Method for detecting CSF MTBR-TAU and application thereof

Abstract

Provided herein are methods and compositions for detecting and measuring MTBR-tau species in CSF, and uses thereof for detecting pathological characteristics and / or clinical symptoms of tauopathies, which can be used for diagnosing, staging, or selecting treatments suitable for a given disease staging, or modifying a given treatment regimen.

Description

Technical Field

[0001] The present disclosure relates to the detection of microtubule binding region (MTBR)-tau peptides in cerebrospinal fluid (CSF), and the use of tau peptides for detecting pathological features or clinical symptoms of primary tauopathy, which can be used for diagnosis, staging, or selection of treatment for primary tauopathy. Background Art

[0002] The accumulation of tau protein as insoluble aggregates in the brain is a hallmark of neurodegenerative diseases classified as tauopathies, such as Alzheimer's disease. Tau pathology appears to propagate and spread across brain regions through the intercellular transmission of certain pathological tau species in a prion-like manner, although the spread process and nature of these species (e.g., monomers, oligomers, and fibrils) 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 over a 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). Therefore, identifying the specific pathological tau species involved in tau spread is challenging.

[0003] Mass spectrometry (MS) studies have shown that the microtubule-binding region of tau (MTBR-tau) is enriched in aggregates in Alzheimer's disease brains (Taniguchi-Watanabe et al., 2016; Roberts et al., 2020). Cryo-electron microscopy (cryo-EM) has demonstrated that the core structure of tau aggregates contains subsegments of MTBR-tau, and the specific 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 crucial for tau aggregation. However, these studies used postmortem brain tissue. Little is known about the pathophysiology of the corresponding extracellular MTBR-tau species in biological samples (e.g., CSF), which can be used as a surrogate biomarker for brain tau aggregates in living humans.

[0004] CSF is routinely obtained from study participants via lumbar puncture during clinical visits. Previous CSF tau biomarker studies have shown that MTBR-tau is absent in CSF and concentrated in the N-terminal tau and middle domain tau regions (Meredith et al., 2013; Sato et al., 2018). After truncation between the middle domain and the MTBR domain of tau, N-terminal to middle domain tau species appear to be actively secreted from neurons into the extracellular space (Sato et al., 2018). Detection of MTBR-tau species has been reported (Barthélemy et al., 2016b, a), but the relationship with the disease has not yet been characterized. Recently, a tau species containing a cleavage at residue 368 (tau368) within repeat region 4 (R4) was identified in CSF (Blennow et al., 2020). However, given the region not captured by the antibody, truncation, and changes in conformational structure, it is unclear whether tau368 reflects the overall pool of MTBR-tau species. Summary of the Invention

[0005] Provided herein are methods for detecting tau in a cerebrospinal fluid (CSF) sample that may include (a) providing a CSF sample; and (b) detecting and measuring the amount of one or more of a microtubule binding region (MTBR)-tau275 peptide and an MTBR-tau282 peptide.

[0006] Provided herein are methods for detecting 4R tau aggregates in a subject, which may include one or more of the following: (a) purifying endogenous cleavage fragments of tau from a CSF sample from the subject without exposing the endogenous cleavage fragments of tau to a protease in vitro; (b) exposing the purified endogenous cleavage fragments of tau to an endopeptidase to obtain one or more of a proteolytic MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1 and a proteolytic MTBR-tau282 peptide comprising amino acids 282-290 relative to SEQ ID NO: 1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptide and / or the proteolytic MTBR-tau282 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptide and / or the proteolytic MTBR-tau282 peptide is indicative of 4R tau aggregates in the subject. The solution containing the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptide can be desalted prior to step (c), optionally wherein the desalting is performed by solid phase extraction. The purification in step (a) may include contacting the CSF sample with one or more anti-tau epitope binding agents. The epitope binding agent may be an anti-tau antibody or an antigen-binding fragment thereof. The purification in step (a) may include immunoprecipitation, such as immunoprecipitation of N-terminal to middle domain tau, to deplete N-terminal to middle 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 shown in SEQ ID NO: 1. The epitope binding agent may be an anti-tau antibody or an antigenic fragment thereof, optionally selected from the anti-tau antibodies Tau1, HJ8.5 or HJ8.7. The purification in step (a) may include immunoprecipitating one or more MTBR-tau species from the biological sample to capture one or more MTBR-tau species from the biological sample. The one or more captured MTBR-tau species can be an MTBR-tau275 peptide, an MTBR-tau282 peptide, or a combination thereof. Immunoprecipitation of the one or more MTBR-tau species can include contacting the CSF sample with one or more anti-tau epitope binders that specifically bind to an epitope within amino acids 244-368 relative to SEQ ID NO: 1 or within amino acids 316-335 of the sequence shown in SEQ ID NO: 1. The epitope binder can be the anti-tau antibody 77G7 or an antigen-binding fragment thereof. The endopeptidase can be trypsin. The CSF sample can contain an internal standard. The method can further include one or more of detecting and quantifying one or more of beta-amyloid, N-terminal tau, middle domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample.The method can quantify the amount of mid-domain tau, optionally wherein the mid-domain tau comprises tau212-221 (t-tau) relative to SEQ ID NO: 1. One or more of the MTBR-tau275 peptide and the MTBR-tau282 peptide can be normalized to the amount of t-tau, and the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau indicates 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 can indicate 4R tau aggregates in the subject.

