • Another step of the method disclosed herein comprises detecting the amount of digested tau peptides from the processed biological sample. All suitable method of detecting an amount of tau protein are contemplated within the scope of the disclosure. Methods of detecting and quantifying tau peptides are described in detail below. 3.1.1. LC-MS 35 92044717.7 PATENT-PRO Atty.
  • the step of detecting and quantifying comprises performing liquid chromatography - mass spectrometry (LC-MS) with a sample comprising peptides of tau to detect and measure the concentration of at least one peptide of tau.
  • LC-MS liquid chromatography - mass spectrometry
  • the disclosed methods use one or more tau peptide to detect and measure the amount of tau protein present in the biological sample.
  • the proteolytic peptides of tau that indicate the presence of endogenously cleaved fragments of tau may comprise MTBR-275 or MTBR-282.
  • the resulting proteolytic peptides may differ slightly but can be readily determined by a person of ordinary skill in the art. Without wishing to be bound by theory, it is believed that a variation in the amount of a cleaved fragments of tau peptides between two bio-logical samples of the same type reflects a difference in the cleaved fragments of tau that make up those biological samples. As disclosed herein, the amounts of certain proteolytic peptides of cleaved fragments of tau, as well ratios of certain proteolytic peptides of tau, may provide clinically meaningful information to guide treatment decisions.
  • Proteolytic peptides of tau may be separated by a liquid chromatography system interfaced with a high-resolution mass spectrometer.
  • Suitable LC-MS systems may comprise a ⁇ 1.0 mm ID column and use a flow rate less than about 100 ⁇ l/min.
  • a nanoflow LC-MS system is used (e.g., about 50-100 ⁇ m ID column and a flow rate of ⁇ 1 ⁇ L / min, preferably about 100-800 nL/min, more preferably about 200-600 nL/min).
  • an LC-MS system may comprise a 0.05 mM ID column and use a flow rate of about 400 nL/min.
  • Tandem mass spectrometry may be used to improve resolution, as is known in the art, or technology may improve to achieve the resolution of tandem mass spectrometry with a single mass analyzer. Suitable types of mass spectrometers are known in the art.
  • an LC-MS system may comprise a mass spectrometer selected from Orbitrap FusionTM TribridTM Mass Spectrometer, Orbitrap FusionTM LumosTM Mass Spectrometer, Orbitrap TribridTM EclipseTM Mass Spectrometer, or a mass spectrometer with similar or improved ion-focusing and ion- transparency at the quadrupole.
  • Suitable mass spectrometry protocols may be developed by optimizing 36 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web the number of ions collected prior to analysis (e.g., AGC setting using an orbitrap) and/or injection time.
  • tau peptides e.g., an MTBR-tau peptide
  • fragments of tau are detected and quantified using an ELISA.
  • Methods for assessing an amount of protein expression using epitope binding agent-based methods are known in the art and all suitable methods for assessing an amount of protein known to one of skill in the art are contemplated within the scope of the present disclosure.
  • the method to assess an amount of tau peptide is an epitope binding agent-based method.
  • an epitope binding agent-based method of assessing an amount of protein expression comprises contacting a sample comprising a polypeptide with an epitope binding agent specific for the polypeptide under conditions effective to allow for formation of a complex between the epitope binding agent and the polypeptide.
  • Epitope binding agent-based method may occur in solution, or the epitope binding agent or sample may be immobilized on a solid surface.
  • suitable surfaces include microtiter plates, test tubes, beads, resins, and other polymers.
  • An epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art.
  • the epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate.
  • the substrate and the epitope binding agent may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the epitope binding agent may be attached directly using the functional groups or indirectly using linkers.
  • the epitope binding agent may also be attached to the substrate non-covalently.
  • a biotinylated epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • an epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography. Additional methods of attaching epitope binding agents to solid surfaces and methods of synthesizing biomolecules on substrates are well known in the art, i.e., VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No.6,566,495, 37 92044717.7 PATENT-PRO Atty.
  • label refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method.
  • suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase).
  • an epitope binding agent-based method is an immunoassay.
  • Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immunoassays and non-competitive immunoassays. In a competitive immunoassay, an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away and the bound analyte is measured.
  • the antibody is labeled, not the analyte.
  • Non-competitive immunoassays may use one antibody (e.g., the capture antibody is labeled) or more than one antibody (e.g., at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above.
  • the epitope binding agent method is an immunoassay.
  • the epitope binding agent method is selected from the group consisting of an enzyme linked immunoassay (ELISA), a fluorescence based assay, a dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), a radiometric assay, a multiplex immunoassay, and a cytometric bead assay (CBA).
  • ELISA enzyme linked immunoassay
  • DELFIA dissociation enhanced lanthanide fluoroimmunoassay
  • radiometric assay a multiplex immunoassay
  • CBA cytometric bead assay
  • the epitope binding agent-based method is an enzyme linked immunoassay (ELISA).
  • the epitope binding agent-based method is a radioimmunoassay.
  • the epitope binding agent-based method is an immunoblot or Western blot.
  • the epitope binding agent-based method is an array.
  • the epitope 38 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web binding agent-based method is flow cytometry.
  • the epitope binding agent-based method is immunohistochemistry (IHC).
  • IHC uses an antibody to detect and quantify antigens in intact tissue samples.
  • the tissue samples may be fresh-frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC. Methods of preparing tissue block for study by IHC, as well as methods of performing IHC are well known in the art. 4.
  • the present disclosure also encompasses the use of measurements of tau peptides (e.g., endogenously cleaved fragments of tau), in CSF as biomarkers of pathological features and/or clinical symptoms of tauopathies to diagnose, stage, choose treatments appropriate for a given disease stage, and modify a given treatment regimen (e.g., change a dose, switch to a different drug or treatment modality, etc.).
  • the pathological feature may be an aspect of tau pathology (e.g., amount of tau deposition, presence / absence of a post-translational modification, amount of a post-translation modification, etc.).
  • a pathological feature may be tau-independent.
  • the clinical symptom may be dementia, as measured by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.), or any other clinical symptom associated with the tauopathy.
  • a clinically validated instrument e.g., MMSE, CDR-SB, etc.
  • the present disclosure provides a method for measuring tauopathy- related pathology in a subject, the method comprising quantifying one or more tau peptides (e.g., as described herein) in a biological sample obtained from a subject, such as a CSF sample.
  • the method may comprise providing the amount of one or more tau peptides as described herein.
  • the amount(s) of the quantified tau peptides may be a representation of tauopathy-related pathology in the brain of the subject.
  • the tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R-tauopathy.
  • the tauopathy is a 4R-tauopathy.
  • the tauopathy-related pathology may be tau deposition, tau post-translational modification, amyloid plaques in the brain and/or arteries of the brain, or other pathological feature known in the art.
  • the present disclosure provides a method for diagnosing a tauopathy in a subject, the method comprising quantifying or providing the amount of one or more tau peptides (e.g., as described herein)in a biological sample obtained from a subject, such as a CSF sample, and diagnosing a tauopathy when the quantified tau peptide is/are about 1.5 ⁇ or above, where ⁇ is the standard deviation defined by the normal distribution measured in a control population that does not have clinical signs or 39 92044717.7 PATENT-PRO Atty.
  • ⁇ is the standard deviation defined by the normal distribution measured in a control population that does not have clinical signs or 39 92044717.7 PATENT-PRO Atty.
  • the tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R- tauopathy. In specific embodiments, the tauopathy is a 4R-tauopathy.
  • the subject may or may not have clinical symptoms of disease.
  • the present disclosure provides a method for measuring tauopathy disease stability in a subject, the method comprising quantifying or providing the amount of one or more tau peptides (e.g., as described herein)in a first biological sample obtained from a subject and then in a second biological sample obtained from the same subject at a later time (e.g., weeks, months or years later), and calculating the difference between the quantified tau peptide between the samples, wherein a statistically significant increase in the quantified tau peptide in the second sample indicates disease progression, a statistically significant decrease in the quantified tau peptide in the second sample indicates disease improvement, and no change indicates stable disease.
  • a statistically significant increase in the quantified tau peptide in the second sample indicates disease progression
  • a statistically significant decrease in the quantified tau peptide in the second sample indicates disease improvement
  • no change indicates stable disease.
  • the tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R-tauopathy.
  • the tauopathy is a 4R-tauopathy.
  • the subject may or may not have clinical symptoms of disease, and may or may not be receiving a tau therapy.
  • a tau therapy is administered one or more times to the subject in the period of time between collection of the first and second biological sample, and the measure of disease stability is an indication of the effectiveness, or lack thereof, of the tau therapy.
  • the MTBR-tau peptide species to be measured comprises MTBR-tau275/t-tau and/or MTBR-tau282/t-tau.
  • MTBR-tau peptides of tau as described herein can serve to illustrate various aspects discussed above, but such discussions do not limit the scope of the invention.
  • Tau peptides e.g., endogenously cleaved fragments of tau, e.g., MTBR-tau
  • their detection are described in detail in the Examples.
  • these tau peptides contain MTBR-tau275 and/or MTBR- tau282 and are cleaved endogenously at their C-terminus. Measuring the amount of tau peptides is one means by which to measure, in a given sample, the amount of this specific group of tau species. As shown in the Examples, decreases in the amount of 4R isoform specific MTBR-tau275 and MTBR-tau282 endogenously cleaved fragments of tau in the CSF correlate with tau deposition in the brain associated with primary tauopathies, especially FTLD (e.g., FTLD-MAPT) and CBD.4R specific MTBR-tau/t-tau measures have inverse correlation in the CSF and brain.
  • FTLD e.g., FTLD-MAPT
  • CBD.4R specific MTBR-tau/t-tau measures have inverse correlation in the CSF and brain.
  • CSF t-tau as well as pT217/T217 ratio is useful as a diagnostic marker for discriminating between tauopathies (e.g., AD distinguished from NC, PSP, or other FTLD-tau; or CBD distinguished from NC, or other FTLD-tau).
  • the detected amounts of MTBR-tau peptides can therefore be used to measure primary tauopathy pathology, to determine a subject’s tau status, to diagnose a primary tauopathy (e.g., FTLD-MAPT or CBD) stage in subjects, and discriminate between primary tauopathies, among other things.
  • treatments may then be provided to the subject to decrease, or prevent any further increase, in the amount of tau peptide in CSF and/or to decrease, or prevent any further increase, of another clinical sign or symptom of a primary tauopathy.
  • Choice of treatment may be further guided by knowledge of the specific disease stage that is informed by the amount of tau peptides (e.g., endogenously cleaved tau fragments), for instance, therapies designed to prevent A ⁇ deposition, reverse A ⁇ deposition, prevent tau deposition, reverse tau deposition, and improve clinical signs of disease would be used in subjects with different, albeit potentially overlapping, amount of tau peptides.
  • the present disclosure provides a method for detecting a primary tauopathy in a subject, the method comprising providing a CSF obtained from a subject, wherein the CSF is purified for a tau peptide (e.g., an endogenously cleaved fragment of tau); and quantifying, in the sample, the tau peptide.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide, or their ratios (e.g., to each other or each to CSF t-tau), is indicative of primary tauopathy in a brain of a subject.
  • the primary tauopathy is selected from frontotemporal lobar degeneration (FTLD-tau) (e.g., FTLD-MAPT), including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilc grain disease (AGD), globular glial tauopathy (GGT), Chronic Traumatic Encephalopathy (CTE), Pick’s disease (PiD), or Alzheimer’s disease.
  • FTLD-tau frontotemporal lobar degeneration
  • CBD corticobasal degeneration
  • PSP progressive supranuclear palsy
  • ATD argyrophilc grain disease
  • GTT globular glial tauopathy
  • CTE Chronic Traumatic Encephalopathy
  • Pick’s disease PiD
  • Alzheimer’s disease Alzheimer’s disease
  • the primary tauopathy is FTLD-tau or CBD.
  • the FTLD- tau is FTLD-MAPT.
  • the FTLD-MAPT is selected from FTLD-MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, and FTLD-MAPT IVS10+16.
  • the present disclosure provides a method of measuring FTLD-tau– related pathology in a subject, comprising detecting or providing the amount of tau or 4R tau aggregates.
  • the detecting tau or 4R tau aggregates comprises providing a CSF obtained from a subject, wherein the CSF is purified for a tau peptide; and quantifying, in the sample, the tau peptide.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence 41 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide, or their ratios (e.g., to each other or each to CSF t-tau), is indicative of FTLD-tau–related pathology in a brain of a subject.
  • the method comprises quantifying or providing an amount p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), A ⁇ (e.g., A ⁇ 42/A ⁇ 40 ratio), or a combination thereof from a sample taken from a subject.
  • p-tau e.g., tau181, p-tau205, p-tau217, and p-tau231
  • a ⁇ e.g., A ⁇ 42/A ⁇ 40 ratio
  • a subject is diagnosed with FTLD-tau when the quantified tau peptide differs by about 1.5 ⁇ or more from the mean of a control population, where ⁇ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or A ⁇ 42/A ⁇ 40 ratio measurement in CSF (for instance, a cutoff value for CSF A ⁇ 42/A ⁇ 40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379–387) that maximizes sensitivity% + Specificity%).
  • ⁇ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or A ⁇ 42/A
  • the present disclosure provides a method of measuring FTLD- tau–related tau deposition in a brain of a subject, comprising detecting or providing the amount of tau or 4R tau aggregate.
  • the method comprises providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a tau peptide; and quantifying, in the sample, the tau peptide.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide is indicative of FTLD-tau-related tau deposition in a brain of a subject.
  • the FTLD-tau is FTLD-MAPT.
  • the FTLD-MAPT is selected from FTLD- MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, and FTLD-MAPT IVS10+16.
  • the present disclosure provides a method of diagnosing FTLD- tau-related disease, the method comprising providing a CSF obtained from a subject, and quantifying, in the sample, a tau peptide. The method may comprise providing the amount of a tau peptide as disclosed herein.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof.
  • the method further comprises quantifying p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), A ⁇ (e.g., A ⁇ 42/A ⁇ 40 ratio), or a combination thereof.
  • a subject is diagnosed with FTLD- tau-related disease when the quantified tau peptide differs by about 1.5 ⁇ or more from the mean of a control population, where ⁇ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or A ⁇ 42/A ⁇ 40 ratio measurement in CSF (for instance, a cutoff value for CSF 42 92044717.7 PATENT-PRO Atty.
  • the FTLD-tau is FTLD-MAPT.
  • the FTLD-MAPT is selected from FTLD-MAPT P301L, FTLD-MAPT S305I, FTLD- MAPT R406W, or FTLD-MAPT IVS10+16.
  • the present disclosure provides a method of measuring FTLD- tau-related disease progression in a subject, the method comprising providing a first CSF sample and a second CSF sample, wherein each sample is obtained from a single subject, and each sample is purified for tau peptide; and for each sample, quantifying the tau peptide.
  • the amounts of tau peptide in the first and second sample are provided.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof.
  • the method further comprises p-tau or A ⁇ measurement, or a combination thereof.
  • the method further comprising calculating the difference between the quantified tau peptide in the second sample and the first sample, wherein a statistically significant in-crease in the quantified tau peptide in the second sample indicates progression of the subject’s FTLD-tau-related disease.
  • the FTLD-tau is FTLD-MAPT.
  • the FTLD- MAPT is selected from FTLD-MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, or FTLD- MAPT IVS10+16.
  • the present disclosure provides a method of measuring CBD– related tau deposition in a brain of a subject, comprising detecting or providing the amount of tau or 4R tau aggregate.
  • the method comprises providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a tau peptide; and quantifying, in the sample, the tau peptide.
  • the amount of the tau peptide is provided.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide is a representation of CBD–related tau deposition in a brain of a subject.
  • the present disclosure provides a method of diagnosing CBD- related disease, the method comprising providing a CSF obtained from a subject, and quantifying, in the sample, a tau peptide. In one example, the amount of the tau peptide is provided.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof.
  • the method further comprises quantifying p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), A ⁇ (e.g., A ⁇ 42/A ⁇ 40 ratio), or a combination thereof.
  • a subject is diagnosed with CBD-related disease when the 43 92044717.7 PATENT-PRO Atty.
  • Via EFS Web quantified tau peptide differs by about 1.5 ⁇ or more from the mean of a control population, where ⁇ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or A ⁇ 42/A ⁇ 40 ratio measurement in CSF (for instance, a cutoff value for CSF A ⁇ 42/A ⁇ 40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379–387) that maximizes sensitivity% + Specificity%).
  • ⁇ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or A ⁇ 42/A ⁇ 40 ratio measurement in CSF (for
  • the present disclosure provides a method of measuring CBD- related disease progression in a subject, the method comprising providing a first CSF sample and a second CSF sample, wherein each sample is obtained from a single subject, and each sample is purified for a tau peptide; and for each sample, quantifying the tau peptide.
  • the amounts of tau peptide in the first and second samples are provided.
  • the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof.
  • the method further comprises p-tau or A ⁇ measurement, or a combination thereof.
  • the method further comprising calculating the difference between the quantified tau peptide in the second sample and the first sample, wherein a statistically significant in-crease in the quantified tau peptide in the second sample indicates progression of the subject’s CBD-related disease.
  • additional biomarkers can be detected and measured to aid in staging and/or determining the pathology of the subject and/or disease progression of the subject.
  • the method may include detecting and measuring the amount of Ab.
  • an A ⁇ 42/A ⁇ 40 value is determined to aid in measuring amyloid plaque specific abnormalities.
  • each of the preceding methods may include detecting and measuring the amount of tau phosphorylation at specific residues.
  • phosphorylation of tau at 217, 181, 231, 205, 153, 111, 208, and/or any combination thereof are determined to aid in measuring amyloid plaque specific abnormalities.
  • a ⁇ 42/A ⁇ 40 is the first to change, then ptau217/181/231 shortly thereafter and those are associated with amyloid plaques.
  • ptau205 is increased and associated with both Ab plaques and tau tangles, similar to total-tau (n-terminal to mid-domain tau).
  • a composite value of ptau phosphorylation and A ⁇ 42/A ⁇ 40 value can be measured and determined.
  • pT217 x A ⁇ 42/A ⁇ 40 value is determined to aid in measuring amyloid plaque specific abnormalities and is found to be highly sensitive and specific.
  • endogenously cleaved fragment of tau as disclosed herein is increased and 44 92044717.7 PATENT-PRO Atty.
  • a ⁇ 42/A ⁇ 40 is a measure of pre-amyloid plaque abnormalities
  • a ⁇ 42/A ⁇ 40 & ptau217% abnormal is a measure of amyloid plaque abnormalities
  • ptau205 is a measure of amyloid plaque plus neurodegeneration
  • endogenously cleaved fragment of tau as disclosed herein is a measure of tau tangles and clinical onset of dementia.
  • 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.
  • Mathematical operations other than a ratio may also be used.
  • the examples use of an endogenously cleaved fragment of tau as disclosed herein, Ab values, and/or site-specific tau phosphorylation values can be used in various statistical models (e.g., linear regressions, LME curves, LOESS curves, etc.) in conjunction with other known biomarkers (e.g.
  • ROC AUC value 0.7 or greater is set as a threshold (e.g., 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.).
  • PET amyloid-positron emission tomography
  • IB 11 C-Pittsburgh compound B PET imaging of cortical A ⁇ -plaques is commonly used to detect A ⁇ -plaque pathology.
  • SUVR standard uptake value ratio
  • 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.
  • the present disclosure provides a method for treating a subject with a tauopathy, the method comprising quantifying tau in a biological sample obtained from a subject, such as a CSF sample; and providing a tau therapy to the subject to improve a measurement of tauopathy-related pathology or a clinical symptom, wherein the subject has a quantified endogenously cleaved peptide fragment of tau at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably 45 92044717.7 PATENT-PRO Atty.
  • ⁇ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and that is amyloid negative as measured by PET imaging and/or A ⁇ 42/A ⁇ 40 ratio 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 treating a subject.
  • the tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R- tauopathy.
  • the tauopathy is a 4R-tauopathy.
  • the measurement of tauopathy- related pathology may be tau deposition as measured by PET imaging, tau post-translational modification as measured by mass spectrometry or other suitable method, amyloid plaques in the brain or arteries of the brain as measured by PET imaging, amyloid plaques as measured by A ⁇ 42/A ⁇ 40 ratio in CSF, or other pathological features known in the art.
  • the clinical symptom may be dementia, as measured by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.) or other clinical symptoms known in the art for 3R- and 4R- tauopathies.
  • two or more tau species are quantified.
  • Many tau therapies target a specific pathophysiological change.
  • a ⁇ targeting therapies are generally designed to decrease A ⁇ production, antagonize A ⁇ aggregation or increase brain A ⁇ clearance
  • tau targeting therapies are generally designed to alter tau phosphorylation patterns, antagonize tau aggregation (general antagonism of tau or antagonism of a specific tau isoform), or increase NFT clearance; a variety of therapies are designed to reduce CNS inflammation or brain insulin resistance; etc.
  • tauopathies share the same pathophysiological changes. Therefore, the efficacy of these various tau therapies can be improved by administering them to subjects that are correctly identified as having a tau pathology, including determining the stage of disease the subject is in thereby more efficiently altering tau phosphorylation patterns, antagonizing tau aggregation, or increasing NFT clearance based on the specific pathological state of the subject.
  • the treatment may comprise one or more of lecanemab, donanemab, AADvac1, ACI-3024, ACI- 35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, Bepranemab, Dasatinib + Quercetin, E2814, Epothilone D, Gosuranemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, Semorinemab, TPI 287, Tideglusib, Tilavonemab, Zagotenemab, an anti-tau monoclonal anti-body, an anti-tau anti-sense oligonucleotide, an anti-tau small interfering RNA, an tau production inhibitor, and a tau active vaccine
  • the treatment is a pharmaceutical composition comprising a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., a selective serotonin 46 92044717.7 PATENT-PRO Atty.
  • NMDA N-methyl D-aspartate
  • an antidepressant e.g., a selective serotonin 46 92044717.7 PATENT-PRO Atty.
  • EFS Web reuptake inhibitor Via EFS Web reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, etc.), a gamma-secretase inhibitor, a beta-secretase inhibitor, an anti-A ⁇ antibody (including antigen-binding fragments, variants, or derivatives thereof), an anti-tau antibody (including antigen- binding fragments, variants, or derivatives thereof), an anti-TREM2 antibody (including antigen-binding fragments, variants or derivatives thereof, a TREM2 agonist, stem cells, dietary supplements (e.g.
  • TRx0237 methylthionimium chloride, etc.
  • a therapy to improve blood sugar control e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • an anti-inflammatory agent e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • an anti-inflammatory agent e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • an anti-inflammatory agent e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • an anti-inflammatory agent e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • an anti-inflammatory agent e.g., insulin, exenatide, liraglutide pioglitazone, etc.
  • an anti-inflammatory agent e.g., insulin, exenatide, liraglutide
  • a pharmaceutical composition may comprise a kinase inhibitor. Suitable kinase inhibitors may inhibit a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3 ⁇ , 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.
  • the treatment is a pharmaceutical composition comprising a tau targeting therapy, including but not limited to active pharmaceutical ingredients that alter tau phosphorylation patterns, antagonize tau aggregation, or increase clearance of pathological tau isoforms and/or aggregates.
  • the treatment is an anti-A ⁇ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma- secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor.
  • the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing when the detected A ⁇ 42/A ⁇ 40 value deviates from the mean of a healthy control population and optionally pTau217/tau217, ptau205/tau205, pTau181, 47 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web pTau231, and/or a cleaved peptide fragment of the present disclosure do not significantly deviate from the mean of a healthy control population.
  • the detected A ⁇ 42/A ⁇ 40 value deviates significantly below the mean of a healthy control population.
  • the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing and/or reduces a subject's existing plaque load when the detected A ⁇ 42/A ⁇ 40 value and pTau217/tau217 (or pTau217 x A ⁇ 42/A ⁇ 40 composite value) significantly deviates from the mean of a healthy control population and optionally ptau205/tau205, and/or a cleaved peptide fragment of the present disclosure do not significantly deviate from the mean of a healthy control population.