[0007] Provided herein are methods for detecting 4R tau aggregates in a subject, which can include (a) affinity depleting a CSF sample from the subject by contacting the biological sample with one or more affinity depleting agents, to obtain a depleted sample and an enriched sample, wherein the affinity depleting agent comprises one or more epitope binding agents that each bind to one of N-terminal tau, middle domain tau, or C-terminal tau, but do not bind to an antigen within MTBR-tau, wherein the CSF sample comprises endogenous cleavage fragments of tau, wherein the depleted sample comprises N-terminal tau, middle domain tau, and / or C-terminal tau, and wherein the enriched sample is enriched for endogenous cleavage fragments of tau comprising an endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1 and an endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1. (b) immunoprecipitating the enriched sample by contacting the enriched sample with one or more immunoprecipitants to obtain a purified sample, wherein the immunoprecipitant comprises one or more epitope binding agents that bind to MTBR-tau to capture one or more of the endogenous MTBR-tau275 peptide and the endogenous MTBR-tau282 peptide; (c) allowing the endogenous MTBR-tau275 peptide and the endogenous MTBR-tau282 peptide in the purified sample to be separated. (d) detecting and measuring one or more of the proteolytic MTBR-tau275 peptides and the proteolytic MTBR-tau282 peptides by performing LC / MS or immunoassay, wherein the amount of one or more of the proteolytic MTBR-tau275 peptides and the proteolytic MTBR-tau282 peptides indicates 4R tau aggregates in the subject. One or more of the proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides may be desalted prior to step (d), optionally wherein the desalting is performed by solid phase extraction. The one or more affinity depletion agents may comprise one or more anti-tau epitope binders that specifically bind to an epitope within amino acids 1-243 of the sequence shown in SEQ ID NO: 1. The epitope binding agent can be an anti-tau antibody or an antigenic fragment thereof, optionally selected from the anti-tau antibodies Tau1, HJ8.5, or HJ8.7. The one or more immunoprecipitants can comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244-368 of the sequence shown in SEQ ID NO: 1 or within amino acids 316-335 of the sequence shown in SEQ ID NO: 1.The one or more immunoprecipitants 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 beta-amyloid, N-terminal tau, middle domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample. The method may quantify the amount of middle domain tau, optionally wherein the middle domain tau comprises tau212-221 (t-tau) relative to SEQ ID NO: 1. One or more of the MTBR-tau275 peptide and the MTBR-tau282 peptide may be normalized to the amount of t-tau, and wherein the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau indicates 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 indicate 4R tau aggregates in the subject.

[0008] Provided herein is a method for detecting the primary tau disease in a subject, which can include providing one or more amounts of MTBR-tau 275, MTBR-tau 275 / t-tau, MTBR-tau 282 and MTBR-tau 282 / t-tau from the CSF sample of a subject. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the method for detection tau or 4R tau aggregates described above. Primary tau disease can be selected from frontotemporal lobar degeneration (FTLD) (such as FTLD-MAPT) and corticobasal degeneration (CBD). One or more amounts of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau can indicate one or more of FTLD (such as FTLD-MAPT) and CBD relative to the minimizing of threshold value. FTLD-MAPT can be P301L, R406W or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0009] Provided herein is a method for detecting the associated deposition of primary tauopathy in a subject's brain, which can include providing one or more amounts of MTBR-tau 275, MTBR-tau 275 / t-tau, MTBR-tau 282, and MTBR-tau282 / t-tau from a CSF sample of a subject. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the method for detecting tau or 4R tau aggregates described above. The associated deposition of primary tauopathy can be from FTLD (e.g., FTLD-MAPT) or CBD. The amount of one or more of MTBR-tau275 / t-tau and MTBR-Tau282 / t-tau can indicate FTLD (e.g., FTLD-MAPT) or CBD relative to the minimizing of threshold value. FTLD-MAPT can be P301L, R406W, or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0010] Provided herein are methods for diagnosing FTLD (e.g., FTLD-MAPT) in a subject, which may include (a) providing an amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample of the subject; and (b) diagnosing FTLD-MAPT when the amount of one or more of the detected MTBR-tau275 / t-tau and MTBR-tau282 / t-tau is reduced relative to a threshold value. FTLD-MAPT may be P301L, R406W, or S305I. FTLD-MAPT may be symptomatic or asymptomatic.

[0011] Provided herein is a method for measuring FTLD (e.g., FTLD-MAPT) progression in a subject, which may include (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 of a subject; and (b) calculating the difference between the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in a second sample and a first sample, wherein the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in the second sample reduces the progression of the FTLD (e.g., FTLD-MAPT) of the subject compared to the first sample. MTBR-tau275 and MTBR-tau282 may be detected according to the method for detecting tau or 4R tau aggregates described above. FTLD-MAPT may be P301L, R406W, or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0012] Provided herein are methods for diagnosing CBD in a subject, which may include (a) providing an amount of one or more of MTBR-tau 275, MTBR-tau 275 / t-tau, MTBR-tau 282, and MTBR-tau 282 / 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 reduced relative to a threshold value. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the methods for detecting tau or 4R tau aggregates described above.

[0013] Provided herein are methods for measuring the progression of CBD in a subject, which may include (a) providing an amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample of the subject; and (b) calculating the difference between the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in a second sample and a first sample, wherein a decrease in the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in the second sample compared to the first sample indicates the progression of CBD in the subject. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the method for detecting tau or 4R tau aggregates described above.

[0014] Provided herein is a method for treating primary tauopathy in a subject in need, which may include (a) providing one or more amounts of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample of a subject; and (b) if one or more amounts of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau are reduced relative to a threshold value, applying the treatment for changing tau pathology to the subject. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the method for detecting tau or 4R tau aggregates described above.

[0015] Provided herein are methods for treating 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 an amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau reduced relative to a threshold as measured according to the methods described above. The treatment can alter or stabilize the amount of one or more of the detected MTBR-tau275 peptide and MTBR-tau282 peptide. The treatment can 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, Tilavonemab, Zagotenemab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau generation inhibitors and tau active vaccines. Treatment can be selected from anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, γ-secretase inhibitors, β-secretase inhibitors, kinase inhibitors, phosphatase activators, vaccines and tau protein aggregation inhibitors. Kinase inhibitors can be inhibitors of thousand-and-oneamino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. Phosphatase activators can increase the activity of protein phosphatase 2A. The vaccine can be CAD106 or AF20513. The anti-Aβ antibody can be aducanumab or another anti-amyloid antibody that removes plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Figure 5. Demographic characteristics and brain MTBR-tau measurements of participants in the 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 mutation (P301L, S305I, R406W, IVS10+16), HS: hippocampal sclerosis, MND: motor neuron disease, LBD: Lewy body dementia, and SFG: superior frontal gyrus.

[0017] Figure 2 Demographic characteristics and CSF MTBR-tau measurements are shown for participants in the 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, and LBD: dementia with Lewy bodies. CDR plus NACC FTLD-SB: the sum of the Clinical Dementia Rating and the National Alzheimer's Coordinating Center FTLD score. ALS: amyotrophic lateral sclerosis, MCI: mild cognitive impairment, bvFTD: behavioral variant frontotemporal dementia, CBS: corticobasal syndrome, DLB: dementia with Lewy bodies, nfvPPA: nonfluent variant primary progressive aphasia, PPS: pallidopyramidal syndrome, svPPA: semantic variant primary progressive aphasia, PAGF: pure akinesia with freezing of gait, PSP-RS: progressive supranuclear palsy with Richardson syndrome, TES: traumatic encephalopathy, EOAD: early-onset Alzheimer's disease, lvPPA: oligolexic variant primary progressive aphasia, PCA: posterior cortical atrophy, CN: normal cognition. Values in square brackets indicate the amount of available information within the group (if limited).