  • the detected A ⁇ 42/A ⁇ 40 value deviates significantly below the mean of a healthy control population
  • the detected pTau217/tau217 is above the mean of a healthy control population and/or the pTau217 x A ⁇ 42/A ⁇ 40 composite value is above the mean of a healthy control population.
  • the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing and/or reduces a subject's existing plaque load and/or treats or prevents neurodegeneration and/or prevents Tau tangles or related pathology when the detected A ⁇ 42/A ⁇ 40 value, pTau217/tau217 (or pTau217 x A ⁇ 42/A ⁇ 40 composite value), and pTau205/tau205 value significantly deviate from the mean of a healthy control population and optionally a cleaved peptide fragment of the present disclosure do not significantly deviate from the mean of a healthy control population.
  • the detected A ⁇ 42/A ⁇ 40 value deviates significantly below the mean of a healthy control population
  • the detected pTau217/tau217 is above the mean of a healthy control population and/or the pTau217 x A ⁇ 42/A ⁇ 40 composite value is above the mean of a healthy control population and/or the pTau205/tau205 value is above the mean of a healthy control population.
  • the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing and/or reduces a subject's existing plaque load and/or treats or prevents neurodegeneration and/or prevents tau tangles from in-creasing and/or reduces a subject’s existing tangles or related pathology when the detected A ⁇ 42/A ⁇ 40 value, pTau217/tau217 (or pTau217 x A ⁇ 42/A ⁇ 40 composite value), pTau205/tau205 and a cleaved peptide fragment of the present disclosure significantly deviate from the mean of a healthy control population.
  • the detected A ⁇ 42/A ⁇ 40 value deviates significantly below the mean of a healthy control population
  • the detected pTau217/tau217 is above the mean of a healthy control population and/or the pTau217 x
  • a ⁇ 42/A ⁇ 40 composite value is above the mean of a healthy control population
  • the pTau205/tau205 value is above the mean of a healthy control population and/or a cleaved peptide fragment of the present disclosure value is above the mean of a health control population.
  • a pharmaceutical composition administered to a subject may comprise an imaging agent.
  • imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh com-pound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.).
  • functional imaging agents e.g., fluorodeoxyglucose, etc.
  • molecular imaging agents e.g., Pittsburgh com-pound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.
  • a method of a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) selecting the subject into a clinical trial for an A ⁇ therapy when the cleaved peptide fragment of tau value is about the same as a healthy control population and the subject’s A ⁇ 42/A ⁇ 40 value is below the man of a healthy control population.
  • the cleaved peptide fragment of tau comprises one or more of the peptides having an amino acid sequence in Table 1, where the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject.
  • a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) excluding the subject into a clinical trial an A ⁇ therapy when the cleaved peptide fragment of tau value is above the mean of a healthy control population and the subject’s A ⁇ 42/A ⁇ 40 value is about the same or below the mean of a healthy control population.
  • the cleaved peptide fragment of tau comprises one or more of the peptides having an amino acid sequence in Table 1, where the C- terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject.
  • a method of selecting a subject into a clinical trial, in particular a clinical trial for a tau therapy provided all other criteria for the clinical trial have been met.
  • a method of a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) selecting the subject into a clinical trial a tau therapy when the cleaved peptide fragment of tau value is above the mean of a healthy control 49 92044717.7 PATENT-PRO Atty.
  • the cleaved peptide fragment of tau comprises one or more of the peptides comprising an amino acid sequence in Table 1, where the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject.
  • a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) excluding the subject into a clinical trial a tau therapy when the cleaved peptide fragment of tau value is about the same as the mean of a healthy control population.
  • the cleaved peptide fragment of tau comprises one or more of the peptides comprising an amino acid sequence in Table 1, where the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject.
  • the phrase “a control population without brain amyloid plaques as measured by PET imaging” is defined above.
  • 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 pT181 and pT205, pT217 and pT205, or pT181 and pT217.
  • a ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between pT181 and total tau, pT205 and total tau, or pT217 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 ⁇ 4 status, age, sex, cognitive test scores, functional test scores, etc.).
  • tauopathies e.g., FTLD-MAPT, CBD
  • tau therapies can be greatly aided by the methods disclosed herein.
  • Many clinical trials are designed to test the efficacy of imaging agents or therapeutic agents that target a specific pathophysiological change which occurs 50 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web prior to the onset of tau pathology symptoms.
  • the efficacy of these various agents can be improved by administering the agents to subjects that have certain site-specific tau phosphorylation levels, as measured by methods disclosed herein and illustrated.
  • a subject may be enrolled into a treatment arm of the clinical trial.
  • the "treatment” is defined above.
  • 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 pharmaceutical ingredient.
  • 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-A ⁇ 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-3 ⁇ , MARK, CDK5, or Fyn.
  • a pharmaceutical composition may comprise a phosphatase activator.
  • 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 a tauopathy. Alternatively, a subject may exhibit signs or symptoms (e.g., memory loss, misplacing things, changes in mood or behavior, etc.,) but not show sufficient cognitive or functional impairment for a clinical diagnosis.
  • a symptomatic or an asymptomatic subject may have A ⁇ amyloidosis; however, prior knowledge of A ⁇ amyloidosis is not a requisite for treatment.
  • kits can include an agent or composition described herein and, in certain embodiments, instructions for administration or measuring a tau peptide (e.g., endogenously cleaved fragment of tau). Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to systems, assays, epitope biding agents, reagents, internal standards, or software.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of 52 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil- lined interiors, such as aluminum or an alloy.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
  • a control sample or a reference sample as described herein can be a sample from a healthy subject or from a randomized group of subjects.
  • a reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects.
  • a control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.
  • the methods and algorithms of the invention may be enclosed in a controller or processor.
  • methods and algorithms of the present invention can be embodied as a computer implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods.
  • computer program computer program
  • Storage media for containing such computer program include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer.
  • the method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods.
  • the method or 53 92044717.7 PATENT-PRO Atty.
  • EFS Web methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes.
  • the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements.
  • Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above.
  • Example 1 CSF tau microtubule binding region identifies pathological changes in primary tauopathies Abstract: [0168] Despite recent advances in fluid biomarker research in Alzheimer’s disease (AD), there are no fluid biomarkers or imaging tracers with utility for diagnosis and/or theragnosis available for other tauopathies.
  • AD Alzheimer’s disease
  • MTBR-tau275 and MTBR-tau282 4 repeat (4R) isoform specific tau species from microtubule binding region
  • CBD corticobasal degeneration
  • PSP Progressive supranuclear palsy
  • FTLD frontotemporal lobar degeneration
  • AD inversely decrease in the CSF of CBD, FTLD-MAPT and AD compared to control and other FTLD-tau (i.e., Pick’s disease).
  • CSF MTBR-tau measures are reproducible in repeated lumbar punctures, and can distinguish CBD from control (area under the curve of 0.889) and other FTLD-tau such as PSP (area under the curve of 0.886).
  • CSF MTBR- tau275 and MTBR-tau282 may represent the first affirmative biomarkers to aid in the diagnosis of primary tauopathies and facilitate clinical trial designs.
  • Tauopathies are a heterogeneous group of neurodegenerative diseases that all include aggregated tau proteins. The symptomatic phases of these fatal illnesses involve neurological impairments that typically progress over years to decades, leading to substantial medical, social, and financial burden on patients and families.
  • AD Alzheimer’s disease
  • CSF cerebrospinal fluid
  • a ⁇ amyloid-beta
  • total and phosphorylated tau have been used to aid diagnosis 1–8 54 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web and these biomarkers have become useful for assessing the outcome of therapies in clinical trials 9–11 .
  • PET positron emission tomography
  • tauopathies classified as frontotemporal lobar degeneration including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilc grain disease (AGD), globular glial tauopathy (GGT), Chronic Traumatic Encephalopathy (CTE) and Pick’s disease (PiD).
  • CBD corticobasal degeneration
  • PSP progressive supranuclear palsy
  • ATD argyrophilc grain disease
  • GTT globular glial tauopathy
  • CTE Chronic Traumatic Encephalopathy
  • Pick’s disease PiD
  • Accurate diagnoses are challenging without fluid biomarkers for the tauopathies as these disorders fall within a spectrum comprising multiple and overlapping clinical phenotypes.
  • Most tauopathies can only be definitively diagnosed by brain autopsy.
  • Antemortem fluid biomarkers for these tauopathies will be required to improve the accuracy of clinical diagnosis and to facilitate clinical trials for tauopathy therapeutics.
  • tau splicing isoforms are expressed in the adult human brain, including isoforms containing R1, R3, R4 repeat domains (3R) and R1, R2, R3, and R4 repeat domains (4R) in the MTBR 13 .
  • Tauopathies can be classified into 3R, 4R, and 3R/4R mixed tauopathies based on the dominant isoforms found in tau aggregates.
  • Cryogenic electron microscopy (CryoEM) studies demonstrated that there are distinct tau filament structures in AD (3R/4R) 14 , PiD (3R)15, CBD (4R)16, PSP (4R)17, and CTE (3R/4R) 18 .
  • R3 and R4 repeat domains are commonly present in tau aggregates from AD and other tauopathies 14 .
  • 4R isoform-specific R2 repeat domain in addition to R3 and R4 are found in tau aggregates from CBD and PSP 16,17 .
  • Biochemical extraction and mass spectrometry methods recently showed that specific tau peptides such as the residues 243-254 (MTBR-tau243; R1), 299-317 (MTBR-tau299; R2-R3), and 354-369 (MTBR-tau354; R4) differentially enriched in AD brains with disease progression 19 .
  • the truncated tau containing MTBR could be detected and quantified in CSF, and CSF soluble concentrations of MTBR-tau peptides reflected AD clinical severity and correlated strongly with tau PET measures 19 .
  • Others have also reported a correlation between CSF soluble truncated MTBR-tau and insoluble tau aggregates measured by tau PET measures 20 , suggesting that measures of MTBR-tau peptides might serve as fluid biomarkers of tau aggregation in AD.
  • the present inventors hypothesized that 4R isoform specific MTBR-tau species accumulate in the brain of specific subtypes of 4R tauopathies.
  • the homogenate was clarified by centrifugation for 20 min at 11,000g at 4 degC and stored at -80 degC as the whole brain extract.
  • Demographics of the brain donors included in this study are described in FIG.1.
  • Human CSF samples [0176] CSF collection methods are similar across all cohorts examined in this study. CSF collection method from pathologically-confirmed cases was as previously described in the Alzheimer's Disease Neuroimaging Initiative (ADNI) procedures manual. [0177] Demographics of participants in pathologically-confirmed cohort is in FIG.2. CSF collection method from repeated LP studies was collected using the same human tau Stable Isotope Labeling Kinetics protocol as previously described 37 .
  • Mass spectrometry analyses of MTBR-tau [0178] Brain insoluble MTBR-tau was analyzed using filter-aided sample preparation methods as previously described 19,36 . Briefly, the whole brain extract was incubated with 1% sarkosyl for 60 min on ice, followed by ultra-centrifugation at 100000g at 4 degC for 60 min to obtain an insoluble pellet. Insoluble brain fractions were filtered, digested, desalted and injected into mass spectrometer for analyses.
  • CSF MTBR-tau was analyzed as previously described 19 with the following modifications: master mix containing detergent and chaotropic reagents (final 1% NP-40, 5 mM guanidine, protease inhibitor cocktail) and internal standards for tau (15N labeled 2N4R recombinant tau) were prepared in polypropylene tubes prior to CSF addition.0.5 mL of CSF was added and immunoprecipitated with Tau1, HJ8.5, and HJ8.7 anti-tau antibodies with epitopes residing in N- terminal to mid-domain of tau 24,37 . Post- 57 92044717.7 PATENT-PRO Atty.
  • MTBR-tau275/t-tau moderately increased in PSP (approximately 2-fold) compared to FTLD-TDP (p ⁇ 0.01).
  • Brain MTBR- tau282/t-tau had similar increase profile to MTBR-tau275/t-tau (FIG.4C) but moderately (2.6-fold) increased in AD (0.915 ⁇ 0.180) compared to FTLD-TDP (0.348 ⁇ 0.133, p ⁇ 0.05), which was not observed in MTBR-tau275/t-tau.
  • CSF MTBR-tau275 and MTBR- tau282 from truncated tau were normalized by t-tau measured by mid domain tau 212-221 (FIG.5A), similar to normalization methods previously reported in truncated tau and A ⁇ isoform measurements 20,22 .
  • CSF MTBR-tau275/t-tau decreased in CBD (0.00525 ⁇ 0.00117), AD (0.00472 ⁇ 0.00085), and FTLD- MAPT (0.00491 ⁇ 0.00207), compared to NC (0.00657 ⁇ 0.00078, p ⁇ 0.001, p ⁇ 0.0001, p ⁇ 0.01, respectively) and non-tauopathy control, FTLD-TDP (0.00611 ⁇ 0.00115, p ⁇ 0.05, p ⁇ 0.01, p ⁇ 0.05, respectively.
  • FIG.5B This decrease was particularly significant in FTLD-MAPT P301L that has more typical FTLD pathology than in R406W which has many features of AD.
  • CSF MTBR-tau275 also decreased in CBD, AD, and FTLD-MAPT compared to other 4R tauopathies, AGD (0.00759 ⁇ 0.00013) and PSP (0.00669 ⁇ 0.00091, p ⁇ 0.001, p ⁇ 0.0001, p ⁇ 0.01, respectively), and 3R tauopathy, PiD (0.00676 ⁇ 0.00138, p ⁇ 0.05, p ⁇ 0.01, p ⁇ 0.05, respectively).
  • CSF MTBR-tau282/t-tau had similar decrease profiles to CSF MTBR-tau275/t-tau (FIG.5C).
  • CSF MTBR-tau275/t-tau did not change in PSP compared to control or FTLD-TDP even though these ratios increased moderately in the brain.
  • MTBR-tau/t-tau measures reflected brain tau pathology measured by the paired insoluble brain MTBR-tau/t-tau measures
  • CSF MTBR-tau is reproducible in repeated lumbar punctures
  • PSP-RS N 7
  • CBS corticobasal syndrome
  • CV mean coefficient of variation
  • CSF MTBR-tau275/t-tau decreased in two symptomatic FTLD-MAPT P301L mutation carriers (participant #02, #03, 0.00381 ⁇ 0.00021) and a symptomatic FTLD-MAPT R406W mutation carrier (#05, 0.00508) compared to NC (#01, #04, 0.00666 ⁇ 0.00027).
  • CSF MTBR-tau275/t-tau did not change in FTLD-MAPT R406W mutation carriers who were asymptomatic at LPs (#06, #07).
  • the FTLD-MAPT variant IVS10+16 promotes the splicing of tau exon 10, resulting in greater production of 4R over 3R isoforms.
  • CSF MTBR-tau275/t-tau decreased in the two participants who were clinically diagnosed as PSP- RS but later were autopsy-confirmed with CBD (#10 (0.00396), #11 (0.00535)), consistent with pathologically-confirmed CSF cohort results.
  • CSF MTBR-tau275/t-tau did not change in participants clinically diagnosed with PSP-RS (0.00779 ⁇ 0.00052) or CBS (0.00748 ⁇ 0.00187) who are not yet autopsy-confirmed during the repeated measures studies.
  • CSF MTBR-tau in clinically diagnosed primary tauopathies [0188] To estimate the CSF MTBR-tau biomarker performance in clinically diagnosed primary tauopathies, CSF MTBR-tau275/t-tau was measured in an additional independent cohort of 238 primary tauopathies with single LP (FIG.8).
  • This cohort was previously analyzed for CSF t-tau and phosphorylated tau 24 and includes clinically diagnosed cases of AD, sporadic behavioral variant frontotemporal dementia (bvFTD), bvFTD secondary to FTLD-MAPT, PSP-RS, CBS, and CBS-PSP continuum 25 .
  • Individuals with CBS-PSP continuum are defined as patients who initially presented with CBS, but subsequently developed clinical features of PSP-RS as the disease progressed.
  • CSF MTBR- tau275/t-tau decreased in CBS-PSP continuum and FTLD-MAPT compared to cognitively NC (p ⁇ 0.05).
  • CSF MTBR-tau275/t-tau did not statistically change in either AD or clinically diagnosed CBS compared to control or other tauopathies. Diagnostic accuracies of CSF MTBR-tau in primary tauopathies [0189] Finally, diagnostic accuracies of CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau were examined in pathologically-confirmed primary tauopathy cohort. First, CSF t-tau (mid domain peptide 212-221) and phosphorylated tau (pT217/T217) were examined in primary tauopathies for comparison (FIG.12C and FIG.14A).
  • CSF t-tau increased in AD compared to NC and PSP (p ⁇ 0.05) and can differentiate AD from FTLD-tau (PSP, CBD, AGD, PiD, FTLD-MAPT) with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.794 FIG.14B).
  • ROC receiver operating characteristic
  • AUC receiver operating characteristic
  • CSF t-tau does not distinguish among FTLD-tau.
  • CSF pT217/T217 increased in AD compared to NC, FTLD-TDP and FTLD-tau (p ⁇ 0.0001) and can differentiate AD from FTLD-tau with AUC of 0.987 (FIG.14C).
  • AD co- pathology in other neurodegenerative diseases i.e., FTLD-TDP, CBD, PSP
  • CSF pT217/T217 can be used to accurately identify individuals with AD pathology, regardless of co-pathology.
  • FIG.15D, and FIG.15E These results suggest that CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau decrease in CBD independently from AD co-pathology.
  • CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau were examined to determine if they distinguish CBD from control, FTLD-TDP, FTLD-tau as a group and individual tauopathy (FIG.3, FIGS.16A-16L).
  • CSF MTBR-tau275/t-tau and CSF MTBR-tau282/t-tau can distinguish CBD from NC, other FTLD-tau (PSP, PiD, and AGD), PiD, and PSP with AUC of 0.800 to 0.889.
  • CBD can be distinguished from FTLD-TDP with AUC of 0.701 to 0.770.
  • CSF MTBR-tau275/t-tau and CSF MTBR-tau282/t-tau can distinguish CBD from PSP with AUC of 0.859 and 0.886, respectively (FIG.3, FIG.16K, and FIG.16L).
  • CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau were retrospectively assessed by final clinical syndromes in neuropathologically-confirmed cohort to determine if these biomarkers can facilitate antemortem diagnosis of primary tauopathies.
  • FTLD- MAPT e.g., P301L
  • FTLD- MAPT can lead to a very high deposition of 4R tau aggregates in neurons and glia in multiple brain regions, including the hippocampus, neocortex, and substantia nigra 34 .
  • CBD has wispy and fine filamentous inclusions within neuronal cell bodies, whereas PSP neurons tends to harbor a larger proportion of more compact tau aggregates 35 .
  • CBD is commonly associated with abundant cortical astrocytic plaque pathology and neuritic tau pathology in both gray and white matter, while PSP neuronal and astrocytic pathology (i.e., tufted astrocytes) are often restricted to the motor and premotor cortex and subcortical nuclei. It is possible that tau aggregates in PSP may have different physicochemical property from CBD and the status of equilibration between insoluble and soluble forms may be different.
  • CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau may potentially positively identify a subset of primary tauopathies and may be useful in assisting with antemortem differential diagnosis. It was confirmed in our repeated lumbar puncture study that CSF MTBR-tau/t-tau measures are reproducible and stable over 4 months, which will reliably provide biomarker values in clinic or clinical trials settings.
  • CSF MTBR-tau275/t-tau did not change in CBS, and there was a higher overlap between CBS and PSP-RS, which might be attributable to lack of one-to-one relationship between clinical syndromes and neuropathological diagnosis in FTLD.
  • 63 92044717.7 PATENT-PRO Atty 63 92044717.7 PATENT-PRO Atty.
  • CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau biomarkers may be able to identify individuals with CBD regardless of clinical syndromes (i.e., CBS, bvFTD, and PSP-RS) with as high as 83% accuracy, which is higher than approximately 25 to 50% diagnostic accuracies of CBD without these biomarkers.
  • Additional limitations of this study include the following. Tauopathies with shorter duration or during asymptomatic stage may not yet show decrease in these biomarkers.
  • the 77G7 MTBR-tau antibody used for sequential immunoprecipitation in the study may also be targeting specific pools of truncated tau, and there may be future technical advancements and analytical method developments that may unveil additional or new populations of tau species in biofluids that reflect qualitative and quantitative aspects of tau pathology in primary tauopathies.
  • [0199] 2. Nakamura, A. et al. High performance plasma amyloid- ⁇ biomarkers for Alzheimer’s disease. Nature 554, 249–254 (2016).
  • [0200] 3. Thijssen, E. H. et al. Diagnostic value of plasma phosphorylated tau181 in Alzheimer’s disease and frontotemporal lobar degeneration. Nat. Med.26, 387–397 (2020).
  • [0201] 4. Janelidze, S. et al. Plasma P-tau181 in Alzheimer’s disease: relationship to other biomarkers, differential diagnosis, neuropathology and longitudinal progression to Alzheimer’s dementia. Nat. Med. 26, 379–386 (2020).
  • Reduced CSF p-Tau181 to Tau ratio is a biomarker for FTLD-TDP. Neurology 81, 1945–1952 (2013). 66 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0226] 29. Wagshal, D. et al. Divergent CSF ⁇ alterations in two common tauopathies: Alzheimer’s disease and progressive supranuclear palsy. J. Neurol. Neurosurg. Psychiatry 86, 244–250 (2015). [0227] 30. Schöll, M. et al. Biomarkers for tau pathology. Mol. Cell. Neurosci.97, 18–33 (2019).

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Abstract

Provided herein are methods and compositions for detecting and measuring MTBR-tau species in CSF, and uses thereof for detecting pathological features and/or clinical symptoms of tauopathies, which may be used to diagnose, stage, or choose treatments appropriate for a given disease stage, or modify a given treatment regimen.