[0018] Figure 3Figure 5 Diagnostic accuracy of the 4R-specific CSF MTBR-tau assay for differentiating CBD from FTLD-tau and controls. 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, and CI: confidence interval.

[0019] Figures 4A-4C We showed that 4R-specific insoluble brain MTBR-tau was enriched in CBD, FTLD-MAPT, AD, and PSP. Figure 4A Schematic diagram depicting quantified total tau (t-tau, 181-190) and peptides specific for the 4R isoform of tau (MTBR-tau) in the 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 ( Figure 4B ) and MTBR-tau282 / t-tau( Figure 4C Both MTBR-tau species were most enriched in CBD (n=12) and FTLD-MAPT (n=8). PSP (n=16) and AD (n=7) showed intermediate enrichment. AGD (n=1), PiD (n=3), and FTLD-TDP (n=12) showed no changes in MTBR-tau275 or MTBR-tau282 compared to NC (n=3). Red (n=9) and blue (n=1) solid circles indicate AD and PSP co-pathology, respectively. 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 (P301L, S305I, R406W, IVS10+16), SFG: superior frontal gyrus. Significance in statistical tests: ****P<0.0001, **P<0.01, *P<0.05. Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum. Differences in biomarker values were assessed using one-way ANOVA. Two-sided p<0.05 was considered statistically significant, and multiple comparisons were corrected using the Benjamini-Hochberg false discovery rate (FDR) method, with the FDR set to 5%.

[0020] Figures 5A-5C 4R-specific reduction of CSF MTBR-tau in CBD, FTLD-MAPT, and AD was demonstrated. Figure 5A Schematic diagram depicting quantification of peptides of total tau (t-tau, 212-221), truncated tau, and the 4R isoform-specific microtubule binding region (MTBR-tau) in the 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 ( Figure 5B ) and MTBR-tau282 / t-tau( Figure 5C ) were significantly decreased in CBD (n=18), AD (n=10), and FTLD-MAPT (n=5). FTLD-MAPT P301L (red, n=2), R406W (blue, n=2), and S305I (green, n=1) were decreased in MTBR-tau / t-tau measurements, in this order. Statistical significance: ****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, and FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations. Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. Differences in biomarker values were assessed using one-way ANOVA. Two-sided p < 0.05 was considered statistically significant, and the Benjamini-Hochberg false discovery rate (FDR) method was used to correct for multiple comparisons, with the FDR set at 5%.

[0021] Figures 6A-6F CSF-soluble MTBR-tau was shown to be associated with brain-insoluble MTBR-tau aggregates. MTBR-tau275 / t-tau ( Figure 6A ) and MTBR-tau282 / t-tau( Figure 6B ) were negatively correlated in tauopathies, FTLD-TDP, and controls (N=54, Spearman r=-0.27, p=0.049, -0.45, p=0.0006, respectively). MTBR-tau275 / t-tau ( Figure 6C) and MTBR-tau282 / t-tau( Figure 6D ) had a higher correlation in the 4R tauopathies (CBD, PSP, and AGD, N=29) (Spearman r=-0.61, p=0.0004, r=-0.75, p<0.0001, respectively). MTBR-tau275 / t-tau ( Figure 6E ) and MTBR-tau282 / t-tau( Figure 6F ) were associated with CBD (N=12, Spearman r=-0.25, p=0.43, r=-0.31, p=0.33, respectively). Gray shading indicates the 95% confidence interval of the linear regression. 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.

[0022] Figure 7 Show that 4R CSF MTBR-tau determination is reproducible and stable in repeated lumbar punctures. CSF MTBR-tau275 / t-tau was measured in 25 participants (#01-#25), who underwent 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 reduced CSF MTBR-tau275 / t-tau. Data are expressed as mean ± SD. CBS: corticobasal syndrome, PSP-RS: progressive supranuclear palsy with Richardson syndrome, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation.

[0023] Figure 8Figure 3 shows a decrease in CSF MTBR-tau275 / t-tau in the clinically diagnosed CBS-PSP continuum and FTLD-MAPT. Compared with NC (n=88), CSF MTBR-tau275 / t-tau was reduced in the clinically diagnosed CBS-PSP continuum (n=7) and genetically confirmed FTLD-MAPT (red: n=3, P301L, blue: n=5, R406W). Differences in biomarker values were assessed using a one-way ANOVA. Two-sided p<0.05 was considered statistically significant, and the Benjamini-Hochberg false discovery rate (FDR) method was used to correct for multiple comparisons, with the FDR set to 5%. *P<0.05. The box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. NC: normal control, bvFTD: behavioral variant frontotemporal dementia (n=28), PSP-RS: progressive supranuclear palsy with Richardson syndrome (n=16), CBS: corticobasal syndrome (n=15), AD: Alzheimer's disease (n=80), FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation (n=8).

[0024] Figure 9 Demographic characteristics and CSF MTBR-tau measures of participants in the repeat lumbar puncture cohort are shown.

[0025] Figure 10 CSF MTBR-tau 275 / t-tau biomarker positivity based on clinical syndrome in the pathologically confirmed cohort is shown.

[0026] Figure 11 CSF MTBR-tau 282 / t-tau biomarker positivity based on clinical syndrome in the pathologically confirmed cohort is shown.

[0027] Figures 12A-12C showed that CSF MTBR-tau and t-tau concentrations do not reflect CBD and FTLD-MAPT pathology. CSF MTBR-tau275 ( Figure 12A ) and MTBR-tau282( Figure 12B ) concentrations were not altered by tauopathy (total n = 112, including 29 NC). FTLD-MAPT included P301L (red, n = 2), R406W (blue, n = 2), and S305I (green, n = 1). CSF t-tau ( Figure 12C) were increased in autopsy-confirmed AD (n=10). Significance in statistical tests: *P<0.05. Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. Differences in biomarker values were assessed using one-way ANOVA. Two-sided p<0.05 was considered statistically significant, and multiple comparisons were corrected using the Benjamini-Hochberg false discovery rate (FDR) method, with the FDR set to 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.

[0028] Figures 13A-13B CSF MTBR-tau / tau was shown to be associated with disease duration in CBD. CSF MTBR-Tau275 / t-tau ( Figure 13A ) and MTBR-tau282 / t-tau( Figure 13B ) was negatively correlated with duration of disease (i.e., the interval between age of onset and CSF collection) in CBD (n=18, Spearman r=-0.37, p=0.13 and r=-0.39, p=0.11, respectively). Grey shading indicates the 95% confidence interval of the linear regression. CBD: corticobasal degeneration.

[0029] Figures 14A-14C We show that CSF pT217 / T217 identifies AD from other tauopathies. Figure 14A CSF pT217 / T217 was shown to be 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 (solid purple, n=9), which had AD co-pathology, had higher CSF pT217 / T217. FTLD-MAPT included P301L (red, n=2), R406W (blue, n=2), and S305I (green, n=1). CSF pT217 / T217 was decreased in FTLD-TDP (n=21) compared with PiD (n=5), PSP (n=22), CBD (n=18), and FTLD-MAPT (n=5) (p<0.05-0.01). CSF t-tau ( Figure 14B ) and CSF pT217 / T217( Figure 14C) can distinguish AD (n=10) from FTLD-MAPT (n=5), with AUCs equal to 0.794 and 0.987, respectively. Differences in biomarker values were assessed using one-way ANOVA. Two-sided p<0.05 was considered statistically significant, and multiple comparisons were corrected using the Benjamini-Hochberg false discovery rate (FDR) method, with the FDR set at 5%. Significance in statistical tests: ****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 mutations. AUC: area under the curve.

[0030] Figures 15A-15E CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau were not associated with amyloid pathology in primary tauopathies. Figure 15A )CSF pT217 / T217 was positively correlated with AD Thal stage (Spearman r = 0.52, p < 0.0001). CSF MTBR-tau275 / t-tau ( Figure 15B and Figure 15D ) and CSF MTBR-tau282 / t-tau( Figure 15C and Figure 15E ) compared with all tauopathy cohorts ( Figure 15B and Figure 15C , n = 79) or CBD only ( Figure 15D and Figure 15E , n=17) were not correlated with AD Thal stage measured in autopsy brains.