Description

PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web METHODS TO DETECT CSF MTBR-TAU AND USES THEREOF FIELD OF THE TECHNOLOGY [001] The present disclosure relates to detection of microtubule-binding region (MTBR)-tau peptides in cerebral spinal fluid (CSF), and use of tau peptides for detecting pathological features or clinical symptoms of a primary tauopathy, which may be used to diagnose, stage, or choose treatments appropriate for the primary tauopathy. BACKGROUND [002] Accumulation of tau protein in the brain as insoluble aggregates is a hallmark of neurodegenerative diseases classified as tauopathies, such as Alzheimer’s disease. Tau pathology appears to propagate and spread across brain regions by cell-to-cell transmission of certain pathological tau species in a prion-like manner, although the spreading process and nature of these species (e.g., monomeric, oligomeric, and fibril species) are uncertain (Frost et al., 2009; Goedert et al., 2010, 2017; Sanders et al., 2014; Wu et al., 2016; Mirbaha et al., 2018; Lasagna-Reeves et al., 2012). Tau has six different isoforms of the full-length protein, and more than one hundred potential post-translational modification sites, including phosphorylation sites and multiple truncation sites (Meredith et al., 2013; Sato et al., 2018; Barthélemy et al., 2019; Cicognola et al., 2019; Blennow et al., 2020). Thus, identifying specific pathological tau species involved in tau spread is challenging. [003] Mass spectrometry (MS) studies suggest that the microtubule-binding region of tau (MTBR-tau) is enriched in aggregates in Alzheimer’s disease brain (Taniguchi-Watanabe et al., 2016; Roberts et al., 2020). Cryogenic electron microscopy (Cryo-EM) demonstrates that the core structure of tau aggregates contains a sub-segment of MTBR-tau and the particular conformation depends on the tauopathy (Fitzpatrick et al., 2017; Falcon et al., 2018, 2019; Zhang et al., 2020). These findings strongly suggest that MTBR-tau is critical for tau aggregation. However, these studies used postmortem brain tissue. Little is known about the pathophysiology of corresponding extracellular MTBR-tau species in biological samples such as CSF, which may serve as a surrogate biomarker of brain tau aggregates in living humans. [004] CSF is routinely obtained from study participants via lumbar puncture during clinical visits. Previous CSF tau biomarker studies suggested that MTBR-tau is missing in CSF and focused on N- terminal tau and mid-domain tau regions (Meredith et al., 2013; Sato et al., 2018). N-terminus to mid- domain tau species appear to be actively secreted from neurons into the extracellular space after truncation between the mid-domain and the MTBR-domain of tau (Sato et al., 2018). Detection of 1 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web MTBR-tau species were reported (Barthélemy et al., 2016b, a) but have not been characterized in relationship to disease. Recently, a tau species containing a cleavage at residue 368 (tau368) within the repeat region 4 (R4) was identified in CSF (Blennow et al., 2020). It is unclear, however, whether tau368 reflects the overall pool of MTBR-tau species given the variations in regions, truncations and conformational structures not captured by antibodies. SUMMARY OF THE INVENTION [005] Provided herein is a method of detecting tau in a cerebrospinal fluid (CSF) sample, which may comprise (a) providing a CSF sample; and (b) detecting and measuring the amount of one or more of Microtubule Binding Region (MTBR)-tau275 peptides and MTBR-tau282 peptides. [006] Provided herein is a method of detecting 4R tau aggregates in a subject, which may comprise one or more of (a) purifying endogenously cleaved fragments of tau from a CSF sample from the subject, without contacting the endogenously cleaved fragments of tau in vitro with a protease; (b) contacting the purified endogenously cleaved fragments of tau with an endopeptidase to obtain one or more of proteolytic MTBR-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and proteolytic MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides is indicative of 4R tau aggregates in the subject. A solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptides may be desalted before step (c), optionally wherein desalting is by solid phase extraction. The purifying in step (a) may comprise contacting the CSF sample with one or more anti-tau epitope binding agents. The epitope binding agent may be an anti-tau antibody or antigen-binding fragment thereof. The purifying in step (a) may comprise immunoprecipitation, such as immunoprecipitation of N-terminal to mid-domain tau to deplete N-terminal to mid-domain tau from the CSF sample. The one or more anti-tau epitope binding agents may specifically bind to an epitope within amino acids 1-243 of the sequence set forth in SEQ ID NO:1. The epitope binding agent may be an anti- tau antibody or antigen fragment thereof, optionally selected from anti-tau antibody Tau1, HJ8.5, or HJ8.7. The purifying in step (a) may comprise immunoprecipitation of one or more MTBR-tau species from the biological sample to capture the one or more MTBR-tau species from the biological sample. The one or more captured MTBR-tau species may be a MTBR-tau275 peptide, MTBR-tau282 peptide, or combination thereof. The immunoprecipitation of one or more MTBR-tau species may comprise contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to an 2 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web epitope within amino acids 244-368 relative to SEQ ID NO:1, or within amino acids 316-335 of the sequence set forth in SEQ ID NO:1. The epitope binding agent may be anti-tau antibody 77G7 or an antigen-binding fragment thereof. The endopeptidase may be trypsin. The CSF sample may comprise an internal standard. The method may further comprise one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and an ApoE isoform, in the CSF sample. The method may quantify the amount of mid-domain tau, optionally wherein the mid-domain tau comprises tau212-221 relative to SEQ ID NO:1 (t-tau). The one or more of MTBR-tau275 peptides and MTBR-tau282 peptides may be normalized to the amount of t-tau, and the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau is indicative of 4R tau aggregates in the subject. A decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value may be indicative of 4R tau aggregates in the subject. [007] Provided herein is a method of detecting 4R tau aggregates in a subject, which may comprise (a) performing affinity depletion on a CSF sample from the subject by contacting the biological sample with one or more affinity depletion agents comprising one or more epitope binding agents that each binds to one of N-terminal tau, mid-domain tau, or C-terminal tau, but not to an antigen within MTBR-tau, wherein the CSF sample comprises endogenously cleaved fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, mid-domain tau, and/or C-terminal tau, and wherein the enriched sample is enriched for endogenously cleaved fragments of tau comprising one or more of endogenous MTBR-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and endogenous MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; (b) performing immunoprecipitation on the enriched sample by contacting the enriched sample with one or more immunoprecipitation agents comprising one or more epitope binding agents that binds to MTBR-tau to capture the one or more of endogenous MTBR-tau275 pep-tides and endogenous MTBR-tau282 peptides, to obtain a purified sample; (c) contacting the one or more of endogenous MTBR-tau275 peptides and endogenous MTBR-tau282 peptides in the purified sample with an endopeptidase to obtain a sample comprising one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides; and (d) detecting and measuring the one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides by performing LC/MS or an immunoassay, wherein the amount of the one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides is indicative of 4R tau aggregates in the subject. The one or more of proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides may be desalted before step (d), optionally wherein desalting is by solid phase extraction. The one or more affinity depletion agents may comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino 3 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web acids 1-243 of the sequence set forth in SEQ ID NO:1. The epitope binding agent may be an anti-tau antibody or antigen fragment thereof, optionally selected from anti-tau antibody Tau1, HJ8.5, or HJ8.7. The one or more immunoprecipitation agents may comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244-368 of the sequence set forth in SEQ ID NO:1, or within amino acids 316-335 of the sequence set forth in SEQ ID NO:1. The one or more immunoprecipitation agents may comprise the anti-tau antibody 77G7 or an antigen-binding fragment thereof. The endopeptidase may be trypsin. The method may further comprise one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and an ApoE isoform, in the CSF sample. The method may quantify the amount of mid-domain tau, optionally wherein the mid-domain tau comprises tau212-221 relative to SEQ ID NO: 1 (t-tau). The one or more of MTBR-tau275 peptides and MTBR-tau282 peptides may be normalized to the amount of t-tau, and wherein the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t- tau is indicative of 4R tau aggregates in the subject. A decrease in the amount of one or more of MTBR- tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value may be indicative of 4R tau aggregates in the subject. [008] Provided herein is a method for detecting a primary tauopathy in a subject, which may comprise providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR- tau282/t-tau from a CSF sample from a subject. One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. The primary tauopathy may be selected from the group consisting of frontotemporal lobar degeneration (FTLD) (e.g., FTLD-MAPT) and corticobasal degeneration (CBD). A decrease in the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value may be indicative of one or more of FTLD (e.g., FTLD-MAPT) and CBD. The FTLD-MAPT may be P301L, R406W, or S305I. The FTLD-MAPT may be symptomatic or asymptomatic. [009] Provided herein is a method for detecting primary tauopathy-related deposition in a brain of a subject, which may comprise providing the amount of one or more of MTBR-tau275, MTBR-tau275/t- tau, MTBR-tau282, and MTBR-tau282/t-tau from a CSF sample from the subject. One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. The primary tauopathy-related deposition may be from FTLD (e.g., FTLD-MAPT) or CBD. A decrease in the amount of one or more of MTBR-tau275/t-tau and MTBR- tau282/t-tau relative to a threshold value may be indicative of FTLD (e.g., FTLD-MAPT) or CBD). The 4 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web FTLD-MAPT may be P301L, R406W, or S305I. The FTLD-MAPT may be symptomatic or asymptomatic. [010] Provided herein is a method of diagnosing FTLD (e.g., FTLD-MAPT) in a subject, which may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR-tau282/t-tau from a CSF sample from the subject; and (b) diagnosing FTLD-MAPT when the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau detected is decreased relative to a threshold value. The FTLD-MAPT may be P301L, R406W, or S305I. The FTLD-MAPT may be symptomatic or asymptomatic. [011] Provided herein is a method of measuring FTLD (e.g., FTLD-MAPT) progression in a subject, which may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR-tau282/t-tau from a CSF sample from the subject; and (b) calculating a difference between amounts of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample and the first sample, wherein a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample as compared to the first sample indicates progression of the subject’s FTLD (e.g., FTLD-MAPT). One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. The FTLD-MAPT may be P301L, R406W, or S305I. The FTLD-MAPT may be symptomatic or asymptomatic. [012] Provided herein is a method of diagnosing CBD in a subject, which may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR-tau282/t- tau from a CSF sample from the subject; and (b) diagnosing CBD when the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau detected is decreased relative to a threshold value. One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. [013] Provided herein is a method of measuring CBD progression in a subject, which may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR-tau282, and MTBR- tau282/t-tau from a CSF sample from the subject; and (b) calculating a difference between amounts of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample and the first sample, wherein a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample as compared to the first sample indicates progression of the subject’s CBD. One or more of MTBR-tau275 and MTBR-tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. 5 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [014] Provided herein is a method of treating a primary tauopathy in a subject in need thereof, which may comprise (a) providing the amount of one or more of MTBR-tau275, MTBR-tau275/t-tau, MTBR- tau282, and MTBR-tau282/t-tau from a CSF sample from the subject; and (b) administering to the subject a treatment that alters tau pathology if the amount of the one or more of MTBR-tau275/t-tau and MTBR- tau282/t-tau is decreased relative to the thresh-old value. One or more of MTBR-tau275 and MTBR- tau282 may be detected according to the method of detecting tau or 4R tau aggregates described hereinabove. [015] Provided herein is a method of treating a primary tauopathy in a subject in need thereof, comprising administering to the subject a treatment that alters tau pathology, wherein the subject has been identified as having a decreased amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau, as measured according to a method as described hereinabove, relative to the threshold value. The treatment may alter or stabilize the amount of the detected one or more of MTBR-tau275 peptides and MTBR-tau282 peptides. The treatment may be selected from the group consisting of lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, Bepranemab, Dasatinib + Quercetin, E2814, Epothilone D, Gosuranemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, Semorinemab, TPI 287, Tideglusib, Tila-vonemab, Zagotenemab, an anti-tau monoclonal antibody, an anti-tau anti-sense oligonucleotide, an anti-tau small interfering RNA, an tau production inhibitor, and a tau active vaccine. The treatment may be selected from the group consisting of anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, gamma-secretase inhibitors, beta-secretase inhibitors, a kinase inhibitor, a phosphatase activator, a vaccine, and a tau protein aggregation inhibitor. The kinase inhibitor may be an inhibitor of a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. The phosphatase activator may increase the activity of protein phosphatase 2A. The vaccine may be CAD106 or AF20513. The anti-Aβ antibody may be aducanumab or another anti-amyloid anti-body that removes plaques. BRIEF DESCRIPTION OF THE DRAWINGS [016] FIG 1 shows demographics and brain MTBR-tau measures of participants in primary tauopathy cohort. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD - MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16), HS: 6 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web hippocampal sclerosis, MND: motor neuron disease, LBD: Lewy body dementia, SFG: superior frontal gyrus. [017] FIG.2 shows demographics and CSF MTBR-tau measures of participants in pathologically- confirmed primary tauopathy cohort. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16), HS: hippocampal sclerosis, MND: motor neuron disease, LBD: Lewy body dementia. CDR plus NACC FTLD-SB: Clinical Dementia Rating plus National Alzheimer's Coordinating Center FTLD sum of boxes. ALS: amyotrophic lateral sclerosis, MCI: mild cognitive impairment, bvFTD: behavioral variant of frontotemporal dementia, CBS: corticobasal syndrome, DLB: dementia with Lewy bodies, nfvPPA: nonfluent variant primary progressive aphasia, PPS: pallidopyramidal syndrome, svPPA: semantic variant of primary progressive aphasia, PAGF: pure akinesia with gait freezing, PSP-RS: Progressive supranuclear palsy with Richardson's Syndrome, TES: traumatic encephalopathy syndrome, EOAD: early-onset Alzheimer's disease, lvPPA: logopenic variant primary progressive aphasia, PCA: posterior cortical atrophy, CN: cognitively normal. Values in square brackets indicate the number of available information within the group if limited. [018] FIG.3 shows diagnostic accuracies of 4R specific CSF MTBR-tau to distinguish CBD from FTLD-tau and control. Abbreviations include CBD: corticobasal degeneration, NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, FTLD-tau: frontotemporal lobar degeneration with TAR DNA-binding protein, PSP: progressive supranuclear palsy, AGD: argyrophilic grain disease, PiD: Pick's disease, AUC: area under the curve, CI: confidence interval. [019] FIGS.4A-4C show 4R specific insoluble brain MTBR-tau is enriched in CBD, FTLD-MAPT, AD and PSP. FIG.4A depicts a schematic of the quantified peptides of total-tau (t-tau, 181-190) and 4R isoform specific microtubule binding region of tau (MTBR-tau) in R2 region (grey bars, MTBR-tau275 and MTBR-tau282). The relative abundance of each MTBR-tau was normalized to the t-tau peptide. MTBR-tau275/t-tau (FIG.4B) and MTBR-tau282/t-tau (FIG.4C) were measured in the tauopathy patient’s insoluble brain fractions from SFG (circle, n=54) and insula (triangle, n=8). Both MTBR-tau species were most enriched in CBD (n=12) and FTLD-MAPT (n=8). PSP (n=16) and AD (n=7) had moderate enrichment. AGD (n=1), PiD (n=3) and FTLD-TDP (n=12) did not change in MTBR-tau275 or MTBR-tau282 compared to NC (n=3). The red (n=9) and blue (n=1) filled circles indicate AD and PSP co-pathology, respectively. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar 7 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD - MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16), SFG: superior frontal gyrus. Significance in statistical test: ****P<0.0001, **p<0.01, *p<0.05. The box plots show the minimum, 25 percentile, median, 75 percentile, and maximum. Differences in biomarker values were assessed with one-way ANOVAs. A two-sided p<0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%. [020] FIGS.5A-5C show 4R specific CSF MTBR-tau decreases in CBD, FTLD-MAPT, and AD. FIG.5A depicts a schematic of the quantified peptides of total-tau (t-tau, 212-221), truncation, and 4R isoform specific microtubule binding region of tau (MTBR-tau) in R2 region (grey bars, MTBR-tau275 and MTBR-tau282). The relative abundance of each MTBR-tau was normalized to the t-tau peptide. CSF MTBR-tau275/t-tau (FIG.5B) and MTBR-tau282/t-tau (FIG.5C) significantly decreased in CBD (n=18), AD (n=10), and FTLD-MAPT (n=5) compared to NC (n=29), FTLD-TDP (n=21) and other FTLD-tau. FTLD-MAPT P301L (red, n=2), R406W (blue, n=2), and S305I (green, n=1) decreased in MTBR-tau/t-tau measurements in this order. Significance in statistical test: ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD- MAPT: frontotemporal lobar degeneration with MAPT mutations. The box plots show the minimum, 25 percentile, median, 75 percentile, and maximum. Differences in biomarker values were assessed with one-way ANOVAs. A two-sided p<0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%. [021] FIGS.6A-6F show CSF soluble MTBR-tau correlates with brain insoluble MTBR-tau aggregates. MTBR-tau275/t-tau (FIG.6A) and MTBR-tau282/t-tau (FIG.6B) from paired CSF and brain inversely correlated in tauopathies, FTLD-TDP and control (N=54, Spearman r=-0.27, p=0.049, -0.45, p=0.0006, respectively). MTBR-tau275/t-tau (FIG.6C) and MTBR-tau282/t-tau (FIG.6D) from paired CSF and brain had higher correlations (Spearman r= -0.61, p=0.0004, r=-0.75, p<0.0001, respectively) in 4R tauopathies (CBD, PSP and AGD, N=29). MTBR-tau275/t-tau (FIG.6E) and MTBR-tau282/t-tau (FIG.6F) from paired CSF and brain correlated in CBD (N=12, Spearman r= -0.25, p=0.43, r=-0.31, p=0.33, respectively). Gray shadow represents 95% confidential intervals for linear regression. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR 8 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations. [022] FIG.7 shows 4R CSF MTBR-tau assay is reproducible and stable in repeated lumbar punctures. CSF MTBR-tau275/t-tau was measured in 25 participants (#01-#25) who had 3 to 5 repeated lumbar punctures within 4 months. FTLD-MAPT P301L (red, n = 2, #02, 03), symptomatic FTLD-MAPT R406W (blue, n = 1, #05), and pathologically-confirmed CBD (green, n = 2, #10 and #11) had decreased CSF MTBR-tau275/t-tau. Data are presented as mean values + /− SD. CBS: corticobasal syndrome, PSP- RS: progressive supranuclear palsy-Richardson’s syndrome, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations. [023] FIG.8 shows CSF MTBR-tau275/t-tau decreases in clinically diagnosed CBS-PSP continuum and FTLD-MAPT. CSF MTBR-tau275/t-tau decreased in clinically diagnosed CBS-PSP continuum (n = 7), and genetically confirmed FTLD-MAPT (red: n = 3, P301L, blue: n = 5, R406W) compared to NC (n = 88). Differences in biomarker values were assessed with one-way ANOVAs. A two-sided p < 0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%. *P < 0.05. The box plots show the minimum, 25 percentile, median, 75 percentile, and maximum. NC: normal control, bvFTD: behavioral variant frontotemporal dementia (n = 28), PSP-RS: progressive supranuclear palsy-Richardson’s syndrome (n = 16), CBS: corticobasal syndrome (n = 15), AD: Alzheimer’s disease (n = 80), FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations (n = 8). [024] FIG.9 shows demographic and CSF MTBR-tau measures of participants in repeated lumbar puncture cohort. [025] FIG.10 shows CSF MTBR-tau275/t-tau biomarker positivity based on clinical syndromes in pathologically-confirmed cohort [026] FIG.11 shows CSF MTBR-tau282/t-tau biomarker positivity based on clinical syndromes in pathologically-confirmed cohort. [027] FIGS.12A-12C show that CSF MTBR-tau and t-tau concentrations do not reflect CBD and FTLD-MAPT pathologies. CSF MTBR-tau275 (FIG.12A) and MTBR-tau282 (FIG.12B) concentrations do not change with tauopathies (n=112 total including 29 NC). FTLD-MAPT includes P301L (red, n=2), R406W (blue, n=2) and S305I (green, n=1). CSF t-tau (FIG.12C) increased in autopsy 9 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web confirmed AD (n=10) compared to NC (n=29) and PSP (n=22, p<0.05). Significance in statistical test: *P<0.05. The box plots show the minimum, 25 percentile, median, 75 percentile, and maximum. Differences in biomarker values were assessed with one-way ANOVAs. A two-sided p<0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick’s disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation. [028] FIGS.13A-13B show CSF MTBR-tau/tau correlates with disease duration in CBD. CSF MTBR- tau275/t-tau (FIG.13A) and MTBR-tau282/t-tau (FIG.13B) negatively correlates with duration of the disease in CBD (i.e., interval between age of onset and CSF collection, n=18, Spearman r=-0.37, p=0.13 and r=-0.39, p=0.11, respectively). Gray shadow represents 95% confidential intervals for linear regression. CBD: corticobasal degeneration. [029] FIGS.14A-14C show CSF pT217/T217 identifies AD from other tauopathies. FIG.14A shows CSF pT217/T217 increased in AD (n=10) compared to NC (n=29), FTLD-tau (i.e., PiD (n=5), PSP (n=22), CBD (n=18), and FTLD-MAPT (n=5)), and FTLD-TDP (n=21) (p<0.0001). PSP, CBD, and FTLD-TDP with AD co-pathology (filled purple, n=9) had higher CSFpT217/T217. FTLD-MAPT includes P301L (red, n=2), R406W (blue, n=2) and S305I (green, n=1). CSF pT217/T217 decreased in FTLD-TDP (n=21) compared to PiD (n=5), PSP (n=22), CBD (n=18) and FTLD-MAPT (n=5, p<0.05- 0.01). CSF t-tau (FIG.14B) and CSF pT217/T217 (FIG.14C) can distinguish AD (n=10) from FTLD- MAPT (n=5) with AUC=0.794, 0.987, respectively. Differences in biomarker values were assessed with one-way ANOVAs. A two-sided p<0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%. Significance in statistical test: ****P<0.0001, **P<0.01, *P<0.05. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick’s disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation. AUC: area under the curve. [030] FIGS.15A-15E show CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau do not correlate with amyloid pathology in primary tauopathies. (FIG.15A) CSF pT217/T217 positively correlates with AD Thal Phase (Spearman r=0.52, p<0.0001). CSF MTBR-tau275/t-tau (FIGS.15B and FIG. 15D) and CSF MTBR-tau282/t-tau (FIG.15C and FIG.15E) do not correlate with AD Thal Phase measured in 10 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web autopsied brains of all tauopathy cohort (FIG.15B and FIG.15C, n=79) or CBD only (FIG.15D and FIG.15E, n=17). [031] FIGS.16A-16L ROC curves for 4R specific CSF MTBR-tau to distinguish CBD from control and other tauopathies. CSF MTBR-tau275/t-tau (FIG.16A, FIG.16C, FIG.16E, FIG.16G, FIG. 16I, and FIG.16K) and MTBR-tau282/t-tau (FIG.16B, FIG.16D, FIG.16F, FIG. 16H, FIG.16J, and FIG.16L) can distinguish CBD from NC (FIG.16A and FIG.16B), FTLD-TDP (FIG.16C and FIG.16D), FTLD-tau (i.e. PSP, PiD and AGD, FIG.16E and FIG.16F), PiD (FIG.16G and FIG.16H), and PSP (FIG.16I and FIG.16J). AUC improves when AD co-pathology cases are excluded from CBD and PSP (FIG.16K and FIG.16L). Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick’s disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer’s disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation. AUC: area under the curve, ROC: receiver operating characteristic. [032] FIGS.17A-17E show retrospective clinical syndromes and CSF MTBR-tau markers in pathologically-confirmed cohort. CSF MTBR-tau275/t-tau (FIG.17A and FIG.17C) and MTBR- tau282/t-tau (FIG.16B and FIG.16D) by pathological diagnoses (FIG.16A and FIG.16B) and clinical syndromes (FIG.16C and FIG.16D) are shown (total n=112). The box plots show the minimum, 25 percentile, median, 75 percentile, and maximum. Dotted lines show cutoff of 0.00563 and 0.01220, respectively for CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau. FIG.16E depicts a schematic of relationship between clinical syndrome and pathological diagnoses in FTLD. Red and black lines show CSF MTBR-tau biomarkers positive and negative, respectively. Biomarker positivity was determined by the median of each disease group. Abbreviations include NC: normal control, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain disease, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I), bvFTD: behavioral variant of frontotemporal dementia, CBS: corticobasal syndrome, nfvPPA: nonfluent variant primary progressive aphasia, PAGF: pure akinesia with gait freezing, PSP-RS: Progressive supranuclear palsy with Richardson's Syndrome, CN: cognitively normal. DETAILED DESCRIPTION [033] Discussed below are components of aspects of methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, 11 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web etc., of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these components may not be explicitly disclosed, each is specifically contemplated and described herein. [034] Other aspects and iterations of the invention are described more thoroughly below. 1. Definitions [035] So that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below. [036] The term "a" or "an" entity refers to one or more of that entity; for example, a "polypeptide subunit" is understood to represent one or more polypeptide subunits. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. [037] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). [038] 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%, 2%,1%, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities. [039] By "specifically binds," it is meant that a binding molecule, e.g., an antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain, and that the binding entails some recognition between the antigen-binding domain and the epitope. According to this definition, a binding 12 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web molecule is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. Thus, the term “specifically binds,” as used herein with regards to epitope binding agents, means that an epitope binding agent does not cross react to a significant extent with other epitopes on the protein of interest (e.g., Tau), or on other proteins in general. [040] The term “antibody”, as used herein, is used in the broadest sense and encompasses various antibody and antibody-like structures, including but not limited to full-length monoclonal, polyclonal, and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as heavy chain antibodies and antibody fragments provided they exhibit the desired antigen-binding activity. The domain(s) of an antibody that is involved in binding an antigen is referred to as a “variable region” or “variable domain,” and is described in further detail below. A single variable domain may be sufficient to confer antigen-binding specificity. Preferably, but not necessarily, antibodies useful in the discovery are produced recombinantly. Antibodies may or may not be glycosylated, though glycosylated antibodies may be preferred. An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by methods known in the art. [041] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; single- chain forms of antibodies and higher or-der variants thereof; single-domain antibodies, and multi-specific antibodies formed from antibody fragments. [042] Single-chain forms of antibodies, and their higher order forms, may include, but are not limited to, single-domain antibodies, single chain variant fragments (scFvs), divalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra-scFvs), diabodies, and triabodies and tetrabodies. ScFv’s are comprised of heavy and light chain variable regions connected by a linker. In most instances, but not all, the linker may be a peptide. A linker peptide is preferably from about 5 to 30 amino acids in length, or from about 10 to 25 amino acids in length. Typically, the linker allows for stabilization of the variable domains without interfering with the proper folding and creation of an active binding site. In preferred embodiments, a linker peptide is rich in glycine, as well as serine or threonine. ScFvs can be used to facilitate phage display or can be used for flow cytometry, immunohistochemistry, or as targeting domains. Methods of making and using scFvs are known in the art. ScFvs may also be conjugated to a human constant domain (e.g., a heavy constant domain is derived from an IgG do-main, such as lgG1, lgG2, lgG3, or lgG4, or a heavy chain constant domain derived from IgA, IgM, or IgE). Diabodies, 13 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web triabodies, and tetrabodies and higher order variants are typically created by varying the length of the linker peptide from zero to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be generated using self-assembling units linked to the variable domain. [043] A “single-domain antibody” refers to an antibody fragment consisting of a single, monomeric variable antibody domain. [044] Multi-specific antibodies include bi-specific antibodies, tri-specific, or anti-bodies of four or more specificities. Multi-specific antibodies may be created by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains can be covalently linked. [045] An antibody of the disclosure may be a Dual-affinity Re-targeting Antibody (DART). The DART format is based on the diabody format that separates cognate variable domains of heavy and light chains of the two antigen-binding specificities on two separate polypeptide chains. Whereas the 2 polypeptide chains associate noncovalently in the diabody format, the DART format provides additional stabilization through a C-terminal disulfide bridge. DARTs can be produced in high quantity and quality and reveal exceptional stability in both formulation buffer and human serum. [046] "Monoclonal antibody" refers to an antibody that is derived from a single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone. "Monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be produced using hybridoma techniques well known in the art, as well as recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies and other technologies readily known in the art. Furthermore, the monoclonal antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art. [047] A “heavy chain antibody” refers to an antibody that consists of two heavy chains. A heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from a cartiliaginous fish. [048] A "humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response in humans following administration but retains similar binding specificity and affinity as the starting non-human antibody. A humanized antibody binds 14 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web to the same or similar epitope as the non-human antibody. The term “humanized antibody” includes an antibody that is composed partially or fully of amino acid sequences derived from a human antibody germline by altering the sequence of an antibody having non-human hypervariable regions (“HVR”). The simplest such alteration may consist simply of substituting the constant region of a human antibody for the murine constant region, thus resulting in a human/murine chimera which may have sufficiently low immunogenicity to be acceptable for pharmaceutical use. Preferably, the variable region of the antibody is also humanized by techniques that are by now well known in the art. For example, the framework regions of a variable region can be substituted by the corresponding human framework regions, while retaining one, several, or all six non-human HVRs. Some framework residues can be substituted with corresponding residues from a non-human VL domain or VH domain (e.g., the non-human antibody from which the HVR residues are derived), e.g., to restore or improve specificity or affinity of the humanized antibody. Substantially human framework regions have at least about 75% homology with a known human framework sequence (i.e., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity). HVRs may also be randomly mutated such that binding activity and affinity for the antigen is maintained or enhanced in the context of fully human germline framework regions or framework regions that are substantially human. As mentioned above, it is sufficient for use in the methods of this discovery to employ an antibody fragment. Further, as used herein, the term "humanized antibody" refers to an antibody comprising a substantially human framework region, at least one HVR from a nonhuman antibody, and in which any constant region present is substantially human. Substantially human constant regions have at least about 90% with a known human constant sequence (i.e., about 90%, about 95%, or about 99% sequence identity). Hence, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences. [049] The term “Aβ” refers to peptides derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Aβ that may have toxic effects: Aβ 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 Aβ may be attributable to any or all of the above noted forms, as well as to others not described specifically. For example, two such Aβ isoforms include Aβ40 and Aβ42; with the Aβ42 isoform being particularly fibrillogenic or insoluble and associated with disease states. The term “Aβ” typically refers to 15 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web a plurality of Aβ species without discrimination among individual Aβ species. Specific Aβ species are identified by the size of the peptide, e.g., Aβ42, Aβ40, Aβ38 etc. [050] As used herein, the term “Aβ42/Aβ40 value” or “Aβ42/Aβ40 ratio” means the ratio of the amount of Aβ42 in a sample obtained from a subject compared to the amount of Aβ40 in the same sample. [051] As used herein, 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. [052] As used herein, the term “control population,” “normal population” or a sample from a “healthy” subject refers to a subject, or group of subjects, who are clinically determined to not have a tauopathy (including but not limited to Alzheimer’s disease, FTLD (e.g., FTLD-MAPT), and CBD), based on qualitative or quantitative test results. [053] The term “isoform”, as used herein, refers to any of several different forms of the same protein variants, arising due to 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. [054] The term “tau” refers to a plurality of isoforms encoded by the gene MAPT (or homolog thereof), as well as species thereof that are C-terminally truncated in vivo, N-terminally truncated in vivo, post- translationally modified in vivo, or any combination thereof. As used herein, the terms “tau” and “tau protein” and “tau species” may be used interchangeably. In many animals, including but not limited to humans, non-human primates, rodents, fish, cattle, frogs, goats, and chicken, tau is encoded by the gene MAPT. In animals where the gene is not identified as MAPT, a homolog may be identified by methods well known in the art. [055] In humans, there are six 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), one insert (1N), or no inserts (0N). 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: R1, R2, 16 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web R3, and R4) or three repeats of the microtubule binding domain (exon 10 excluded: R1, R3, and R4). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau may be phosphorylated, ubiquinated, glycosylated, and glycated. Human tau also may or may not be proteolytically processed in vivo at the C-terminus, at the N-terminus, or at the C-terminus and the N- terminus. Accordingly, the term “human tau” encompasses the 2N3R, 2N4R, 1N3R, 1N4R, 0N3R, and 0N4R isoforms, as well as species thereof that are C-terminally truncated in vivo, N-terminally truncated in vivo, post-translationally modified in vivo, or any combination thereof. Alternative splicing of the gene encoding tau similarly occurs in other animals. [056] The terms “tau-441” or “full length tau” as used herein, refers to the longest human tau isoform (2N4R), which is 441 amino acids in length. The amino acid sequence of tau-441 is provided as: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSET SDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM VSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSG DRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKS KIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCG SLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTD HGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:1). The N-terminus (N term) tau, mid-domain tau, MTBR-tau, and C-terminus (C term) tau are described hereinbelow for this isoform. These regions will vary in a predictable way for other tau isoforms (e.g., 2N3R, 1 NR4, 1 N3R, 0N4R, and 0N3R). Accordingly, when amino acid positions are identified relative to tau-441, a skilled artisan will be able to determine the corresponding amino acid position for the other isoforms. [057] The term “N-terminal tau,” as used herein, refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the N-terminus of tau (e.g., amino acids 1-103 of tau-441). [058] The term “mid-domain tau,” as used herein, refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the mid-domain of tau (e.g., amino acids 104- 243 of tau-441). [059] The term “MTBR tau” or “MTBR-tau” as used herein, refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the microtubule binding region (MTBR) of tau (e.g., amino acids 244-368 of tau-441). 17 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [060] The term “C-terminal tau,” as used herein, refers to a tau peptide, or a plurality of tau proteins, that comprise(s) two or more, or all of the amino acids of the C-terminus of tau (e.g., amino acids 369- 441 of tau-441, etc.). [061] A disease associated with tau deposition in the brain is referred to herein as a “tauopathy.” The term “tau deposition” is inclusive of all forms pathological tau deposits including but not limited to neurofibrillary tangles, neuropil threads, and tau aggregates in dystrophic neurites. Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy (PSP), 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 (CBD), argyrophilic grain disease (AGD), Frontotemporal lobar degeneration (FTLD), Alzheimer’s disease (AD), and frontotemporal dementia (FTD). [062] Tauopathies are classified by the predominance of tau isoforms found in the pathological tau deposits. Those tauopathies with tau deposits predominantly composed of tau with three MTBRs are referred to as “3R-tauopathies.” Pick’s disease is a non-limiting example of a 3R-tauopathy. For clarification, pathological tau deposits of some 3R-tauopathies may be a mix of 3R and 4R tau isoforms with 3R isoforms predominant. Intracellular neurofibrillary tangles (i.e., tau deposits) in brains of subjects with Alzheimer’s disease are generally thought to contain both approximately equal amounts of 3R and 4R isoforms. Those tauopathies with tau deposits predominantly composed of tau with four MTBRs are referred to as “4R-tauopathies.” PSP, CBD, and AGD are non-limiting examples of 4R-tauopathies, as are some forms of FTLD. Notably, pathological tau deposits in brains of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, show a mix of 3R and 4R isoforms. [063] A clinical sign of a tauopathy may be aggregates of tau in the brain, including but not limited to neurofibrillary tangles. Methods for detecting and quantifying tau aggregates in the brain are known in the art (e.g., tau PET using tau-specific ligands such as [18F]THK5317, [18F]THK5351 , [18F]AV1451 , [11C]PBB3, [18F]MK-6240, [18F]RO-948, [18F]P I-2620, [18F]GTP1 , [18F]PM-PBB3, and [18F]JNJ64349311 , [18F]JNJ-067), etc.). [064] 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 18 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 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. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of disease. [065] The phrase “tau therapy” collectively refers to any imaging agent, therapeutic treatment, and/or a prophylactic or preventative measure contemplated for, or used with, subjects at risk of developing a tauopathy, or subjects clinically diagnosed as having a tauopathy. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.). Non-limiting examples of therapeutic agents 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. lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract, etc.), antagonists of the serotonin receptor 6, p38alpha MAPK inhibitors, recombinant granulocyte macrophage colony-stimulating factor, passive immunotherapies, active vaccines (e.g. CAD106, AF20513, etc. ), tau protein aggregation inhibitors (e.g. TRx0237, methylthionimium chloride, etc.), therapies to improve blood sugar control (e.g., insulin, exenatide, liraglutide pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and/or CB2 endocannabinoid receptor partial agonists, b-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT 1A and 1 D receptor agonists, Glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA 19 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof. [066] Accumulation of aggregated tau in brain is a hallmark of neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal lobar degeneration with tau (FTLD-tau) including corticobasal degeneration (CBD). Biomarkers in cerebrospinal fluid (CSF) and blood provide the accurate performance for identifying AD. However, there are no biomarkers to affirmatively identify non-AD tauopathies. Since some FTLD-tau such as CBD brains accumulate tau species from 4-repeat (4R) tau- specific domain in the microtubule-binding region (MTBR), the present disclosure examined if CSF levels of these MTBR-tau species altered in the FTLD-tau CSF as a reflection of accumulating tau aggregates in brain. A method quantifying various tau species in CSF was designed and applied for two discovery studies to analyze CSF from individuals with AD and frontotemporal dementia (FTD) syndrome including genetically-confirmed as well as pathologically-unconfirmed cases. The specific MTBR-tau species level against total tau (t-tau) specifically decreased in CSF from individuals with symptomatic AD and FTD syndrome compared to the cases with asymptomatic AD and healthy control. Notably, pathogenic FTLD-tau mutants (i.e., P301L and R406W) also decreased the specific CSF MTBR- tau level significantly, suggesting that the change may recapitulate tau pathology in FTLD-tau. Finally, the MTBR-tau species in all pathologically- or genetically-confirmed CSF were analyzed as a validation study. The levels of specific MTBR-tau species normalized by t-tau differentiated CBD and FTD with some specific MAPT mutations (P301L and R406W) from other FTLD-tau such as progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), Pick's disease (PiD) and “FTLD unrelated to tau (e.g., FTLD-TDP43)”, suggesting that the specific MTBR-tau species in CSF could serve as the first affirmative biomarker to recapitulate tau pathology in non-AD tauopathies. [067] The present disclosure provides methods for measuring MTBR-tau peptides in a biological sample (e.g., by mass spectrometry or immunoassay). [068] The present disclosure further contemplates in each of the methods described herein, determining the presence/absence of one or more protein in the biological sample and/or measuring the concentration of one or more additional protein in the biological sample. In some embodiments, the one or more protein may be a protein depleted from the biological sample prior to purification of tau. For instance, in certain embodiments, N-terminal tau and/or mid-domain tau species may be identified and/or quantified separately from tau species (e.g., MTBR-tau, C-terminal tau) quantified by the methods disclosed herein. 20 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web In some embodiment, post-translation modifications such as phosphorylation of tau at specific residues can be measure and quantified in addition to the endogenously cleaved fragment of tau. Alternatively, or in addition, Aβ (e.g. Aβ42/Aβ40), ApoE, or any other protein of interest may be identified and/or quantified either by processing a portion of the biological sample in parallel, by depleting the protein of interest from the biological sample prior to utilization in the methods disclosed herein, or by depleting the protein of interest from the biological sample during the sample processing steps disclosed herein. [069] The biological sample, suitable internal standards, and the steps of, purifying tau, optionally depleting one or more proteins, optionally cleaving purified tau with a protease, and detecting and measuring the endogenously cleaved peptide fragment of tau are described in more detail below. 2. Methods of Detecting Fragments of Tau [070] The present disclosure provides methods for detecting tau (e.g., peptide fragments of tau) in a cerebrospinal fluid (CSF) sample. The detection may comprise confirming the presence of tau, such as for measuring the peptide fragment of tau or quantifying the amount of the peptide fragment of tau. The presence, measurement, or quantified amount of a peptide fragment of tau may be indicative of aggregated insoluble tau or tangles (e.g., neurofibrillary tangles) associated with a tauopathy. The detection and measuring may be performed by mass spectrometry or an immunoassay. The biological sample may be obtained from a subject having or suspected of having a tauopathy. [071] A peptide fragment of tau (e.g., fragment of tau) as described herein may be endogenously cleaved (for example, cleaved in cells or tissues of a subject). In some embodiments, a peptide fragment of tau comprises amino acids of a 4R tau splicing isoform of tau. In some embodiments, a peptide fragment of tau comprises an R2 region fragment of tau. In some embodiments, a peptide fragment of tau comprises amino acids of a portion of the R2 region of tau. In some embodiments, a peptide fragment of tau comprises a MTBR-tau275 peptide, as described herein. In some embodiments, a peptide fragment of tau (e.g., an MTBR-tau275 peptide) comprises or consists of amino acids 275-280 of tau-441. In some embodiments, a peptide fragment of tau (e.g., an MTBR-tau275 peptide) comprises or consists of amino acids 275-280 as set forth SEQ ID NO:1. In some embodiments, a peptide fragment of tau comprises or consists of the amino acid sequence VQIINK (SEQ ID NO:2). In some embodiments, a peptide fragment of tau comprises a MTBR-tau282 peptide, as described herein. In some embodiments, a peptide fragment of tau (e.g., an MTBR-tau282 peptide) comprises or consists of amino acids 282-290 of tau-441. In some embodiments, a peptide fragment of tau (e.g., an MTBR-tau282 peptide) comprises or consists of amino 21 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web acids 282-290 as set forth SEQ ID NO:1. In some embodiments, a peptide fragment of tau comprises or consists of the amino acid sequence LDLSNVQSK (SEQ ID NO:3). [072] Detecting tau (e.g., tau peptide fragments) in a CSF sample may comprise providing a CSF sample and purifying the tau peptide from the sample. In some embodiments, the CSF sample comprises endogenously cleaved tau. An endogenously cleaved fragment of tau may not be a tryptic tau peptide. The purification may be performed without first exposing the sample to additional in-vitro proteolytic cleavage. A pre-clearing step (e.g., protein precipitation and/or immunodepleting) before or after purifying the endogenously cleaved fragment of tau may not be performed. An endogenously cleaved fragment of tau may be further cleaved in vitro with a protease before or after purification. In some embodiments, the protease is trypsin. [073] A sample as described herein (e.g., a CSF sample comprising endogenously cleaved fragments of tau) may be subject to at least one step or two steps of immunodepletion and/or immunoprecipitation. Each round of immunodepletion or immunoprecipitation may be performed using one or more anti-tau or anti-MTBR-tau antibodies described herein, or a combination thereof. After each immunodepletion or immunoprecipitation step, the purified tau peptides from one or more of the immunoprecipitated sample or the immunodepleted sample may be further cleaved with one or more proteases described herein. [074] Liquid chromatography–mass spectrometry (LC/MS) or an immunoassay with a sample comprising a purified peptide fragment of tau (e.g., an endogenously cleaved fragment of tau) may be used to detect and measure the concentration (relative or absolute) of at least one fragment of tau. One or more fragments of tau may be used to detect and measure the amount of tau, such as the amount of insoluble tau aggregates, present in the biological sample. The immunoassay may comprise contacting a sample comprising a purified peptide fragment of tau with an anti-MTBR-tau antibody disclosed herein. 2.1. Detecting tau in a CSF sample [075] As contemplated herein, detecting tau (e.g., a tau peptide fragment) in a CSF sample may comprise one or more of confirming the presence of a tau peptide, measuring a tau peptide, and/or quantifying a tau peptide (e.g., an amount of a tau peptide) in a CSF sample. In some embodiments, a method as described herein comprises detecting and measuring the amount of proteolytic MTBR-tau275 peptides, proteolytic MTBR-tau282 peptides, or both. [076] In one aspect, provided herein is a method of detecting a tau peptide in a CSF sample. The method comprising the steps of: (a) providing a CSF sample (e.g., a CSF sample comprising 22 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web endogenously cleaved tau fragments); (b) detecting and measuring the amount of one or more of MTBR- tau peptides (e.g., MTBR-tau275 peptides and MTBR-tau282 peptides). The one or more of MTBR-tau peptides may be detected by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay of the sample to detect and/or measure the amount of the one or more of MTBR-tau peptides. [077] In one example, a method of the present disclosure further comprises purifying the one or more of MTBR-tau peptides from the CSF sample, wherein the one or more of MTBR-tau peptides is optionally further cleaved in vitro with one or more proteases before or after purification. In some embodiments, the one or more of MTBR-tau peptides are not cleaved in vitro (e.g., not endogenously) before purification. [078] In another example, a method of the present disclosure further comprises decreasing in the CSF sample by affinity depletion or immunoprecipitation of at least one peptide of tau, wherein the at least one peptide of tau that is affinity depleted or immunoprecipitated is not the one or more of MTBR-tau peptides for detection and/or measurement (e.g., not MTBR-tau275 or MTBR-tau282). In some embodiments, the affinity depletion or immunoprecipitation is of N-terminal tau, mid-domain tau, and optionally, C-terminal tau, or a combination thereof. While not required, in each of the methods as described herein, removing additional proteins from the CSF sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant can be performed before or after purification of the of one or more of MTBR-tau peptides. [079] In another example, a method of the present disclosure further enriching one or more of MTBR- tau peptides comprising purifying one or more of the MTBR-tau peptides from the affinity depleted sample. The enriched one or more of MTBR-tau peptides may be enriched for MTBR-tau275 peptide and/or MTBR-tau282 peptide. The MTBR-tau275 peptide and/or MTBR-tau282 peptide may be detected and/or measured using an immunoassay, which may comprise the use of anti-tau antibody 77G7, or an antigen-binding fragment thereof. The sample comprising enriched MTBR-tau275 peptide and/or MTBR- tau282 peptide may be proteolytically cleaved with a protease. The resultant cleavage may be desalted (e.g., by solid phase extraction) to obtain a sample comprising the one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and/or MTBR-tau282 peptide). [080] In some embodiments, the method further comprises desalting (e.g., by solid phase extraction) to obtain a sample comprising proteolytic peptides of tau (e.g., endogenously cleaved tau) prior to purification. In some embodiments, the method comprises detecting and/or measuring the amount of 23 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web MTBR-tau275 peptides. In some embodiments, the method comprises detecting and/or measuring the amount of MTBR-tau282 peptides. [081] In another aspect, provided herein is a method of detecting 4R tau aggregates in a subject. The method comprising one or more of the following steps: (a) purifying endogenously cleaved fragments of tau from a CSF sample from the subject, without contacting the endogenously cleaved fragments of tau in-vitro with a protease; (b) contacting the purified endogenously cleaved fragments of tau with an endopeptidase to obtain one or more of proteolytic MTBR-tau275 peptides comprising amino acids 275- 280 relative to SEQ ID NO:1 and proteolytic MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR- tau282 peptides is indicative of 4R tau aggregates in the subject. In some embodiments, the method further comprises desalting a solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR- tau282 peptides is before step (c). In some embodiments, the desalting is by solid phase extraction. In some embodiments, the purifying in step (a) comprises contacting the CSF sample with one or more anti- tau epitope binding agents. [082] In some embodiments, the purifying in step (a) comprises immunoprecipitation. In some embodiments, the immunoprecipitation of the purification step (a) is for immunoprecipitation of N- terminal to mid-domain tau (e.g., to deplete N-terminal to mid-domain tau from the CSF sample). In some embodiments, the immunoprecipitation of N-terminal to mid-domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid- domain tau. In some embodiments, the immunoprecipitation of the purification step (a) is for immunoprecipitation of N-terminal tau (e.g., to deplete N-terminal tau from the CSF sample). In some embodiments, the immunoprecipitation of N-terminal tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal tau. In some embodiments, the immunoprecipitation of the purification step (a) is for immunoprecipitation of mid-domain tau (e.g., to deplete mid-domain tau from the CSF sample). In some embodiments, the immunoprecipitation of mid- domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to mid-domain tau. In some embodiments, the immunoprecipitation of purification step (a) is for immunoprecipitation of one or more MTBR-tau species from the CSF sample (e.g., to capture the one or more MTBR-tau species from the biological sample). In some embodiments, the 24 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web immunoprecipitation of one or more MTBR-tau species comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species. [083] In another aspect, provided herein is a method of detecting 4R tau aggregates in a subject, the method comprising one or more of the following steps: (a) performing affinity depletion on a cerebrospinal fluid (CSF sample from the subject by contacting the biological sample with one or more affinity depletion agents comprising one or more epitope binding agents that each binds to one of N- terminal tau, mid-domain tau, or C-terminal tau, but not to an antigen within MTBR-tau, wherein the CSF sample comprises endogenously cleaved fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, mid-domain tau, and/or C-terminal tau, and wherein the enriched sample is enriched for endogenously cleaved fragments of tau comprising one or more of endogenous MTBR-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and endogenous MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; (b) performing immunoprecipitation on the enriched sample by contacting the enriched sample with one or more immunoprecipitation agents comprising one or more epitope binding agents that binds to MTBR-tau to capture the one or more of endogenous MTBR-tau275 peptides and endogenous MTBR-tau282 peptides, to obtain a purified sample; (c) contacting the one or more of endogenous MTBR-tau275 peptides and endogenous MTBR-tau282 peptides in the purified sample with an endopeptidase to obtain a sample comprising one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides; and (d) detecting and measuring the one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides by performing liquid chromatography- mass spectrometry (LC/MS) or an immunoassay, wherein the amount of the one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides is indicative of 4R tau aggregates in the subject. In some embodiments, the sample comprising one or more of proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides is desalted before step. In some embodiments, the desalting is by solid phase extraction. [084] In some embodiments, the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau. In some embodiments, the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to N-terminal tau. In some embodiments, the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to mid-domain tau. In some embodiments, the one or more immunoprecipitation agents comprises one or more anti-tau epitope 25 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web binding agents that specifically bind to one or more MTBR-tau species (e.g., MTBR-tau275 and/or MTBR-tau282). [085] A biological sample comprising endogenously cleaved fragments of tau is contacted with one or more, or all of, HJ8.5, HJ8.7, and Tau1. In one example, the biological sample is contacted with Tau1, HJ8.5, and HJ8.7. A sample depleted of tau peptides using the aforementioned antibodies may be proteolytically cleaved in vitro using one or more proteases disclosed herein, and the resulting in vitro cleaved tau peptides (proteolytic fragments) may be contacted with an antibody that binds to the one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and/or MTBR-tau282 peptide, such as anti-tau. In some embodiments, the protease is trypsin. 