[0031] Figures 16A-16L : ROC curves of 4R-specific CSF MTBR-tau for differentiating CBD from controls and other tauopathies. CSF MTBR-tau275 / t-tau( Figure 16A 、 Figure 16C 、 Figure 16E 、 Figure 16G 、 Figure 16I and Figure 16K ) and MTBR-tau282 / t-tau( Figure 16B 、 Figure 16D 、 Figure 16F 、 Figure 16H 、 Figure 16J and Figure 16L ) can distinguish CBD from NC ( Figure 16A and Figure 16B )、FTLD-TDP( Figure 16C and Figure 16D ), FTLD-tau (ie, PSP, PiD, and AGD, Figure 16E and Figure 16F )、PiD( Figure 16G and Figure 16H ) and PSP( Figure 16I and Figure 16J When AD co-pathology cases were excluded from CBD and PSP, the AUC improved ( Figure 16K and Figure 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, and FTLD-MAPT, frontotemporal lobar degeneration with MAPT mutations. AUC, area under the curve; ROC, receiver operating characteristic.

[0032] Figures 17A-17E Shown are retrospective clinical syndromes and CSF MTBR-tau markers in a pathologically confirmed cohort. Figure 16A and Figure 16B ) and clinical syndromes ( Figure 16C and Figure 16D ) shows CSF MTBR-tau275 / t-tau ( Figure 17A and Figure 17C ) and MTBR-Tau282 / t-tau( Figure 16B and Figure 16D (Total n = 112). Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. Dashed lines show the cutoff values of CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau, which were 0.00563 and 0.01220, respectively. Figure 16EA schematic diagram depicting the relationship between clinical syndromes and pathological diagnoses in FTLD. Red and black lines indicate CSF MTBR-tau biomarker positivity and negativity, respectively. Biomarker positivity was determined by the median value for 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 mutation (P301L, S305I), bvFTD: behavioral variant frontotemporal dementia, CBS: corticobasal syndrome, nfvPPA: nonfluent variant primary progressive aphasia, PAGF: pure akinesia with freezing of gait, PSP-RS: progressive supranuclear palsy with Richardson syndrome, and CN: cognitively normal. DETAILED DESCRIPTION

[0033] Discussed below are components of various aspects of the methods disclosed herein. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups of these materials are disclosed, while specific reference to each different individual and collective combination and arrangement of these components may not be explicitly disclosed, each combination and arrangement is specifically contemplated and described herein.

[0034] Other aspects and iterations of the invention are described more fully below. 1. Definition

[0035] In order to make the present invention more easily understandable, first some terms are defined.Unless defined otherwise, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art generally understood by embodiments of the present invention.Many methods and materials similar to, modifying or equivalent to those described herein can be used for implementing embodiments of the present invention without excessive experimentation, and preferred materials and methods are described herein.When describing and claiming embodiments of the present invention, the following terms will be used according to the definition of the following elaboration.

[0036] 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. Thus, the terms "a" (or "a"), "one or more" and "at least one" are used interchangeably herein.

[0037] Furthermore, "and / or" when used herein should be considered to specifically disclose each of the specified features or components, with or without the additional features or components. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone).

[0038] As used herein, the term "about" refers to variations in numerical quantities that may occur, for example, by typical measurement techniques and equipment, with respect to any quantifiable variable, including but not limited to mass, volume, time, distance, and amount. In addition, given the solid and liquid handling procedures used in the real world, there are certain unintentional errors and variations that may be due to differences in the manufacture, source, or purity of the ingredients used to make the composition or implement the method, etc. The term "about" also encompasses these variations, which may be up to ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about," the claims include equivalents to these quantities.

[0039] "Specific binding" refers to the binding of a binding molecule, such as an antibody or antigen binding fragment thereof, to an epitope via its antigen binding domain, and the binding requires some recognition between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it is easier to bind to the epitope via its antigen binding domain than to a random, unrelated epitope. Thus, the term "specific binding" as used herein with respect to an epitope binding agent means that the epitope binding agent does not cross-react to a significant extent with other epitopes on the target protein (e.g., Tau) or generally on other proteins.

[0040] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibodies 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 that they exhibit the desired antigen-binding activity. The domains of an antibody that are involved in binding to an antigen are referred to as "variable regions" or "variable domains" and are described in further detail below. A single variable domain may be sufficient to provide antigen-binding specificity. Preferably, but not necessarily, the antibodies useful in the present invention are recombinantly produced. The antibodies may or may not be glycosylated, although glycosylated antibodies may be preferred. An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by methods known in the art.

[0041] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to 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-order variants thereof; single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0042] The single-chain form of an antibody and its higher-order form may include, but are not limited to, single-domain antibodies, single-chain variant fragments (scFv), divalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), double-chain antibodies (diabody) and three-chain antibodies (triabody) and four-chain antibodies (tetrabody). scFv comprises a heavy chain and a light chain variable region connected by a joint. In most cases (but not all cases), the joint can be a peptide. The length of the joint peptide is preferably about 5 to 30 amino acids, or about 10 to 25 amino acids. Generally, the joint enables the stabilization of the variable domain without interfering with the correct folding and generation of the active binding site. In a preferred embodiment, the joint peptide is rich in glycine and serine or threonine. scFv can be used to promote phage display or can be used for flow cytometry, immunohistochemistry or as a targeting domain. The method of making and using scFv is known in the art. scFv can also be conjugated to human constant domains (e.g., heavy chain constant domains derived from IgG domains, such as IgG1, IgG2, IgG3, or IgG4, or derived from heavy chain constant domains of IgA, IgM, or IgE). Diabodies, triabodies, and tetrabodies, as well as higher order variants, are typically produced by changing 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 produced using self-assembly units connected to variable domains.

[0043] "Single-domain antibodies" refer to antibody fragments consisting of a single monomeric variable antibody domain.

[0044] Multispecific antibodies include bispecific antibodies, trispecific antibodies, or antibodies with more than four specificities. Multispecific antibodies can be produced 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.

[0045] The antibodies disclosed herein can be dual affinity retargeting antibodies (DARTs). The DART format is based on a diabody format that separates the homologous variable domains of the heavy and light chains of the two antigen-binding specificities on two separate polypeptide chains. Although the two polypeptide chains are non-covalently associated in the diabody format, the DART format provides additional stabilization through a C-terminal disulfide bridge. DART can be produced in high quantity and quality and shows excellent stability in formulation buffer and human serum.

[0046] "Monoclonal antibody" refers to an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone). "Monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology well known in the art, as well as recombinant technology, phage display technology, synthetic technology, or a combination of such technology and other technologies readily known in the art. In addition, monoclonal antibodies can be labeled with a detectable marker, immobilized on a solid phase, and / or conjugated to a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[0047] A "heavy chain antibody" is an antibody composed of two heavy chains. Heavy chain antibodies can be IgG-like antibodies from camels, llamas, alpacas, sharks, etc., or IgNARs from cartilaginous fish.

[0048] "Humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of non-human antibodies eliciting an immune response in humans after administration, but retains a binding specificity and affinity similar to the starting non-human antibody. A humanized antibody binds to the same or similar epitope as a non-human antibody. The term "humanized antibody" includes antibodies that are partially or completely composed of an amino acid sequence derived from a human antibody germline by changing the sequence of an antibody with a non-human hypervariable region ("HVR"). The simplest such change can simply consist of replacing the mouse constant region with the constant region of a human antibody, thereby producing a human / mouse chimera that has sufficiently low immunogenicity to make it acceptable for pharmaceutical use. Preferably, the variable region of the antibody is also humanized by techniques currently known in the art. For example, the framework region of the variable region can be replaced by the corresponding human framework region 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), for example to restore or improve the specificity or affinity of the humanized antibody. The substantially human framework regions have at least about 75% homology to known human framework sequences (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 so that binding activity and affinity to the antigen are maintained or enhanced in the context of fully human germline framework regions or substantially human framework regions. As mentioned above, for use in the methods of the present invention, it is sufficient to employ antibody fragments. In addition, as used herein, the term "humanized antibody" refers to an antibody comprising a substantially human framework region, at least one HVR from a non-human antibody, and any constant region present therein being substantially human. The substantially human constant region has at least about 90% (i.e., about 90%, about 95%, or about 99% sequence identity) to known human constant sequences. Thus, except for possible HVRs, all portions of a humanized antibody are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences.