2.2.Epitope binding agents [086] In some embodiments of the methods described herein, the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau bind to an epitope within amino acids 1-243 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau bind to an epitope within amino acids 1-243 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau to mid-domain tau bind to an epitope within the amino acid sequence of: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSET SDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM VSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSG DRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRL (SEQ ID NO:4). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid- domain tau is selected from anti-tau antibodies HJ8.5, HJ8.7, or Tau1, or an antigen-binding fragment thereof. [087] In some embodiments of the methods described herein, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within amino acids 1-103 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within amino acids 1-103 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within the amino acid sequence of: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSET SDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTA (SEQ ID NO:5). 26 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [088] In some embodiments of the methods described herein, the one or more anti-tau epitope binding agents that specifically bind to mid-domain tau bind to an epitope within amino acids 104-243 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to mid-domain tau bind to an epitope within amino acids 104-243 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to mid-domain tau bind to an epitope within the amino acid sequence of: EEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQA NATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SPSSAKSRL (SEQ ID NO:6). [089] In some embodiments of the methods described herein, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species specifically bind to an epitope within amino acids 244-368 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope within amino acids 244-368 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope of the amino acid sequence of: QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGN (SEQ ID NO:7). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species specifically bind to an epitope within amino acids 316-335 of tau (e.g., of tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope within amino acids 316-335 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species bind to an epitope of the amino acid sequence of: SKVTSKCGSLGNIHHKPGGG (SEQ ID NO:8). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to the one or more MTBR-tau species is anti-tau antibody 77G7, or an antigen-binding fragment thereof. In some embodiments, the immunoprecipitation of one or more MTBR-tau species is for immunoprecipitation of MTBR-tau275 peptides and/or MTBR-tau282 peptides. In some embodiments, the one or more captured MTBR-tau species comprises MTBR-tau275 peptides and/or MTBR-tau282 peptides. [090] A CSF sample comprising tau (e.g., endogenously cleaved fragments of tau) is contacted with one or more, or all of: anti-tau antibody HJ8.5 or an antigen-binding fragment thereof; anti-tau antibody 27 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web HJ8.7 or an antigen-binding fragment thereof; and/or anti-tau antibody Tau1 or an antigen-binding fragment thereof. In some embodiments, a CSF sample is contacted with anti-tau antibodies Tau1, HJ8.5, and HJ8.7, or antigen-binding fragments thereof. A sample depleted of tau peptides using the aforementioned antibodies may be contacted with an antibody that binds to MTBR-tau275 and MTBR- tau282, such as anti-tau antibody 77G7 or an antigen-binding fragment thereof. 2.3. Biological samples [091] Suitable biological samples include a cerebrospinal fluid (CSF) sample obtained from a subject. In some embodiments, the subject is a human. A human subject may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment. In various embodiments, a human subject may be a healthy subject, a subject at risk of developing a neurodegenerative disease, a subject with signs and/or symptoms of a neurodegenerative disease, or a subject diagnosed with a neurodegenerative disease. In further embodiments, the neurodegenerative disease may be a tauopathy, which may be a primary tauopathy. In specific examples, the tauopathy may be frontotemporal lobar degeneration (FTLD-tau) (e.g., FTLD-MAPT), including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilc grain disease (AGD), globular glial tauopathy (GGT), Chronic Traumatic Encephalopathy (CTE), Pick’s disease (PiD), or Alzheimer’s disease. In specific embodiments, the primary tauopathy is FTLD-tau or CBD. In specific embodiments, the FTLD-tau is FTLD-MAPT. In specific embodiments, the FTLD-MAPT is selected from FTLD- MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, and FTLD-MAPT IVS10+16. In other embodiments, the subject is a laboratory animal. In a further embodiment, the subject is a laboratory animal genetically engineered to express human tau and optionally one or more additional human protein (e.g., human Aβ, human ApoE, etc.). [092] CSF may have been obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple CSF samples contemporaneously collected from the subject may be pooled. Once collected, CSF samples may have been 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.). CSF samples may be used immediately or may be frozen and stored indefinitely. Prior to use in the methods disclosed herein, the CSF sample may also have been modified, if needed or desired, to include protease inhibitors, isotope labeled internal standards, detergent(s) and chaotropic agent(s), and/or to optionally deplete other analytes (e.g., proteins peptides, metabolites). 28 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [093] The size of the sample used can and will vary depending upon the sample type, the health status of the subject from whom the sample was obtained, and the analytes to be analyzed (in addition to tau). CSF samples volumes may be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In a specific example, the size of the sample may be about 0.05 mL to about 1 mL CSF. [094] In some embodiments, a single sample is obtained from a subject. Alternatively, samples may be obtained from a subject over time. As such, more than one sample may be collected from a subject over time. For instance, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more samples may be collected from a subject over time. When more than on sample is collected from a subject over time, samples may be collected every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days. In some embodiments, a sample is collected a month apart, 3 months apart, 6 months apart, 1 year apart, 2 years apart, 5 years apart, 10 years apart, 20 years apart or more. 2.4. Purifying tau [095] Another step of the methods disclosed herein comprises purifying tau (e.g., a peptide fragment of tau), in particular, endogenously cleaved and/or proteolytically cleaved fragments of tau. In some examples, the cleaved fragment of tau is N-terminal-independent, mid-domain-independent, and/or C- terminal-independent. The purified tau may be partially purified or completely purified. [096] In some embodiments, a method of the present disclosure comprises purifying tau peptides (e.g., an endogenously cleaved tau fragment) by affinity purification. Affinity purification refers to methods that enrich for a protein of interest by virtue of its specific binding properties to a molecule. Typically, the molecule is a ligand attached to a solid support, such as a bead, resin, tissue culture plate, etc. (referred to as an immobilized ligand). Immobilization of a ligand to a solid support may also occur after the ligand- protein inter-action occurs. Suitable ligands include antibodies, aptamers, and other epitope-binding agents. Purifying a tau peptide by affinity purification comprises contacting a sample comprising an endogenously cleaved fragment tau with a suitable immobilized ligand, one or more wash steps, and elution of the cleaved fragment tau from the immobilized ligand. [097] In some embodiments, a method of the present disclosure comprises purifying tau peptides (e.g., an endogenously cleaved tau fragment) tau by affinity purification using at least one epitope-binding agent that specifically binds to an epitope within amino acids 225-258 of tau-441, inclusive, or within amino acids 235-258 of tau-441, inclusive, or within amino acids 235-242, inclusive, (or within similarly defined regions for other full-length isoforms). In various embodiments, one, two, three or more epitope- 29 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web binding agents may be used. When two or more epitope-binding agents are used, they may be used sequentially or simultaneously. Non-limiting examples of suitable epitope-binding agents are disclosed herein. In various embodiments, one, two, three or more epitope-binding agents may be used. When two or more epitope-binding agents are used, they may be used sequentially or simultaneously. [098] In some embodiments, a method of the present disclosure comprises purifying tau peptides by solid phase extraction. Purifying by solid phase extraction comprises contacting a sample comprising tau with a solid phase comprising a sorbent that adsorbs tau, one or more wash steps, and elution of tau from the sorbent. Suitable sorbents include reversed-phase sorbents. Suitable re-versed phase sorbents are known in the art and include, but are not limited to alkyl-bonded silicas, aryl-bonded silicas, styrene/divinylbenzene materials, N- vinylpyrrolidone/divinylbenzene materials. In an exemplary embodiment, the reversed phase material is a polymer comprising N-vinylpyrrolidone and divinylbenzene or a polymer comprising styrene and divinylbenzene. In an exemplary embodiment, a sorbent is Oasis HLB (Waters). Prior to contact with the supernatant comprising tau, the sorbent is typically preconditioned per manufacturer’s instructions or as is known in the art (e.g., with a water miscible organic solvent and then the buffer comprising the mobile phase). In addition, the supernatant may be optionally acidified, as some reversed-phase materials retain ionized analytes more strongly than others. The use of volatile components in the mobile phases and for elution is preferred, as they facilitate sample drying. In exemplary embodiments, a wash step may comprise the use of a liquid phase comprising about 0.05% v/v trifluoroacetic acid (TFA) to about 1% v/v TFA, or an equivalent thereof. In some examples, the wash may be with a liquid phase comprising about 0.05% v/v to about 0.5% v/v TFA or about 0.05% v/v to about 0.1% v/v TFA. In some examples, the wash may be with a liquid phase comprising about 0.1% v/v to about 1.0% v/v TFA or about 0.1% v/v to about 0.5% v/v TFA. Bound tau is then eluted with a liquid phase comprising about 20% v/v to about 50% v/v acetonitrile (ACN), or an equivalent thereof. In some examples, tau is may be eluted with a liquid phase comprising about 20% v/v to about 40% v/v ACN, or about 20% v/v to about 30% v/v ACN. In some examples, tau is may be eluted with a liquid phase comprising about 30% v/v to about 50% v/v ACN, or about 30% v/v to about 40% v/v ACN. The eluate may be dried by methods known in the art (e.g., vacuum drying (e.g., speed-vac), lyophilization, evaporation un-der a nitrogen stream, etc.). 2.5. Depleting one or more proteins [099] Methods of the present disclosure may comprise a step wherein one or more protein is depleted from a sample. The term “deplete” means to diminish in quantity or number. Accordingly, a sample 30 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web depleted of a protein may have any amount of the protein that is measurably less than the amount in the original sample, including no amount of the protein. [0100] Protein(s) may be depleted from a sample by a method that specifically targets one or more protein, for example by affinity depletion, solid phase extraction, or other method known in the art. Targeted depletion of a protein, or multiple proteins, may be used in situations where down-stream analysis of that protein is desired (e.g., identification, quantification, analysis of post-translation modifications, etc.). For instance, Aβ peptides may be identified and quantified by methods known in the art following affinity depletion of Aβ with a suitable epitope-binding agent. As another non-limiting example, apolipoprotein E (ApoE) status may be determined by methods known in the art following affinity depletion of ApoE and identification of the ApoE isoform. Targeted depletion may also be used to isolate other proteins for subsequent analysis including, but not limited to, apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, an interleukin, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, NFL, prion protein, pNFH, and DJ-1. Targeted depletion of certain tau peptide fragments is also used herein to enrich for other tau peptides and/or eliminate proteins that cofound the mass spectrometry analysis. For instance, in certain embodiments of the present disclosure, N-terminal tau, mid-domain tau, and/or C-terminal MTBR-tau are depleted from a sample prior to further sample processing for analysis. Downstream analysis of the depleted tau proteins may or may not occur, but both options are contemplated by the methods of the present disclosure. [0101] In some embodiments, targeted depletion may occur by affinity depletion. Affinity depletion refers to methods that deplete a protein of interest from a sample by virtue of its specific binding properties to a molecule. Typically, the molecule is a ligand attached to a solid support, such as a bead, resin, tissue culture plate, etc. (referred to as an immobilized ligand). Immobilization of a ligand to a solid support may also occur after the ligand-protein interaction occurs. Suitable ligands include antibodies, aptamers, and other epitope-binding agents. The molecule may also be a polymer or other material that selectively absorbs a protein of interest. As a non-limiting example, polyhydroxymethylene substituted by fat oxethylized alcohol (e.g., PHM-L LIPOSORB, Sigma Aldrich) may be used to selectively absorb lipoproteins (including ApoE) from serum. Two or more affinity depletion agents may be combined to sequentially or simultaneously deplete multiple proteins. [0102] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using at least one epitope-binding agent that specifically binds to an epitope within amino acids 1 to 243 of tau-441, inclusive (or within a similarly defined region for 0N or 1N 31 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web isoforms). In various embodiments, one, two, three or more epitope-binding agents may be used. When two or more epitope-binding agents are used, they may be used sequentially or simultaneously. [0103] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within the N-terminus of tau (e.g., amino acids 1 to 103 of tau-441, inclusive), and an epitope-binding agent that specifically binds to an epitope within the mid-domain of tau (e.g., amino acids 104 to 243 of tau-441, inclusive). The epitope-binding agents may be used sequentially or simultaneously. [0104] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 of tau-441, inclusive, and an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive (or within similarly defined regions for 0N or 1N isoforms). The epitope-binding agents may be used sequentially or simultaneously. [0105] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 103 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive(or within a similarly defined region for 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope-binding agents may be used sequentially or simultaneously. [0106] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope-binding agents may be used sequentially or simultaneously. [0107] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 103 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); 32 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web and an epitope-binding agent that specifically binds to an epitope of amyloid be-ta. The epitope-binding agents may be used sequentially or simultaneously. [0108] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); and an epitope-binding agent that specifically binds to an epitope of amyloid be-ta. The epitope-binding agents may be used sequentially or simultaneously. [0109] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243 of tau-441, inclusive (or within a similarly defined region for 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope-binding agents may be used sequentially or simultaneously. [0110] In some embodiments, a method of the present disclosure comprises affinity depleting one or more protein from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 260 to 441 of tau-441, inclusive. [0111] In each of the above embodiments, the epitope binding agent may comprise an antibody or an aptamer. In some embodiments, the epitope-binding agent that specifically binds to that specifically binds to an epitope within amino acids 1 to 103 of tau-441, inclusive, is HJ8.5, or is an epitope-binding agent that binds the same epitope as HJ8.5 and/or competitively inhibits HJ8.5. In some embodiments, the epitope-binding agent that specifically binds to that specifically binds to an epitope within amino acids 104 to 221 of tau-441, inclusive, is Tau1, or is an epitope-binding agent that binds the same epitope as Tau1 and/or competitively inhibits Tau1. [0112] In some embodiments, the epitope-binding agent that specifically binds to mid-domain tau is selected from HJ34.8, 77G7, RD3, RD4, UCB1017, PT76, E2815, or 7G6, or is an epitope-binding agent that binds the same epitope as HJ34.8, 77G7, RD3, RD4, UCB1017, PT76, E2815, or 7G6, and/or competitively inhibits HJ34.8, 77G7, RD3, RD4, UCB1017, or PT76 (e.g., as described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, and antibodies E2814 and 7G6 described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, as well as other epitope-binding agents that specifically bind the same epitopes as those antibodies. Methods for identifying epitopes to which an antibody specifically binds, and assays to evaluate competitive inhibition between two antibodies, are known in the art. 33 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0113] Alternatively, protein(s) may be depleted from a sample by a more general method, for example by ultrafiltration or protein precipitation with an acid, an organic solvent or a salt. Generally speaking, these methods are used to reliably reduce high abundance and high molecular weight proteins, which in turn enriches for low molecular weight and/or low abundance proteins and peptides (e.g., tau, Aβ, etc.). [0114] In some embodiments, proteins may be depleted from a sample by precipitation. Briefly, precipitation comprises adding a precipitating agent to a sample and thoroughly mixing, incubating the sample with precipitating agent to precipitate proteins, and separating the precipitated proteins by centrifugation or filtration. The resulting supernatant may then be used in downstream applications. The amount of the reagent needed may be experimentally determined by methods known in the art. Suitable precipitating agents include perchloric acid, trichloroacetic acid, acetonitrile, methanol, and the like. In an exemplary embodiment, proteins are depleted from a sample by acid precipitation. In a further embodiment, proteins are depleted from a sample by acid precipitation using perchloric acid. [0115] As a non-limiting example, proteins may be depleted from a sample by acid precipitation using perchloric acid. As used herein, “perchloric acid” refers to 70% perchloric acid unless otherwise indicated. In some embodiments, perchloric acid is added to a final concentration of about 1% v/v to about 15% v/v. In other embodiments, perchloric acid is added to a final concentration of about 1% v/v to about 10% v/v. In other embodiments, perchloric acid is added to a final concentration of about 1% v/v to about 5% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3% v/v to about 15% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3% v/v to about 10% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3% v/v to about 5% v/v. In other embodiments, perchloric acid is added to a final concentration of 3.5% v/v to about 15% v/v, 3.5% v/v to about 10% v/v, or 3.5% v/v to about 5% v/v. In other embodiments, perchloric acid is added to a final concentration of about 3.5% v/v. Following addition of the perchloric acid, the sample is mixed well (e.g., by a vortex mixer) and held at a cold temperature, typically for about 10 minutes or longer, to facilitate precipitation. For example, samples may be held for about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes. In other example, samples may be held for about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes. In other examples, samples may be held for about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes. In other examples, samples are held for about 30 minutes. The sample is then centrifuged at a cold temperature to pellet the precipitated protein, and the supernatant (i.e., the acid soluble fraction), comprising soluble tau, is transferred to a fresh vessel. As used in the above context, a “cold temperature” refers to a temperature of 10°C or less. For instance, a cold temperature 34 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C. In some embodiments, a narrower temperature range may be preferred, for ex-ample, about 3°C to about 5°C, or even about 4°C. In certain embodiments, a cold temperature may be achieved by placing a sample on ice. [0116] Two or more methods from one or both of the above approaches may be combined to sequentially or simultaneously deplete multiple proteins. For instance, one or more proteins may be selectively depleted (targeted depletion) followed by depletion of high abundance / molecular weight proteins. Alternatively, high abundance / molecular weight proteins may be first depleted followed by targeted depletion of one or more proteins. In still another alternative, high abundance / molecular weight proteins may be first depleted followed by a first round of target-ed depletion of one or more proteins and then a second round of targeted depletion of one or more different protein(s) than targeted in the first round. Other iterations will be readily apparent to a skilled artisan. 3. Protease digestion of purified tau fragments [0117] Another step of the methods disclosed herein optionally comprises digesting purified tau fragments (e.g., endogenously cleaved tau) with a protease (e.g., thereby producing a proteolytic tau peptide). The digestion may be in vitro. Digesting purified tau with a protease comprises contacting a sample comprising purified tau with a protease under conditions suitable to digest tau. When affinity purification is used, digestion may occur after eluting tau from the immobilized ligand or while tau is bound. Suitable proteases include but are not limited to trypsin, Lys-N, Lys-C, Asp-C, Arg-N, and Arg-C. For detecting MTBR-tau275 of MTBR-tau282, the proteases comprise trypsin. The resultant digestion product (e.g., cleavage product) is a composition comprising proteolytic peptides of tau. When the protease is trypsin, the resultant cleavage product comprises tryptic peptides of tau. Following proteolytic cleavage, the resultant digestion product (e.g., cleavage product) may be desalted by solid phase extraction. 3.1. Detection and quantifying fragments of tau [0118] Another step of the method disclosed herein comprises detecting the amount of digested tau peptides from the processed biological sample. All suitable method of detecting an amount of tau protein are contemplated within the scope of the disclosure. Methods of detecting and quantifying tau peptides are described in detail below. 3.1.1. LC-MS 35 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0119] The step of detecting and quantifying comprises performing liquid chromatography - mass spectrometry (LC-MS) with a sample comprising peptides of tau to detect and measure the concentration of at least one peptide of tau. Thus, in practice, the disclosed methods use one or more tau peptide to detect and measure the amount of tau protein present in the biological sample. [0120] The proteolytic peptides of tau that indicate the presence of endogenously cleaved fragments of tau may comprise MTBR-275 or MTBR-282. When using an alternative enzyme for digestion, the resulting proteolytic peptides may differ slightly but can be readily determined by a person of ordinary skill in the art. Without wishing to be bound by theory, it is believed that a variation in the amount of a cleaved fragments of tau peptides between two bio-logical samples of the same type reflects a difference in the cleaved fragments of tau that make up those biological samples. As disclosed herein, the amounts of certain proteolytic peptides of cleaved fragments of tau, as well ratios of certain proteolytic peptides of tau, may provide clinically meaningful information to guide treatment decisions. Thus, methods that allow for detection and quantification of cleaved fragments of tau have utility in the diagnosis and treatment of many neurodegenerative diseases. [0121] Proteolytic peptides of tau may be separated by a liquid chromatography system interfaced with a high-resolution mass spectrometer. Suitable LC-MS systems may comprise a <1.0 mm ID column and use a flow rate less than about 100 µl/min. In preferred embodiments, a nanoflow LC-MS system is used (e.g., about 50-100 µm ID column and a flow rate of < 1 µL / min, preferably about 100-800 nL/min, more preferably about 200-600 nL/min). In an exemplary embodiment, an LC-MS system may comprise a 0.05 mM ID column and use a flow rate of about 400 nL/min. [0122] Tandem mass spectrometry may be used to improve resolution, as is known in the art, or technology may improve to achieve the resolution of tandem mass spectrometry with a single mass analyzer. Suitable types of 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 Fusion™ Tribrid™ Mass Spectrometer, Orbitrap Fusion™ Lumos™ Mass Spectrometer, Orbitrap Tribrid™ Eclipse™ Mass Spectrometer, Q Exactive Mass Spectrometer, each from ThermoFisher Scientific). In an exemplary embodiment, an LC-MS system may comprise a mass spectrometer selected from Orbitrap Fusion™ Tribrid™ Mass Spectrometer, Orbitrap Fusion™ Lumos™ Mass Spectrometer, Orbitrap Tribrid™ Eclipse™ Mass Spectrometer, or a mass spectrometer with similar or improved ion-focusing and ion- transparency at the quadrupole. Suitable mass spectrometry protocols may be developed by optimizing 36 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web the number of ions collected prior to analysis (e.g., AGC setting using an orbitrap) and/or injection time. In an exemplary embodiment, a mass spectrometry protocol outlined in the Examples is used. 3.1.2. Immunoassays [0123] In some embodiments of a method as described herein, tau peptides (e.g., an MTBR-tau peptide) can be measured and quantified by immunoassay. In specific embodiments, fragments of tau are detected and quantified using an ELISA. [0124] Methods for assessing an amount of protein expression using epitope binding agent-based methods are known in the art and all suitable methods for assessing an amount of protein known to one of skill in the art are contemplated within the scope of the present disclosure. [0125] Thus, in some embodiments, the method to assess an amount of tau peptide (e.g., as described herein) is an epitope binding agent-based method. In general, an epitope binding agent-based method of assessing an amount of protein expression comprises contacting a sample comprising a polypeptide with an epitope binding agent specific for the polypeptide under conditions effective to allow for formation of a complex between the epitope binding agent and the polypeptide. Epitope binding agent-based method may occur in solution, or the epitope binding agent or sample may be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtiter plates, test tubes, beads, resins, and other polymers. [0126] An epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. The epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate. The substrate and the epitope binding agent may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the epitope binding agent may be attached directly using the functional groups or indirectly using linkers. [0127] The epitope binding agent may also be attached to the substrate non-covalently. For example, a biotinylated epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, an epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography. Additional methods of attaching epitope binding agents to solid surfaces and methods of synthesizing biomolecules on substrates are well known in the art, i.e., VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No.6,566,495, 37 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web and Rockett and Dix, Xenobiotica 30(2):155-177, both of which are hereby incorporated by reference in their entirety). [0128] Contacting the sample with an epitope binding agent under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough for the epitope binding agent to bind to any antigen present. After this time, the complex will be washed and the complex may be detected by any method well known in the art. Methods of detecting the epitope binding agent- polypeptide complex are generally based on the detection of a label or marker. The term “label”, as used herein, refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase). Methods of detecting an epitope binding agent- polypeptide complex based on the detection of a label or marker are well known in the art. [0129] In some embodiments, an epitope binding agent-based method is an immunoassay. Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immunoassays and non-competitive immunoassays. In a competitive immunoassay, an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away and the bound analyte is measured. In a noncompetitive immunoassay, the antibody is labeled, not the analyte. Non-competitive immunoassays may use one antibody (e.g., the capture antibody is labeled) or more than one antibody (e.g., at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above. [0130] In an embodiment, the epitope binding agent method is an immunoassay. In another embodiment, the epitope binding agent method is selected from the group consisting of an enzyme linked immunoassay (ELISA), a fluorescence based assay, a dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), a radiometric assay, a multiplex immunoassay, and a cytometric bead assay (CBA). In some embodiments, the epitope binding agent-based method is an enzyme linked immunoassay (ELISA). In other embodiments, the epitope binding agent-based method is a radioimmunoassay. In still other embodiments, the epitope binding agent-based method is an immunoblot or Western blot. In alternative embodiments, the epitope binding agent-based method is an array. In another embodiment, the epitope 38 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web binding agent-based method is flow cytometry. In different embodiments, the epitope binding agent-based method is immunohistochemistry (IHC). IHC uses an antibody to detect and quantify antigens in intact tissue samples. The tissue samples may be fresh-frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC. Methods of preparing tissue block for study by IHC, as well as methods of performing IHC are well known in the art. 4. Uses of MTBR-tau peptide measurements [0131] The present disclosure also encompasses the use of measurements of tau peptides (e.g., endogenously cleaved fragments of tau), in CSF as biomarkers of pathological features and/or clinical symptoms of tauopathies to diagnose, stage, choose treatments appropriate for a given disease stage, and modify a given treatment regimen (e.g., change a dose, switch to a different drug or treatment modality, etc.). The pathological feature may be an aspect of tau pathology (e.g., amount of tau deposition, presence / absence of a post-translational modification, amount of a post-translation modification, etc.). Alternatively, or in addition to tau deposition, a pathological feature may be tau-independent. For instance, amyloid beta (Aβ) deposition in the brain or in arteries of the brain when the tauopathy is Alzheimer’s disease. The clinical symptom may be dementia, as measured by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.), or any other clinical symptom associated with the tauopathy. [0132] Accordingly, in one aspect, the present disclosure provides a method for measuring tauopathy- related pathology in a subject, the method comprising quantifying one or more tau peptides (e.g., as described herein) in a biological sample obtained from a subject, such as a CSF sample. The method may comprise providing the amount of one or more tau peptides as described herein. The amount(s) of the quantified tau peptides may be a representation of tauopathy-related pathology in the brain of the subject. The tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R-tauopathy. In specific embodiments, the tauopathy is a 4R-tauopathy. The tauopathy-related pathology may be tau deposition, tau post-translational modification, amyloid plaques in the brain and/or arteries of the brain, or other pathological feature known in the art. The subject may or may not have clinical symptoms of the tauopathy [0133] In another aspect, the present disclosure provides a method for diagnosing a tauopathy in a subject, the method comprising quantifying or providing the amount of one or more tau peptides (e.g., as described herein)in a biological sample obtained from a subject, such as a CSF sample, and diagnosing a tauopathy when the quantified tau peptide is/are about 1.5σ or above, where σ is the standard deviation defined by the normal distribution measured in a control population that does not have clinical signs or 39 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web symptoms of a tauopathy and is amyloid negative as measured by PET imaging and/or Aβ42/Aβ40 ratio measurement in CSF. The tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R- tauopathy. In specific embodiments, the tauopathy is a 4R-tauopathy. The subject may or may not have clinical symptoms of disease. [0134] In another aspect, the present disclosure provides a method for measuring tauopathy disease stability in a subject, the method comprising quantifying or providing the amount of one or more tau peptides (e.g., as described herein)in a first biological sample obtained from a subject and then in a second biological sample obtained from the same subject at a later time (e.g., weeks, months or years later), and calculating the difference between the quantified tau peptide between the samples, wherein a statistically significant increase in the quantified tau peptide in the second sample indicates disease progression, a statistically significant decrease in the quantified tau peptide in the second sample indicates disease improvement, and no change indicates stable disease. The tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R-tauopathy. In specific embodiments, the tauopathy is a 4R-tauopathy. The subject may or may not have clinical symptoms of disease, and may or may not be receiving a tau therapy. In some examples, a tau therapy is administered one or more times to the subject in the period of time between collection of the first and second biological sample, and the measure of disease stability is an indication of the effectiveness, or lack thereof, of the tau therapy. [0135] As contemplated for each of the methods as described herein, the MTBR-tau peptide species to be measured comprises MTBR-tau275/t-tau and/or MTBR-tau282/t-tau. [0136] Exemplary uses of the MTBR-tau peptides of tau as described herein can serve to illustrate various aspects discussed above, but such discussions do not limit the scope of the invention. Tau peptides (e.g., endogenously cleaved fragments of tau, e.g., MTBR-tau) and their detection are described in detail in the Examples. Generally speaking, these tau peptides contain MTBR-tau275 and/or MTBR- tau282 and are cleaved endogenously at their C-terminus. Measuring the amount of tau peptides is one means by which to measure, in a given sample, the amount of this specific group of tau species. As shown in the Examples, decreases in the amount of 4R isoform specific MTBR-tau275 and MTBR-tau282 endogenously cleaved fragments of tau in the CSF correlate with tau deposition in the brain associated with primary tauopathies, especially FTLD (e.g., FTLD-MAPT) and CBD.4R specific MTBR-tau/t-tau measures have inverse correlation in the CSF and brain. Additionally, subjects having a longer duration of the interval between age at disease onset and CSF collection (e.g., who are at a later pathological stage) have larger decrease in the CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau biomarkers. Stated another way, the amount of CSF MTBR-tau endogenously cleaved fragments (e.g., MTBR-tau275 and MTBR- 40 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web tau282) may be indicative of a primary tauopathy pathology. Moreover, CSF t-tau, as well as pT217/T217 ratio is useful as a diagnostic marker for discriminating between tauopathies (e.g., AD distinguished from NC, PSP, or other FTLD-tau; or CBD distinguished from NC, or other FTLD-tau). The detected amounts of MTBR-tau peptides can therefore be used to measure primary tauopathy pathology, to determine a subject’s tau status, to diagnose a primary tauopathy (e.g., FTLD-MAPT or CBD) stage in subjects, and discriminate between primary tauopathies, among other things. [0137] After diagnosing and/or staging disease, treatments may then be provided to the subject to decrease, or prevent any further increase, in the amount of tau peptide in CSF and/or to decrease, or prevent any further increase, of another clinical sign or symptom of a primary tauopathy. Choice of treatment may be further guided by knowledge of the specific disease stage that is informed by the amount of tau peptides (e.g., endogenously cleaved tau fragments), for instance, therapies designed to prevent Aβ deposition, reverse Aβ deposition, prevent tau deposition, reverse tau deposition, and improve clinical signs of disease would be used in subjects with different, albeit potentially overlapping, amount of tau peptides. [0138] In a specific embodiment, the present disclosure provides a method for detecting a primary tauopathy in a subject, the method comprising providing a CSF obtained from a subject, wherein the CSF is purified for a tau peptide (e.g., an endogenously cleaved fragment of tau); and quantifying, in the sample, the tau peptide. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide, or their ratios (e.g., to each other or each to CSF t-tau), is indicative of primary tauopathy in a brain of a subject. In some embodiments, the primary tauopathy is selected from frontotemporal lobar degeneration (FTLD-tau) (e.g., FTLD-MAPT), including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilc grain disease (AGD), globular glial tauopathy (GGT), Chronic Traumatic Encephalopathy (CTE), Pick’s disease (PiD), or Alzheimer’s disease. In specific embodiments, the primary tauopathy is FTLD-tau or CBD. In specific embodiments, the FTLD- tau is FTLD-MAPT. In specific embodiments, the FTLD-MAPT is selected from FTLD-MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, and FTLD-MAPT IVS10+16. [0139] In a specific embodiment, the present disclosure provides a method of measuring FTLD-tau– related pathology in a subject, comprising detecting or providing the amount of tau or 4R tau aggregates. In some embodiments, the detecting tau or 4R tau aggregates comprises providing a CSF obtained from a subject, wherein the CSF is purified for a tau peptide; and quantifying, in the sample, the tau peptide. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence 41 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide, or their ratios (e.g., to each other or each to CSF t-tau), is indicative of FTLD-tau–related pathology in a brain of a subject. In some embodiments, the method comprises quantifying or providing an amount p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), Aβ (e.g., Aβ42/Aβ40 ratio), or a combination thereof from a sample taken from a subject. In some embodiments, a subject is diagnosed with FTLD-tau when the quantified tau peptide differs by about 1.5σ or more from the mean of a control population, where σ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or Aβ42/Aβ40 ratio measurement in CSF (for instance, a cutoff value for CSF Aβ42/Aβ40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379–387) that maximizes sensitivity% + Specificity%). [0140] In another specific embodiment, the present disclosure provides a method of measuring FTLD- tau–related tau deposition in a brain of a subject, comprising detecting or providing the amount of tau or 4R tau aggregate. In some embodiments, the method comprises providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a tau peptide; and quantifying, in the sample, the tau peptide. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide is indicative of FTLD-tau-related tau deposition in a brain of a subject. In specific embodiments, the FTLD-tau is FTLD-MAPT. In specific embodiments, the FTLD-MAPT is selected from FTLD- MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, and FTLD-MAPT IVS10+16. [0141] In another specific embodiment, the present disclosure provides a method of diagnosing FTLD- tau-related disease, the method comprising providing a CSF obtained from a subject, and quantifying, in the sample, a tau peptide. The method may comprise providing the amount of a tau peptide as disclosed herein. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof. In some embodiments, the method further comprises quantifying p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), Aβ (e.g., Aβ42/Aβ40 ratio), or a combination thereof. In some embodiments, a subject is diagnosed with FTLD- tau-related disease when the quantified tau peptide differs by about 1.5σ or more from the mean of a control population, where σ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or Aβ42/Aβ40 ratio measurement in CSF (for instance, a cutoff value for CSF 42 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web Aβ42/Aβ40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379–387) that maximizes sensitivity% + Specificity%). In specific embodiments, the FTLD-tau is FTLD-MAPT. In specific embodiments, the FTLD-MAPT is selected from FTLD-MAPT P301L, FTLD-MAPT S305I, FTLD- MAPT R406W, or FTLD-MAPT IVS10+16. [0142] In another specific embodiment, the present disclosure provides a method of measuring FTLD- tau-related disease progression in a subject, the method comprising providing a first CSF sample and a second CSF sample, wherein each sample is obtained from a single subject, and each sample is purified for tau peptide; and for each sample, quantifying the tau peptide. In one example, the amounts of tau peptide in the first and second sample are provided. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof. In some embodiments, the method further comprises p-tau or Aβ measurement, or a combination thereof. The method further comprising calculating the difference between the quantified tau peptide in the second sample and the first sample, wherein a statistically significant in-crease in the quantified tau peptide in the second sample indicates progression of the subject’s FTLD-tau-related disease. In specific embodiments, the FTLD-tau is FTLD-MAPT. In specific embodiments, the FTLD- MAPT is selected from FTLD-MAPT P301L, FTLD-MAPT S305I, FTLD-MAPT R406W, or FTLD- MAPT IVS10+16. [0143] In another specific embodiment, the present disclosure provides a method of measuring CBD– related tau deposition in a brain of a subject, comprising detecting or providing the amount of tau or 4R tau aggregate. In some embodiments, the method comprises providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a tau peptide; and quantifying, in the sample, the tau peptide. In one example, the amount of the tau peptide is provided. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of the tau peptide is a representation of CBD–related tau deposition in a brain of a subject. [0144] In another specific embodiment, the present disclosure provides a method of diagnosing CBD- related disease, the method comprising providing a CSF obtained from a subject, and quantifying, in the sample, a tau peptide. In one example, the amount of the tau peptide is provided. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof. In some embodiments, the method further comprises quantifying p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), Aβ (e.g., Aβ42/Aβ40 ratio), or a combination thereof. In some embodiments, a subject is diagnosed with CBD-related disease when the 43 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web quantified tau peptide differs by about 1.5σ or more from the mean of a control population, where σ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and/or that is amyloid negative as measured by PET imaging (for instance by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and/or Aβ42/Aβ40 ratio measurement in CSF (for instance, a cutoff value for CSF Aβ42/Aβ40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379–387) that maximizes sensitivity% + Specificity%). [0145] In another specific embodiment, the present disclosure provides a method of measuring CBD- related disease progression in a subject, the method comprising providing a first CSF sample and a second CSF sample, wherein each sample is obtained from a single subject, and each sample is purified for a tau peptide; and for each sample, quantifying the tau peptide. In one example, the amounts of tau peptide in the first and second samples are provided. In some embodiments, the tau peptide comprises one or more of the peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof. In some embodiments, the method further comprises p-tau or Aβ measurement, or a combination thereof. The method further comprising calculating the difference between the quantified tau peptide in the second sample and the first sample, wherein a statistically significant in-crease in the quantified tau peptide in the second sample indicates progression of the subject’s CBD-related disease. [0146] In each of the preceding methods additional biomarkers can be detected and measured to aid in staging and/or determining the pathology of the subject and/or disease progression of the subject. For example, the method may include detecting and measuring the amount of Ab. In some embodiments an Aβ42/Aβ40 value is determined to aid in measuring amyloid plaque specific abnormalities. Aβ42/Aβ40 values which deviate from a normal control population without amyloid pathology begin to occur some 20 years before symptom on set and proved a high sensitivity measure of these changes. In addition to or in the place of an Ab measurement, each of the preceding methods may include detecting and measuring the amount of tau phosphorylation at specific residues. In some embodiments, phosphorylation of tau at 217, 181, 231, 205, 153, 111, 208, and/or any combination thereof, are determined to aid in measuring amyloid plaque specific abnormalities. Aβ42/Aβ40 is the first to change, then ptau217/181/231 shortly thereafter and those are associated with amyloid plaques. Then about 10 years later, ptau205 is increased and associated with both Ab plaques and tau tangles, similar to total-tau (n-terminal to mid-domain tau). In some embodiments, a composite value of ptau phosphorylation and Aβ42/Aβ40 value can be measured and determined. For example, pT217 x Aβ42/Aβ40 value is determined to aid in measuring amyloid plaque specific abnormalities and is found to be highly sensitive and specific. Finally, about 20 years after Aβ42/Aβ40 abnormalities, endogenously cleaved fragment of tau as disclosed herein is increased and 44 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web correlates with tau pathology only (not amyloid pathology). Thus, each marker when measured and combined in the methods disclosed herein provides a more precise ability to determine a subject’s stage, pathology and/or disease progression which has not been available prior to the present disclosure. Aβ42/Aβ40 is a measure of pre-amyloid plaque abnormalities; Aβ42/Aβ40 & ptau217% abnormal is a measure of amyloid plaque abnormalities, ptau205 is a measure of amyloid plaque plus neurodegeneration; and endogenously cleaved fragment of tau as disclosed herein is a measure of tau tangles and clinical onset of dementia. [0147] Alternatively or in addition to the above, additional markers such as a measurement of total tau, in any of the above embodiments, 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. Mathematical operations other than a ratio may also be used. For instance, the examples use of an endogenously cleaved fragment of tau as disclosed herein, Ab values, and/or site-specific tau phosphorylation values can be used in various statistical models (e.g., linear regressions, LME curves, LOESS curves, etc.) in conjunction with other known biomarkers (e.g. MAPT status, APOE ε4 status, age, sex, cognitive test scores, functional test scores, etc.). Selection of measurements and choice of mathematical operations may be optimized to maximize specificity of the method. For instance, 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.). [0148] Brain amyloid plaques in humans are routinely measured by amyloid-positron emission tomography (PET). For instance, 11C-Pittsburgh compound B (PiB) PET imaging of cortical Aβ-plaques is commonly used to detect Aβ-plaque pathology. The standard uptake value ratio (SUVR) of cortical PiB-PET reliably identifies significant cortical Aβ-plaques and is used to classify subjects as PIB positive (SUVR ≥ 1.25) or negative (SUVR < 1.25). Accordingly, in the above embodiments, 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) or 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. [0149] In another aspect, the present disclosure provides a method for treating a subject with a tauopathy, the method comprising quantifying tau in a biological sample obtained from a subject, such as a CSF sample; and providing a tau therapy to the subject to improve a measurement of tauopathy-related pathology or a clinical symptom, wherein the subject has a quantified endogenously cleaved peptide fragment of tau at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably 45 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web at least 1.5 standard deviations or even more preferably at least 2 standard deviations, above or below the mean of a control population (i.e., differs by 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in a control population does not have clinical signs or symptoms of a tauopathy and that is amyloid negative as measured by PET imaging and/or Aβ42/Aβ40 ratio measurement in CSF. In addition to using a threshold (e.g., at least 1 standard deviation above or below the mean), in some embodiments the extent of change above or below the mean may be used as criteria for treating a subject. The tauopathy may be a 3R-tauopathy, a mixed 3R/4R-tauopathy, or a 4R- tauopathy. In specific embodiments, the tauopathy is a 4R-tauopathy. The measurement of tauopathy- related pathology may be tau deposition as measured by PET imaging, tau post-translational modification as measured by mass spectrometry or other suitable method, amyloid plaques in the brain or arteries of the brain as measured by PET imaging, amyloid plaques as measured by Aβ42/Aβ40 ratio in CSF, or other pathological features known in the art. The clinical symptom may be dementia, as measured by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.) or other clinical symptoms known in the art for 3R- and 4R- tauopathies. In further embodiments, two or more tau species are quantified. Many tau therapies target a specific pathophysiological change. For instance, Aβ targeting therapies are generally designed to decrease Aβ production, antagonize Aβ aggregation or increase brain Aβ clearance; tau targeting therapies are generally designed to alter tau phosphorylation patterns, antagonize tau aggregation (general antagonism of tau or antagonism of a specific tau isoform), or increase NFT clearance; a variety of therapies are designed to reduce CNS inflammation or brain insulin resistance; etc. However, not all tauopathies share the same pathophysiological changes. Therefore, the efficacy of these various tau therapies can be improved by administering them to subjects that are correctly identified as having a tau pathology, including determining the stage of disease the subject is in thereby more efficiently altering tau phosphorylation patterns, antagonizing tau aggregation, or increasing NFT clearance based on the specific pathological state of the subject. [0150] The treatment may comprise one or more of lecanemab, donanemab, AADvac1, ACI-3024, ACI- 35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, Bepranemab, Dasatinib + Quercetin, E2814, Epothilone D, Gosuranemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, Semorinemab, TPI 287, Tideglusib, Tilavonemab, Zagotenemab, an anti-tau monoclonal anti-body, an anti-tau anti-sense oligonucleotide, an anti-tau small interfering RNA, an tau production inhibitor, and a tau active vaccine. [0151] In some embodiments the treatment is a pharmaceutical composition comprising a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., a selective serotonin 46 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, etc.), a gamma-secretase inhibitor, a beta-secretase inhibitor, an anti-Aβ antibody (including antigen-binding fragments, variants, or derivatives thereof), an anti-tau antibody (including antigen- binding fragments, variants, or derivatives thereof), an anti-TREM2 antibody (including antigen-binding fragments, variants or derivatives thereof, a TREM2 agonist, stem cells, dietary supplements (e.g. lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, cur-cumin, and grape seed extract, etc.), an antagonist of the serotonin receptor 6, a p38alpha MAPK inhibitor, a recombinant granulocyte macrophage colony-stimulating factor, a passive immunotherapy, an active vaccine (e.g. CAD106, AF20513, etc.), a tau protein aggregation inhibitor (e.g. TRx0237, methylthionimium chloride, etc.), a therapy to improve blood sugar control (e.g., insulin, exenatide, liraglutide pioglitazone, etc.), an anti-inflammatory agent, a phosphodiesterase 9A inhibitor, a sigma-1 receptor agonist, a kinase inhibitor, a phosphatase activator, a phosphatase inhibitor, an angiotensin receptor blocker, a CB1 and/or CB2 endocannabinoid receptor partial agonist, a β-2 adrenergic receptor agonist, a nicotinic acetylcholine receptor agonist, a 5-HT2A inverse agonist, an alpha-2c adrenergic receptor antagonist, a 5-HT 1A and 1D receptor agonist, a Glutaminyl-peptide cyclotransferase inhibitor, a selective inhibitor of APP production, a monoamine oxidase B inhibitor, a glutamate receptor antagonist, a AMPA receptor agonist, a nerve growth factor stimulant, a HMG-CoA reductase inhibitor, a neurotrophic agent, a muscarinic M1 receptor agonist, a GABA receptor modulator, a PPAR-gamma agonist, a microtubule protein modulator, a calcium channel blocker, an antihypertensive agent, a statin, and any combination thereof. In an exemplary embodiment, a pharmaceutical composition may comprise a kinase inhibitor. Suitable kinase inhibitors may inhibit a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. In another exemplary embodiment, a pharmaceutical composition may comprise a phosphatase activator. As a non-limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments the treatment is a pharmaceutical composition comprising a tau targeting therapy, including but not limited to active pharmaceutical ingredients that alter tau phosphorylation patterns, antagonize tau aggregation, or increase clearance of pathological tau isoforms and/or aggregates. In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma- secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor. [0152] In some embodiments, the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing when the detected Aβ42/Aβ40 value deviates from the mean of a healthy control population and optionally pTau217/tau217, ptau205/tau205, pTau181, 47 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web pTau231, and/or a cleaved peptide fragment of the present disclosure do not significantly deviate from the mean of a healthy control population. In an exemplary embodiment, the detected Aβ42/Aβ40 value deviates significantly below the mean of a healthy control population. In some embodiments, the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing and/or reduces a subject's existing plaque load when the detected Aβ42/Aβ40 value and pTau217/tau217 (or pTau217 x Aβ42/Aβ40 composite value) significantly deviates from the mean of a healthy control population and optionally ptau205/tau205, and/or a cleaved peptide fragment of the present disclosure do not significantly deviate from the mean of a healthy control population. In an exemplary embodiment, the detected Aβ42/Aβ40 value deviates significantly below the mean of a healthy control population, the detected pTau217/tau217 is above the mean of a healthy control population and/or the pTau217 x Aβ42/Aβ40 composite value is above the mean of a healthy control population. In some embodiments, the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing and/or reduces a subject's existing plaque load and/or treats or prevents neurodegeneration and/or prevents Tau tangles or related pathology when the detected Aβ42/Aβ40 value, pTau217/tau217 (or pTau217 x Aβ42/Aβ40 composite value), and pTau205/tau205 value significantly deviate from the mean of a healthy control population and optionally a cleaved peptide fragment of the present disclosure do not significantly deviate from the mean of a healthy control population. In an exemplary embodiment, the detected Aβ42/Aβ40 value deviates significantly below the mean of a healthy control population, the detected pTau217/tau217 is above the mean of a healthy control population and/or the pTau217 x Aβ42/Aβ40 composite value is above the mean of a healthy control population and/or the pTau205/tau205 value is above the mean of a healthy control population. In some embodiments, the subject is administered a therapeutic agent or a therapeutic agent is selected which prevents amyloid deposition from increasing and/or reduces a subject's existing plaque load and/or treats or prevents neurodegeneration and/or prevents tau tangles from in-creasing and/or reduces a subject’s existing tangles or related pathology when the detected Aβ42/Aβ40 value, pTau217/tau217 (or pTau217 x Aβ42/Aβ40 composite value), pTau205/tau205 and a cleaved peptide fragment of the present disclosure significantly deviate from the mean of a healthy control population. In an exemplary embodiment, the detected Aβ42/Aβ40 value deviates significantly below the mean of a healthy control population, the detected pTau217/tau217 is above the mean of a healthy control population and/or the pTau217 x Aβ42/Aβ40 composite value is above the mean of a healthy control population and/or the pTau205/tau205 value is above the mean of a healthy control population and/or a cleaved peptide fragment of the present disclosure value is above the mean of a health control population. 48 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0153] In each of the above embodiments, a pharmaceutical composition administered to a subject may comprise an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh com-pound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.). [0154] Another aspect of the present disclosure is a method of selecting a subject into a clinical trial, in particular a clinical trial for an Aβ or tau therapy, provided all other criteria for the clinical trial have been met. In one embodiment, a method of a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) selecting the subject into a clinical trial for an Aβ therapy when the cleaved peptide fragment of tau value is about the same as a healthy control population and the subject’s Aβ42/Aβ40 value is below the man of a healthy control population. In some embodiments, the cleaved peptide fragment of tau comprises one or more of the peptides having an amino acid sequence in Table 1, where the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject. [0155] In another embodiment, a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) excluding the subject into a clinical trial an Aβ therapy when the cleaved peptide fragment of tau value is above the mean of a healthy control population and the subject’s Aβ42/Aβ40 value is about the same or below the mean of a healthy control population. In some embodiments, the cleaved peptide fragment of tau comprises one or more of the peptides having an amino acid sequence in Table 1, where the C- terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject. [0156] In another embodiment, a method of selecting a subject into a clinical trial, in particular a clinical trial for a tau therapy, provided all other criteria for the clinical trial have been met. In one embodiment, a method of a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) selecting the subject into a clinical trial a tau therapy when the cleaved peptide fragment of tau value is above the mean of a healthy control 49 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web population and optionally when the subject’s Aβ42/Aβ40 value is about the same or below the mean of a healthy control population. In some embodiments, the cleaved peptide fragment of tau comprises one or more of the peptides comprising an amino acid sequence in Table 1, where the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject. [0157] In another embodiment, a method of selecting a subject into a clinical trial, in particular a clinical trial for a tau therapy, provided all other criteria for the clinical trial have been met. In one embodiment, a method of a method of selecting a subject into a clinical trial may comprise (a) providing a CSF sample obtained from a subject, wherein the CSF sample is purified for a cleaved peptide fragment of tau; (b) quantifying, in the sample, the cleaved peptide fragment of tau and (c) excluding the subject into a clinical trial a tau therapy when the cleaved peptide fragment of tau value is about the same as the mean of a healthy control population. In some embodiments, the cleaved peptide fragment of tau comprises one or more of the peptides comprising an amino acid sequence in Table 1, where the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, wherein the amount of the cleaved peptide fragment of tau, or their ratios, is a representation of tau deposition in a brain of a subject. The phrase “a control population without brain amyloid plaques as measured by PET imaging” is defined above. [0158] Alternatively or in addition to using a measurement of a cleaved peptide fragment of tau as disclosed herein, site-specific tau phosphorylation, optionally with a measurement of total tau, in any of the above embodiments, 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 pT181 and pT205, pT217 and pT205, or pT181 and pT217. A ratio calculated from the measured phosphorylation level(s) and total tau may be a ratio between pT181 and total tau, pT205 and total tau, or pT217 and total tau. 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 ε4 status, age, sex, cognitive test scores, functional test scores, etc.). [0159] The design of clinical trials for tauopathies (e.g., FTLD-MAPT, CBD) as well as tau therapies can be greatly aided by the methods disclosed herein. Many clinical trials are designed to test the efficacy of imaging agents or therapeutic agents that target a specific pathophysiological change which occurs 50 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web prior to the onset of tau pathology symptoms. As described herein, the efficacy of these various agents can be improved by administering the agents to subjects that have certain site-specific tau phosphorylation levels, as measured by methods disclosed herein and illustrated. Similarly, clinical trials selecting subjects with symptoms of Aβ pathology or tau only pathology would also benefit from being able to accurately discriminate an enrollee’s pathology in order to determine if efficacy is associated with a particular disease state. Accordingly, measuring tau phosphorylation levels as described herein prior to selecting 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. In some instances, methods described herein may be developed and used as a companion diagnostic for a therapeutic agent. [0160] In each of the above embodiments, a subject may be enrolled into a treatment arm of the clinical trial. The "treatment" is defined above. Subjects enrolled in the treatment arm of a clinical trial may be administered a pharmaceutical composition. In some embodiments, a pharmaceutical composition may comprise an imaging agent. Non-limiting examples of 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.). Alternatively, a pharmaceutical composition may comprise an active pharmaceutical ingredient. 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-Aβ 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. lithium water, omega-3 fatty acids with lipoic acid, long chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract, etc.), antagonists of the serotonin receptor 6, p38alpha MAPK inhibitors, recombinant granulocyte macrophage colony- stimulating factor, passive immunotherapies, active vaccines (e.g. CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g. TRx0237, methylthionimium chloride, etc.), therapies to improve blood sugar control (e.g., insulin, exenatide, liraglutide pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and/or CB2 endocannabinoid receptor partial agonists, β-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT 1A and 1D receptor agonists, Glutaminyl- peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, 51 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof. In an exemplary embodiment, a pharmaceutical composition may comprise a kinase inhibitor. Suitable kinase inhibitors may inhibit a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. In another exemplary embodiment, a pharmaceutical composition may comprise a phosphatase activator. As a non- limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A. [0161] In each of the above embodiments, 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 a tauopathy. Alternatively, a subject may exhibit signs or symptoms (e.g., memory loss, misplacing things, changes in mood or behavior, etc.,) but not show sufficient cognitive or functional impairment for a clinical diagnosis. A symptomatic or an asymptomatic subject may have Aβ amyloidosis; however, prior knowledge of Aβ amyloidosis is not a requisite for treatment. In still further embodiments, a subject may have AD. In any of the aforementioned embodiments, a subject may carry one of the gene mutations known to cause an inherited tauopathy. In alternative embodiments, a subject may not carry a gene mutation known to cause an inherited tauopathy. 5. Kits [0162] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration or measuring a tau peptide (e.g., endogenously cleaved fragment of tau). Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to systems, assays, epitope biding agents, reagents, internal standards, or software. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components. [0163] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of 52 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil- lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like. [0164] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit. [0165] A control sample or a reference sample as described herein can be a sample from a healthy subject or from a randomized group of subjects. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample. [0166] The methods and algorithms of the invention may be enclosed in a controller or processor. Furthermore, methods and algorithms of the present invention, can be embodied as a computer implemented method or methods for performing such computer-implemented method or methods, and can also be embodied in the form of a tangible or non-transitory computer readable storage medium containing a computer program or other machine-readable instructions (herein “computer program”), wherein when the computer program is loaded into a computer or other processor (herein “computer”) and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. Storage media for containing such computer program include, for example, floppy disks and diskettes, compact disk (CD)-ROMs (whether or not writeable), DVD digital disks, RAM and ROM memories, computer hard drives and back-up drives, external hard drives, “thumb” drives, and any other storage medium readable by a computer. The method or methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted over a transmission medium such as electrical conductors, fiber optics or other light conductors, or by electromagnetic radiation, wherein when the computer program is loaded into a computer and/or is executed by the computer, the computer becomes an apparatus for practicing the method or methods. The method or 53 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the process or processes. When a general-purpose microprocessor is employed, the computer program code configures the circuitry of the microprocessor to create specific logic circuit arrangements. Storage medium readable by a computer includes medium being readable by a computer per se or by another machine that reads the computer instructions for providing those instructions to a computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above. EXAMPLES [0167] The present invention has multiple aspects, illustrated by the following non-limiting examples. Example 1: CSF tau microtubule binding region identifies pathological changes in primary tauopathies Abstract: [0168] Despite recent advances in fluid biomarker research in Alzheimer’s disease (AD), there are no fluid biomarkers or imaging tracers with utility for diagnosis and/or theragnosis available for other tauopathies. Using immunoprecipitation and mass spectrometry methods, it is shown that 4 repeat (4R) isoform specific tau species from microtubule binding region (MTBR-tau275 and MTBR-tau282) increase in the brains of corticobasal degeneration (CBD), Progressive supranuclear palsy (PSP), frontotemporal lobar degeneration (FTLD)-MAPT and AD but inversely decrease in the CSF of CBD, FTLD-MAPT and AD compared to control and other FTLD-tau (i.e., Pick’s disease). CSF MTBR-tau measures are reproducible in repeated lumbar punctures, and can distinguish CBD from control (area under the curve of 0.889) and other FTLD-tau such as PSP (area under the curve of 0.886). CSF MTBR- tau275 and MTBR-tau282 may represent the first affirmative biomarkers to aid in the diagnosis of primary tauopathies and facilitate clinical trial designs. Introduction: [0169] Tauopathies are a heterogeneous group of neurodegenerative diseases that all include aggregated tau proteins. The symptomatic phases of these fatal illnesses involve neurological impairments that typically progress over years to decades, leading to substantial medical, social, and financial burden on patients and families. For the most common tauopathy, Alzheimer’s disease (AD), cerebrospinal fluid (CSF) biomarkers amyloid-beta (Aβ) and total and phosphorylated tau have been used to aid diagnosis1–8 54 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web and these biomarkers have become useful for assessing the outcome of therapies in clinical trials9–11. Additionally, recent progress in positron emission tomography (PET) imaging now enables the measurement of aggregated Aβ and tau in the brains of living AD patients12. In contrast, such progress is lacking for other tauopathies classified as frontotemporal lobar degeneration (FTLD-tau), including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilc grain disease (AGD), globular glial tauopathy (GGT), Chronic Traumatic Encephalopathy (CTE) and Pick’s disease (PiD). Accurate diagnoses are challenging without fluid biomarkers for the tauopathies as these disorders fall within a spectrum comprising multiple and overlapping clinical phenotypes. Most tauopathies can only be definitively diagnosed by brain autopsy. Antemortem fluid biomarkers for these tauopathies will be required to improve the accuracy of clinical diagnosis and to facilitate clinical trials for tauopathy therapeutics. [0170] Recent structural and biochemical analyses implicate that distinct tau species constitute brain tau aggregates in subtypes of tauopathies. Six tau splicing isoforms are expressed in the adult human brain, including isoforms containing R1, R3, R4 repeat domains (3R) and R1, R2, R3, and R4 repeat domains (4R) in the MTBR13. Tauopathies can be classified into 3R, 4R, and 3R/4R mixed tauopathies based on the dominant isoforms found in tau aggregates. Cryogenic electron microscopy (CryoEM) studies demonstrated that there are distinct tau filament structures in AD (3R/4R)14, PiD (3R)15, CBD (4R)16, PSP (4R)17, and CTE (3R/4R)18. R3 and R4 repeat domains are commonly present in tau aggregates from AD and other tauopathies14. In contrast, 4R isoform-specific R2 repeat domain in addition to R3 and R4 are found in tau aggregates from CBD and PSP16,17. Biochemical extraction and mass spectrometry methods recently showed that specific tau peptides such as the residues 243-254 (MTBR-tau243; R1), 299-317 (MTBR-tau299; R2-R3), and 354-369 (MTBR-tau354; R4) differentially enriched in AD brains with disease progression19. Furthermore, the truncated tau containing MTBR could be detected and quantified in CSF, and CSF soluble concentrations of MTBR-tau peptides reflected AD clinical severity and correlated strongly with tau PET measures19. Others have also reported a correlation between CSF soluble truncated MTBR-tau and insoluble tau aggregates measured by tau PET measures20, suggesting that measures of MTBR-tau peptides might serve as fluid biomarkers of tau aggregation in AD. [0171] As a solution, the present inventors hypothesized that 4R isoform specific MTBR-tau species accumulate in the brain of specific subtypes of 4R tauopathies. It was investigated if these changes are reflected in the CSF, and whether the changes can distinguish different subtypes of primary tauopathies. Tau peptides 275-280 (MTBR-tau275) and 282-290 (MTBR-tau282) were specifically monitored, which are in the R2 region and specific to 4R tau splicing isoforms. Differential diagnostic abilities of MTBR- 55 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web tau275 and MTBR-tau282 normalized to total tau were tested, to distinguish effects in FTLD-tau, FTLD- TDP, control, and within different subtypes of tauopathies. Methods: Human Studies [0172] The retrospective study of pathologically-confirmed cohort included participants seen at UCSF or participating research sites under the following projects: Hillblom Healthy Aging Study, UCSF Alzheimer’s Disease Research Center Program Project Grant (P30AG062422), ARTFL-LEFFTDS Longitudinal Frontotemporal Lobar Degeneration (ALLFTD, U19AG063911), and the Four Repeat Tauopathy Neuroimaging Initiative (4RTNI, R01AG038791). Collection and use of biospecimens was approved by the institutional review board at ALLFTD or each research center from which the individual was recruited, and this study was approved by the Biospecimen Resource Committee at UCSF. Participants provided written informed consent at the time of recruitment. Participants underwent a standardized clinical evaluation that included collection of demographic data, structured participant/informant interview, functional assessment, neurologic examination, and neuropsychological testing. CSF draws were performed at the same visit as clinical evaluation; primary clinical syndrome was determined based on all available data at the time of clinical evaluation by an experienced neurologist or panel of neurologists, following established diagnostic criteria. Consent to undergo autopsy was provided by the patient or their surrogate, following principles as outlined in the Declaration of Helsinki. [0173] Repeated lumbar puncture (LP) study (NCT03545126) was approved by the Institutional Review Board at Washington University in St. Louis, MO, USA and University College London (UCL), UK. All participants or their delegates consented to the collection and sharing of biofluid samples and brain autopsy, and were compensated. Exclusion criteria included any contraindications for LPs or lumbar catheters, including a bleeding disorder, active anticoagulation, and active infection. [0174] The study of clinically diagnosed cohort was approved by the Ethics Committee of the Montpellier University Hospital, France (CSF-Neurobank #DC-2008-417 at the certified NFS 96-900 CHU resource center BB-0033-00031 (http://www.biobanques.eu). Authorization to handle personal data was granted by the French Data Protection Authority (CNIL) under the number 1709743 v0. 56 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web Human brain samples [0175] Neuropathological diagnoses of human brain donations were rendered according to established diagnostic criteria at Washington University and UCSF. Frozen human brain tissue samples selected for this study were processed as previously described19,36. Briefly, frozen brain tissues were sliced using a cryostat and sonicated in ice-cold buffer containing 25 mM Tris-hydrochloride (pH 7.4), 150 mM sodium chloride, 10 mM EDTA, 10 mM EGTA, phosphatase inhibitor cocktail, and protease inhibitor cocktail. The homogenate was clarified by centrifugation for 20 min at 11,000g at 4 degC and stored at -80 degC as the whole brain extract. Demographics of the brain donors included in this study are described in FIG.1. Human CSF samples [0176] CSF collection methods are similar across all cohorts examined in this study. CSF collection method from pathologically-confirmed cases was as previously described in the Alzheimer's Disease Neuroimaging Initiative (ADNI) procedures manual. [0177] Demographics of participants in pathologically-confirmed cohort is in FIG.2. CSF collection method from repeated LP studies was collected using the same human tau Stable Isotope Labeling Kinetics protocol as previously described37. Demographics of participants in the repeat lumbar puncture study are described in FIG.9. CSF collection method and demographics for clinically diagnosed cohort were previously described24. Mass spectrometry analyses of MTBR-tau [0178] Brain insoluble MTBR-tau was analyzed using filter-aided sample preparation methods as previously described19,36. Briefly, the whole brain extract was incubated with 1% sarkosyl for 60 min on ice, followed by ultra-centrifugation at 100000g at 4 degC for 60 min to obtain an insoluble pellet. Insoluble brain fractions were filtered, digested, desalted and injected into mass spectrometer for analyses. [0179] CSF MTBR-tau was analyzed as previously described19 with the following modifications: master mix containing detergent and chaotropic reagents (final 1% NP-40, 5 mM guanidine, protease inhibitor cocktail) and internal standards for tau (15N labeled 2N4R recombinant tau) were prepared in polypropylene tubes prior to CSF addition.0.5 mL of CSF was added and immunoprecipitated with Tau1, HJ8.5, and HJ8.7 anti-tau antibodies with epitopes residing in N- terminal to mid-domain of tau24,37. Post- 57 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web immunoprecipitated samples depleted of N-terminal to mid-domain of tau were sequentially immunoprecipitated with 77G7 anti-tau antibody to the MTBR (the residue 316-335) to measure MTBR- tau species. After washing, samples were digested with trypsin, desalted, and analyzed by Orbitrap Eclipse mass spectrometer (Thermo Scientific). The mass spectrometry methods used for measuring MTBR-tau were as described previously with some modifications. To account for individual variability in t-tau concentrations, the ratio of MTBR-tau275 or MTBR-tau282 normalized to mid domain tau peptides (181-190 and 212-221 for brain and CSF analyses, respectively) was used, which are common to all isoforms. Statistics [0180] All statistical analyses were performed using GraphPad Prism software (v9.3.1). Differences in biomarker values were assessed with one-way ANOVAs unless otherwise specified. A two-sided p<0.05 was considered statistically significant and corrected for multiple comparisons using Benjamini-Hochberg false discovery rate (FDR) method with FDR set at 5%38. P-values reported in tables and figures are corrected by the Benjamini-Hochberg method (FDR=5%). Spearman correlations were used to assess associations between CSF tau biomarkers and neuropathological changes. Results: 4R specific brain MTBR-tau increases in primary tauopathies [0181] First, frozen brain tissues from 59 individuals with autopsy-confirmed AD, FTLD-tau, or FTLD with TAR DNA-binding protein aggregates (FTLD-TDP) and 3 normal control (NC) p<0.0001, p<0.01, respectively), 4R tauopathies, AGD (0.200) and PSP (0.489 ± 0.229, p<0.0001, p<0.05, respectively), and 3R/4R mixed tauopathy, AD (0.319 ± 0.047, p<0.0001, p<0.01, respectively). MTBR-tau275/t-tau moderately increased in PSP (approximately 2-fold) compared to FTLD-TDP (p<0.01). Brain MTBR- tau282/t-tau had similar increase profile to MTBR-tau275/t-tau (FIG.4C) but moderately (2.6-fold) increased in AD (0.915 ± 0.180) compared to FTLD-TDP (0.348 ± 0.133, p<0.05), which was not observed in MTBR-tau275/t-tau. These results suggest that 4R specific MTBR-tau species are enriched in the insoluble fraction of SFG/insular cortex brain tissue in a subset of 4R tauopathies such as CBD and FTLD-MAPT, and moderately increased in a different PSP (4R tauopathy) and AD (3R/4R mixed tauopathy). 58 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 4R specific CSF MTBR-tau decreases in primary tauopathies [0182] Next, CSF from 29 NC, - FTLD-MAPT and 78 autopsy-confirmed cases of AD, primary tauopathies, and FTLD-TDP were analyzed for MTBR-tau275 and MTBR-tau282 (FIG.2). CSF MTBR- tau275 and MTBR-tau282 concentrations did not separate different tauopathies (FIGS.12A and 12B due to individual variability in t-tau concentrations (FIG.12C). Therefore, CSF MTBR-tau275 and MTBR- tau282 from truncated tau were normalized by t-tau measured by mid domain tau 212-221 (FIG.5A), similar to normalization methods previously reported in truncated tau and Aβ isoform measurements20,22. CSF MTBR-tau275/t-tau decreased in CBD (0.00525 ± 0.00117), AD (0.00472 ± 0.00085), and FTLD- MAPT (0.00491 ± 0.00207), compared to NC (0.00657 ± 0.00078, p<0.001, p<0.0001, p<0.01, respectively) and non-tauopathy control, FTLD-TDP (0.00611 ± 0.00115, p<0.05, p<0.01, p<0.05, respectively. FIG.5B). This decrease was particularly significant in FTLD-MAPT P301L that has more typical FTLD pathology than in R406W which has many features of AD. CSF MTBR-tau275 also decreased in CBD, AD, and FTLD-MAPT compared to other 4R tauopathies, AGD (0.00759 ± 0.00013) and PSP (0.00669 ± 0.00091, p<0.001, p<0.0001, p<0.01, respectively), and 3R tauopathy, PiD (0.00676 ± 0.00138, p<0.05, p<0.01, p<0.05, respectively). CSF MTBR-tau282/t-tau had similar decrease profiles to CSF MTBR-tau275/t-tau (FIG.5C). Interestingly, CSF MTBR-tau275/t-tau did not change in PSP compared to control or FTLD-TDP even though these ratios increased moderately in the brain. [0183] To assess if the soluble CSF MTBR-tau/t-tau measures reflected brain tau pathology measured by the paired insoluble brain MTBR-tau/t-tau measures, MTBR-tau/t-tau from antemortem CSF and brain from the same individuals were analyzed for correlation (N=54, FIG.6A and FIG.6B). MTBR-tau275/t- tau and MTBR-tau282/t-tau from the CSF and brain correlated moderately (r=-0.27, p=0.049, and r=- 0.45, p=0.0006 respectively) across all disease groups (N=54), and strongly (r=-0.61, p=0.0004, and r=0.75, p<0.0001, FIG.6C and FIG.6D) in 4R tauopathies (PSP, CBD, and AGD, N=29). This suggests that 4R specific MTBR-tau species have inverse correlation in the CSF and brain in 4R tauopathies. In CBD, MTBR-tau275/t-tau and MTBR-tau282/t-tau from the CSF and brain correlated moderately but no statistical significance was obtained (N=12, r=-0.25, p=0.43, and r=-0.31, p=0.33 respectively. FIG.3E, F). One CBD participant who had no cognitive impairment (Clinical Dementia Rating plus National Alzheimer's Coordinating Center FTLD sum of boxes23 (CDR plus NACC FTLD-SB) = 0) had less changes in MTBR-tau275/t-tau and MTBR-tau282/t-tau in both brain and CSF (brain MTBR-tau275/t-tau = 0.321, brain MTBR-tau282/t-tau = 0.499, CSF MTBR-tau275/t-tau = 0.0071, CSF MTBR-tau282/t-tau = 0.0134). This may suggest that the changes of these biomarkers depend on the severity of the disease. 59 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0184] To assess if CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau decrease with disease stage, the correlations between these biomarkers and duration (i.e., interval between age at onset (AAO) and CSF collection) were investigated (FIG.2, FIG.13B). Average duration across diseases were 5 ± 4 years (N=81), and 4 ± 1 years for CBD only (N=18). In CBD, there were negative correlations between duration and CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau (r=-0.37 and -0.39, respectively) although statistical significances were not observed (p=0.13 and 0.11, respectively). This result suggests that participants with longer duration who are at later pathological stages have larger degrees of decrease in the CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau biomarkers. CSF MTBR-tau is reproducible in repeated lumbar punctures [0185] To evaluate reproducibility and stability of the CSF MTBR-tau measurements within the same individual, CSF MTBR-tau275/t-tau was examined in an independent cohort of 25 participants who underwent repeated lumbar punctures (LPs, 3-5 times) within approximately 4 months as a part of an ongoing study examining protein turnover kinetics37 (FIG.9, FIG.7). These participants include individuals clinically diagnosed with PSP-Richardson’s syndrome (PSP-RS N=7) or corticobasal syndrome (CBS, a clinical syndrome associated with heterogenous neuropathological substrates including AD, CBD, PSP, and FTLD-TDP. N=9), with 2 participants having autopsy-confirmed CBD. They also include 7 MAPT mutation (P301L, R406W, and IVS10+16) carriers who are either symptomatic or asymptomatic, and 2 non-carrier family members who are normal controls (NC). The mean coefficient of variation (CV) for CSF MTBR-tau275/t-tau in repeated LPs was 12 ± 7%, establishing the high reproducibility and stability of CSF MTBR-tau measures within 4 months. [0186] Consistent with the FTLD-MAPT cases analyzed with the pathologically-confirmed cohort, CSF MTBR-tau275/t-tau decreased in FTLD-MAPT mutation carriers in the repeated LP cohort. Interestingly, CSF MTBR-tau275/t-tau decreased in two symptomatic FTLD-MAPT P301L mutation carriers (participant #02, #03, 0.00381 ± 0.00021) and a symptomatic FTLD-MAPT R406W mutation carrier (#05, 0.00508) compared to NC (#01, #04, 0.00666 ± 0.00027). However, CSF MTBR-tau275/t-tau did not change in FTLD-MAPT R406W mutation carriers who were asymptomatic at LPs (#06, #07). The FTLD-MAPT variant IVS10+16 promotes the splicing of tau exon 10, resulting in greater production of 4R over 3R isoforms. Indeed, symptomatic FTLD-MAPT IVS10+16 mutation carriers (#08, #09, 0.00921 ± 0.00053) had 1.38-fold higher CSF MTBR-tau275/t-tau compared to NC, indicating that an increase in 4R isoforms is reflected in the CSF. 60 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0187] CSF MTBR-tau275/t-tau decreased in the two participants who were clinically diagnosed as PSP- RS but later were autopsy-confirmed with CBD (#10 (0.00396), #11 (0.00535)), consistent with pathologically-confirmed CSF cohort results. However, CSF MTBR-tau275/t-tau did not change in participants clinically diagnosed with PSP-RS (0.00779 ± 0.00052) or CBS (0.00748 ± 0.00187) who are not yet autopsy-confirmed during the repeated measures studies. Average duration across diseases were 5 ± 3 years (N=21), and 4 ± 2 for CBS only (N=9), which were similar to pathologically-confirmed cohort. CSF MTBR-tau in clinically diagnosed primary tauopathies [0188] To estimate the CSF MTBR-tau biomarker performance in clinically diagnosed primary tauopathies, CSF MTBR-tau275/t-tau was measured in an additional independent cohort of 238 primary tauopathies with single LP (FIG.8). This cohort was previously analyzed for CSF t-tau and phosphorylated tau24 and includes clinically diagnosed cases of AD, sporadic behavioral variant frontotemporal dementia (bvFTD), bvFTD secondary to FTLD-MAPT, PSP-RS, CBS, and CBS-PSP continuum25. Individuals with CBS-PSP continuum are defined as patients who initially presented with CBS, but subsequently developed clinical features of PSP-RS as the disease progressed. CSF MTBR- tau275/t-tau decreased in CBS-PSP continuum and FTLD-MAPT compared to cognitively NC (p<0.05). However, CSF MTBR-tau275/t-tau did not statistically change in either AD or clinically diagnosed CBS compared to control or other tauopathies. Diagnostic accuracies of CSF MTBR-tau in primary tauopathies [0189] Finally, diagnostic accuracies of CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau were examined in pathologically-confirmed primary tauopathy cohort. First, CSF t-tau (mid domain peptide 212-221) and phosphorylated tau (pT217/T217) were examined in primary tauopathies for comparison (FIG.12C and FIG.14A). CSF t-tau increased in AD compared to NC and PSP (p<0.05) and can differentiate AD from FTLD-tau (PSP, CBD, AGD, PiD, FTLD-MAPT) with a receiver operating characteristic (ROC) area under the curve (AUC) of 0.794 FIG.14B). However, CSF t-tau does not distinguish among FTLD-tau. CSF pT217/T217 increased in AD compared to NC, FTLD-TDP and FTLD-tau (p<0.0001) and can differentiate AD from FTLD-tau with AUC of 0.987 (FIG.14C). AD co- pathology in other neurodegenerative diseases (i.e., FTLD-TDP, CBD, PSP) also increased CSF pT217/T217. These results suggest that CSF pT217/T217 can be used to accurately identify individuals with AD pathology, regardless of co-pathology. 61 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0190] Effect of amyloid on CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau in primary tauopathies were further assessed using AD Thal phase. CSF pT217/T217 strongly correlated with AD Thal phase (r=0.52, p<0.0001. FIG.15A). However, CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau did not correlate with AD Thal phase in whole cohort (r=-0.22, p=0.06 and r=-0.24, p=0.04, respectively. FIG.15B, and FIG.15C) or in CBD (r=-0.14, p=0.60 and r=-0.07, p=0.78, respectively. FIG.15D, and FIG.15E). These results suggest that CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau decrease in CBD independently from AD co-pathology. [0191] Diagnostic accuracies of CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau were examined to determine if they distinguish CBD from control, FTLD-TDP, FTLD-tau as a group and individual tauopathy (FIG.3, FIGS.16A-16L). CSF MTBR-tau275/t-tau and CSF MTBR-tau282/t-tau can distinguish CBD from NC, other FTLD-tau (PSP, PiD, and AGD), PiD, and PSP with AUC of 0.800 to 0.889. CBD can be distinguished from FTLD-TDP with AUC of 0.701 to 0.770. When AD co-pathology cases were excluded, CSF MTBR-tau275/t-tau and CSF MTBR-tau282/t-tau can distinguish CBD from PSP with AUC of 0.859 and 0.886, respectively (FIG.3, FIG.16K, and FIG.16L). [0192] Lastly, CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau were retrospectively assessed by final clinical syndromes in neuropathologically-confirmed cohort to determine if these biomarkers can facilitate antemortem diagnosis of primary tauopathies. Numbers of individuals who had CSF MTBR- tau275/t-tau and MTBR-tau282/t-tau lower than cutoff (0.00563 and 0.01220 respectively, defined in FIG.3 to differentiate CBD and PSP) within each clinical syndromes applications of these biomarkers include distinguishing CBD from PSP among PSP-RS, as 0/11 (0%) and 2/11 (18%) PSP had lower than cutoff for CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau, respectively. Discussion: [0193] Despite the long quest for antemortem biomarkers of FTLD-tau pathology, to date, no fluid biomarkers have been identified that can differentiate subgroups of tauopathies other than AD. Previous studies showed that CSF phosphorylated tau at T181 decreased in PSP and FTLD-TDP relative to controls26–30. CSF pT217/T217 was recently demonstrated to be increased in FTLD-MAPT R406W compared to control without Aβ pathology24. However, phosphorylated tau changes most significantly in AD and thus these studies may not have captured key pathological changes in primary tauopathies. In this study, focused was on MTBR-tau that constitutes the core regions of tau aggregates in the brains and also exists in CSF as truncated C-terminal tau peptide19. Using biochemical purification and quantitative mass spectrometry methods, it was shown that 4R isoform specific MTBR-tau275 and MTBR-tau282 that are 62 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web normalized to t-tau decrease in the CSF soluble tau as they increase in brain insoluble tau in primary tauopathies, especially CBD and FTLD-MAPT P301L. MTBR-tau/t-tau measures are inversely correlated in the CSF and brain, suggesting that there may be an equilibrium or a unidirectional transfer between soluble CSF MTBR-tau and insoluble brain MTBR-tau in these primary tauopathies. This study provides the possibility of the first fluid biomarker that reflects brain pathology in primary tauopathies. [0194] One of the interesting findings of this study was that changes in 4R isoform specific MTBR- tau275 and MTBR-tau282 were only observed in a subset of 4R and 3R/4R mixed tauopathies. As expected, these 4R isoform specific measures did not change in 3R tauopathy (PiD) or non-tauopathy FTLD (FTLD-TDP). However, CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau specifically decreased in CBD and FTLD-MAPT but not PSP and AGD even though all are categorized as 4R tauopathies. This may be due to higher variability in the degree of neocortical pathology in PSP and AGD cases compared to CBD and most FTLD-MAPT cases. [0195] Many cases of PSP have 4R tau aggregates primarily in subcortical regions, such as the thalamus and brainstem31, and AGD pathology is most severe within the medial temporal lobe, even in advanced stages32. In contrast, CBD has more abundant and widespread tau pathology in the cerebrum33. FTLD- MAPT (e.g., P301L) can lead to a very high deposition of 4R tau aggregates in neurons and glia in multiple brain regions, including the hippocampus, neocortex, and substantia nigra34. An alternative explanation is that CBD has wispy and fine filamentous inclusions within neuronal cell bodies, whereas PSP neurons tends to harbor a larger proportion of more compact tau aggregates35. CBD is commonly associated with abundant cortical astrocytic plaque pathology and neuritic tau pathology in both gray and white matter, while PSP neuronal and astrocytic pathology (i.e., tufted astrocytes) are often restricted to the motor and premotor cortex and subcortical nuclei. It is possible that tau aggregates in PSP may have different physicochemical property from CBD and the status of equilibration between insoluble and soluble forms may be different. Together, it is speculated that the quantity or total burden of 4R tau pathology in the whole brain may reflect changes in CSF 4R specific MTBR-tau. [0196] CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau may potentially positively identify a subset of primary tauopathies and may be useful in assisting with antemortem differential diagnosis. It was confirmed in our repeated lumbar puncture study that CSF MTBR-tau/t-tau measures are reproducible and stable over 4 months, which will reliably provide biomarker values in clinic or clinical trials settings. In the clinically diagnosed cohorts without autopsy confirmation, CSF MTBR-tau275/t-tau did not change in CBS, and there was a higher overlap between CBS and PSP-RS, which might be attributable to lack of one-to-one relationship between clinical syndromes and neuropathological diagnosis in FTLD. However, 63 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web from retrospective clinical syndrome analyses in pathologically-confirmed cohort, it is shown that CSF MTBR-tau275/t-tau and MTBR-tau282/t-tau biomarkers may be able to identify individuals with CBD regardless of clinical syndromes (i.e., CBS, bvFTD, and PSP-RS) with as high as 83% accuracy, which is higher than approximately 25 to 50% diagnostic accuracies of CBD without these biomarkers. [0197] Additional limitations of this study include the following. Tauopathies with shorter duration or during asymptomatic stage may not yet show decrease in these biomarkers. Future studies targeting larger cohorts with different severity and longitudinal samples with clinical measures would help address whether CSF MTBR-tau can capture disease at earlier stage of primary tauopathies. The lack of orthogonal measures to identify brain tau pathology in living patients (i.e., tau PET imaging with a tracer specific to primary tauopathies) limits our ability to assess correlation between CSF and brain MTBR-tau antemortem. Moderate size of the cohorts with small sample sizes for subgroups such as AGD and PiD can limit interpretation. The 77G7 MTBR-tau antibody used for sequential immunoprecipitation in the study may also be targeting specific pools of truncated tau, and there may be future technical advancements and analytical method developments that may unveil additional or new populations of tau species in biofluids that reflect qualitative and quantitative aspects of tau pathology in primary tauopathies. Overall, these findings advance our knowledge of heterogeneous pathophysiology in primary tauopathies and open avenues for therapeutics development and clinical trial targeting primary tauopathies. References: [0198] 1. Ovod, V. et al. 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Utility of the global CDR® plus NACC FTLD rating and development of scoring rules: Data from the ARTFL/LEFFTDS Consortium. Alzheimers Dement.16, 106–117 (2020). [0221] 24. Sato, C. et al. MAPT R406W increases tau T217 phosphorylation in absence of amyloid pathology. Ann. Clin. Transl. Neurol.8, 1817–1830 (2021). [0222] 25. Ling, H. Clinical Approach to Progressive Supranuclear Palsy. J. Mov. Disord.9, 3–13 (2016). [0223] 26. Irwin, D. J. et al. Ante mortem cerebrospinal fluid tau levels correlate with postmortem tau pathology in frontotemporal lobar degeneration. Ann. Neurol.82, 247–258 (2017). [0224] 27. Borroni, B. et al. Csf p-tau181/tau ratio as biomarker for TDP pathology in frontotemporal dementia. Amyotroph. Lateral Scler. Front. Degener.16, 86–91 (2015). [0225] 28. Hu, W. T. et al. Reduced CSF p-Tau181 to Tau ratio is a biomarker for FTLD-TDP. Neurology 81, 1945–1952 (2013). 66 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web [0226] 29. Wagshal, D. et al. Divergent CSF τ alterations in two common tauopathies: Alzheimer’s disease and progressive supranuclear palsy. J. Neurol. Neurosurg. Psychiatry 86, 244–250 (2015). [0227] 30. Schöll, M. et al. Biomarkers for tau pathology. Mol. Cell. Neurosci.97, 18–33 (2019). [0228] 31. Kovacs, G. G. et al. Distribution patterns of tau pathology in progressive supranuclear palsy. Acta Neuropathol. (Berl.) 140, 99–119 (2020). [0229] 32. Togo, T. et al. Argyrophilic Grain Disease Is a Sporadic 4-Repeat Tauopathy. J. Neuropathol. Exp. Neurol. 61, 547–556 (2002). [0230] 33. Kouri, N., Whitwell, J. L., Josephs, K. A., Rademakers, R. & Dickson, D. W. Corticobasal degeneration: a pathologically distinct 4R tauopathy. Nat. Rev. Neurol.7, 263–272 (2011). [0231] 34. Spillantini, M. G., Crowther, R. A., Kamphorst, W., Heutink, P. & van Swieten, J. C. Tau Pathology in Two Dutch Families with Mutations in the Microtubule-Binding Region of Tau. Am. J. Pathol. 153, 1359–1363 (1998). [0232] 35. 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Claims

PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web CLAIMS What is claimed is: 1. A method of detecting tau in a cerebrospinal fluid (CSF) sample, the method comprising: (a) providing a CSF sample; and (b) detecting and measuring the amount of one or more of Microtubule Binding Region (MTBR)-tau275 peptides and MTBR-tau282 peptides. 2. A method of detecting 4R tau aggregates in a subject, the method comprising: (a) purifying endogenously cleaved fragments of tau from a CSF sample from the subject, without contacting the endogenously cleaved fragments of tau in vitro with a protease; (b) contacting the purified endogenously cleaved fragments of tau with an endopeptidase to obtain one or more of proteolytic Microtubule Binding Region (MTBR)-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and proteolytic MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptides and/or proteolytic MTBR-tau282 peptides is indicative of 4R tau aggregates in the subject. 3. The method of claim 2, wherein a solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptides is desalted before step (c), optionally wherein desalting is by solid phase extraction. 4. The method of claim 2, wherein the purifying in step (a) comprises contacting the CSF sample with one or more anti-tau epitope binding agents. 5. The method of any one of claims 2 to 4, wherein the epitope binding agent is an anti-tau antibody or antigen-binding fragment thereof. 6. The method of any one of claims 2 to 5, wherein the purifying in step (a) comprises immunoprecipitation. 68 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 7. The method of any one of claims 2 to 6, wherein the purifying in step (a) comprises immunoprecipitation of N-terminal to mid-domain tau to deplete N-terminal to mid-domain tau from the biological sample. 8. The method of claim 7, wherein the immunoprecipitation of N-terminal to mid-domain tau comprises contacting the biological sample with one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 1-243 of the sequence set forth in SEQ ID NO:1. 9. The method of any one of claims 4 to 9, wherein the epitope binding agent is one or more of: (a) anti-tau antibody Tau1 or an antigen-binding fragment thereof; (b) anti-tau antibody HJ8.5 or an antigen-binding fragment thereof; and (c) anti-tau antibody HJ8.7 or an antigen-binding fragment thereof. 10. The method of any one of claims 2 to 9, wherein the purifying in step (a) comprises immunoprecipitation of one or more MTBR-tau species from the biological sample to capture the one or more MTBR-tau species from the biological sample. 11. The method of claim 10, wherein the one or more captured MTBR-tau species comprises MTBR- tau275 peptides and/or MTBR-tau282 peptides. 12. The method of claim 10 or 11, wherein the immunoprecipitation of one or more MTBR-tau species comprises contacting the biological sample with one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244-368 relative to SEQ ID NO:1. 13. The method of any one of claims 10 to 12, wherein the immunoprecipitation of one or more MTBR-tau species comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 316-335 of the sequence set forth in SEQ ID NO:1. 14. The method of any one of claims 10 to 13, wherein the epitope binding agent is anti-tau antibody 77G7 or an antigen-binding fragment thereof. 15. The method of any one of claims 2 to 14, wherein the endopeptidase comprises trypsin. 16. The method of any one of claims 2 to 15, wherein the CSF sample comprises an internal standard. 69 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 17. The method of any one of claims 1 to 16, further comprising one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and an ApoE isoform, in the CSF sample. 18. The method of claim 17, wherein mid-domain tau is quantified, wherein the mid-domain tau comprises tau212-221 relative to SEQ ID NO: 1 (t-tau). 19. The method of claim 18, wherein the amount of the one or more of MTBR-tau275 peptides and MTBR-tau282 peptides is normalized to the amount of t-tau, and the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau is indicative of 4R tau aggregates in the subject. 20. The method of claim 19, wherein a decrease in the amount of the one or more of MTBR-tau275/t- tau and MTBR-tau282/t-tau relative to a threshold value is indicative of 4R tau aggregates in the subject. 21. A method of detecting 4R tau aggregates in a subject, the method comprising (a) performing affinity depletion on a cerebrospinal fluid (CSF sample from the subject by contacting the biological sample with one or more affinity depletion agents comprising one or more epitope binding agents that each binds to one of N-terminal tau, mid-domain tau, or C-terminal tau, but not to an antigen within Microtubule Binding Region (MTBR) tau, wherein the CSF sample comprises endogenously cleaved fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N- terminal tau, mid-domain tau, and/or C-terminal tau, and wherein the enriched sample is enriched for endogenously cleaved fragments of tau comprising one or more of endogenous MTBR-tau275 peptides comprising amino acids 275-280 relative to SEQ ID NO:1 and endogenous MTBR-tau282 peptides comprising amino acids 282-290 relative to SEQ ID NO:1; (b) performing immunoprecipitation on the enriched sample by contacting the enriched sample with one or more immunoprecipitation agents comprising one or more epitope binding agents that binds to MTBR-tau to capture the one or more of endogenous MTBR- tau275 peptides and endogenous MTBR-tau282 peptides, to obtain a purified sample; (c) contacting the one or more of endogenous MTBR-tau275 peptides and endogenous MTBR-tau282 peptides in the purified sample with an endopeptidase to obtain a sample comprising one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR- tau282 peptides; and 70 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web (d) detecting and measuring the one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC/MS) or an immunoassay, wherein the amount of the one or more of proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides is indicative of 4R tau aggregates in the subject. 22. The method of claim 21, wherein the sample comprising one or more of proteolytic MTBR- tau275 and proteolytic MTBR-tau282 peptides is desalted before step (d), optionally wherein desalting is by solid phase extraction. 23. The method of claim 21 or 22, wherein the one or more affinity depletion agents comprises one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 1-243 of the sequence set forth in SEQ ID NO:1. 24. The method of any one of claims 21 to 23, wherein the epitope binding agent is an anti-tau antibody or antigen-binding fragment thereof. 25. The method of any one of claims 21 to 24, wherein the epitope binding agent is one or more of (a) anti-tau antibody Tau1 or an antigen-binding fragment thereof; (b) anti-tau antibody HJ8.5 or an antigen-binding fragment thereof; and (c) anti-tau antibody HJ8.7 or an antigen-binding fragment thereof. 26. The method of any one of claims 21 to 25, wherein the one or more immunoprecipitation agents comprises one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244-368 of the sequence set forth in SEQ ID NO:1. 27. The method of claim 26, wherein the one or more immunoprecipitation agents comprises one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 316- 335 of the sequence set forth in SEQ ID NO:1. 28. The method of any one of claims 21 to 27, wherein the one or more immunoprecipitation agents comprises the anti-tau antibody 77G7 or an antigen-binding fragment thereof. 29. The method of any one of claims 21 to 28, wherein the endopeptidase comprises trypsin. 71 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 30. The method of any one of claims 21 to 29, further comprising one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and an ApoE isoform, in the CSF sample. 31. The method of claim 30, wherein mid-domain tau is quantified, wherein the mid-domain tau comprises tau212-221 relative to SEQ ID NO: 1 (t-tau). 32. The method of claim 31, wherein the amount of the one or more of MTBR-tau275 peptides and MTBR-tau282 peptides is normalized to the amount of t-tau, and wherein the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau is indicative of 4R tau aggregates in the subject. 33. The method of claim 32, wherein a decrease in the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value is indicative of 4R tau aggregates in the subject. 34. A method for detecting a primary tauopathy in a subject, the method comprising detecting tau or 4R tau aggregates according to the method of any one of claims 1 to 33. 35. The method of claim 34, wherein the primary tauopathy is selected from the group consisting of frontotemporal lobar degeneration (FTLD)-MAPT and corticobasal degeneration (CBD). 36. The method of claim 35, wherein a decrease in the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value is indicative of one or more of frontotemporal lobar degeneration (FTLD)-MAPT and corticobasal degeneration (CBD). 37. A method for detecting primary tauopathy-related deposition in a brain of a subject, the method comprising detecting tau or 4R tau aggregates according to the method of any one of claims 1 to 33. 38. The method of claim 37, wherein the primary tauopathy-related deposition is from frontotemporal lobar degeneration (FTLD)-MAPT or corticobasal degeneration (CBD). 39. The method of claim 38, wherein a decrease in the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau relative to a threshold value is indicative of frontotemporal lobar degeneration (FTLD)-MAPT or corticobasal degeneration (CBD). 72 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 40. A method of diagnosing frontotemporal lobar degeneration (FTLD)-MAPT in a subject, comprising (a) detecting tau or 4R tau aggregates in a CSF sample from the subject according to the method of any one of claims 1 to 33, or providing the amount of one or more of MTBR- tau275/t-tau and MTBR-tau282/t-tau from the CSF sample; and (b) diagnosing FTLD-MAPT when the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau detected is decreased relative to a threshold value. 41. A method of measuring FTLD-MAPT progression in a subject, comprising: (a) detecting tau or 4R tau aggregates in a first CSF sample and a second CSF sample from the subject according to the method of any one of claims 1 to 33, or providing the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau from the CSF sample; and (b) calculating a difference between amounts of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample and the first sample, wherein a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample as compared to the first sample indicates progression of the subject’s FTLD- MAPT. 42. A method of diagnosing corticobasal degeneration (CBD) in a subject, comprising (a) detecting tau or 4R tau aggregates in a CSF sample from the subject according to the method of any one of claims 1 to 33, or providing the amount of one or more of MTBR- tau275/t-tau and MTBR-tau282/t-tau from the CSF sample; and (b) diagnosing CBD when the amount of one or more of MTBR-tau275/t-tau and MTBR- tau282/t-tau detected is decreased relative to a threshold value. 43. A method of measuring CBD progression in a subject, comprising: (a) detecting tau or 4R tau aggregates in a first CSF sample and a second CSF sample from the subject according to the method of any one of claims 1 to 33, or providing the amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau from the CSF sample; and (b) calculating a difference between amounts of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample and the first sample, wherein a decrease in the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau in the second sample as compared to the first sample indicates progression of the subject’s CBD. 44. A method of treating a primary tauopathy in a subject in need thereof, comprising 73 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web (a) detecting tau or 4R tau aggregates in a CSF sample from the subject according to the method of any one of claims 1 to 33, or providing the amount of one or more of MTBR- tau275/t-tau and MTBR-tau282/t-tau from the CSF sample; and (b) administering to the subject a treatment that alters tau pathology if the amount of the one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau is decreased relative to the threshold value. 45. A method of treating a primary tauopathy in a subject in need thereof, comprising administering to the subject a treatment that alters tau pathology, wherein the subject has been identified as having a decreased amount of one or more of MTBR-tau275/t-tau and MTBR-tau282/t-tau, optionally as measured according to the method of any one of claims 1 to 33, relative to the threshold value. 46. The method of claim 44 or 45, wherein the treatment alters or stabilizes the amount of the detected one or more of MTBR-tau275 peptides and MTBR-tau282 peptides. 47. The method of any one of claims 44 to 46, wherein the treatment is selected from the group consisting of lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, Bepranemab, Dasatinib + Quercetin, E2814, Epothilone D, Gosuranemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX- 07010, PNT001, PRX005, RG7345, Rember TM, Semorinemab, TPI 287, Tideglusib, Tilavonemab, Zagotenemab, an anti-tau monoclonal antibody, an anti-tau anti-sense oligonucleotide, an anti-tau small interfering RNA, an tau production inhibitor, and a tau active vaccine. 48. The method of any one of claims 44 to 47, wherein the treatment is selected from the group consisting of anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, gamma-secretase inhibitors, beta-secretase inhibitors, a kinase inhibitor, a phosphatase activator, a vaccine, and a tau protein aggregation inhibitor. 49. The method of claim 48, wherein the kinase inhibitor is an inhibitor of a thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. 50. The method of claim 48, wherein the phosphatase activator increases the activity of protein phosphatase 2A. 74 92044717.7 PATENT-PRO Atty. Docket No.047563-773107 (019962-WO) Via EFS Web 51. The method of claim 48, wherein the vaccine is CAD106 or AF20513. 52. The method of claim 48, wherein the anti-Aβ antibody is aducanumab or another anti-amyloid antibody that removes plaques. 75 92044717.7
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