[0049] The term "Aβ" refers to a peptide derived from a region at the carboxyl terminus of a larger protein called the amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Aβ that can have toxic effects: Aβ peptides are typically 37-43 amino acid sequences in length, although they can have truncations and modifications that alter their overall size. They can be found as monomers, oligomers, and aggregates, in soluble and insoluble compartments, inside or outside cells, and can be complexed with other proteins or molecules. The adverse or toxic effects of Aβ can be attributed to any or all of the above forms, as well as other forms not specifically described. For example, two such Aβ subtypes include Aβ40 and Aβ42; of which the Aβ42 subtype is particularly fibrillogenic or insoluble and is associated with disease states. The term "Aβ" generally refers to multiple Aβ species, without distinguishing between individual Aβ species. Specific Aβ species are identified by the size of the peptide, such as Aβ42, Aβ40, Aβ38, etc.

[0050] As used herein, the term "Aβ42 / Aβ40 value" or "Aβ42 / Aβ40 ratio" refers to the ratio of the amount of Aβ42 in a sample obtained from a subject compared to the amount of Aβ40 in the same sample.

[0051] As used herein, the term "subject" refers to a mammal, preferably a human. Mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be awaiting medical care or treatment, may be currently receiving medical care or treatment, or may have already received medical care or treatment.

[0052] As used herein, the terms "control population," "normal population," or sample from a "healthy" subject refer 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.

[0053] As used herein, the term "isoform" refers to any of several different forms of the same protein variant that arise due to alternative splicing of the mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein, genetic variation, and somatic recombination. The terms "isoform" and "variant" are used interchangeably.

[0054] The term "tau" refers to a variety of subtypes encoded by gene MAPT (or its homologues), and species of its C-terminal truncation in vivo, N-terminal truncation in vivo, post-translational modification in vivo, or any combination thereof. As used herein, the terms "tau" and "tau protein" and "tau species" are used interchangeably. In many animals (including but not limited to humans, non-human primates, rodents, fish, cattle, frogs, goats, and chickens), tau is encoded by gene MAPT. In animals where gene is not identified as MAPT, homologues can be identified by methods well known in the art.

[0055] In humans, there are six tau isoforms resulting from 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 each encode a 29-amino acid insert (designated N) at the N-terminus, and full-length human tau isoforms may have two inserts (2N), one insert (1N), or no insert (0N). All full-length human tau isoforms also have three repeats of the microtubule-binding domain (designated R). The inclusion of exon 10 at the C-terminus results in the inclusion of a fourth microtubule-binding domain encoded by exon 10. Therefore, full-length human tau isoforms may contain repeats of four microtubule-binding domains (including exon 10: R1, R2, R3, and R4) or repeats of three microtubule-binding domains (excluding exon 10: R1, R3, and R4). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated and saccharified. Human tau may or may not be proteolytically processed at the C-terminus, at the N-terminus, or at the C-terminus and N-terminus in vivo. Therefore, the term "human tau" encompasses 2N3R, 2N4R, 1N3R, 1N4R, ON3R and ON4R subtypes, and species thereof that are truncated at the C-terminus in vivo, truncated at the N-terminus in vivo, post-translational modifications in vivo, or any combination thereof. Variable splicing of genes encoding tau similarly occurs in other animals.

[0056] As used herein, the term "tau-441" or "full-length tau" 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: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPS LPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 1). N-terminal (N-terminal) tau, middle domain tau, MTBR-tau and C-terminal (C-terminal) tau are described below for this subtype. For other tau subtypes (e.g., 2N3R, 1NR4, 1N3R, 0N4R and 0N3R), these regions will vary in a predictable manner. Thus, when an amino acid position is determined relative to tau-441, the skilled artisan will be able to determine the corresponding amino acid position for the other isoforms.

[0057] As used herein, the term "N-terminal tau" refers to a tau peptide or tau proteins comprising two or more or all of the amino acids at the N-terminus of tau (eg, amino acids 1-103 of tau-441).

[0058] As used herein, the term "middle domain tau" refers to a tau peptide or multiple tau proteins comprising two or more or all of the amino acids of the middle domain of tau (eg, amino acids 104-243 of tau-441).

[0059] As used herein, the term "MTBR tau" or "MTBR-tau" refers to a tau peptide or multiple tau proteins comprising 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).

[0060] As used herein, the term "C-terminal tau" refers to a tau peptide or multiple tau proteins comprising two or more or all of the amino acids at the C-terminus of tau (eg, amino acids 369-441 of tau-441, etc.).

[0061] Diseases associated with tau deposition in the brain are referred to herein as “tauopathies.” The term “tau deposition” includes all forms of pathological tau deposition, including but not limited to tau aggregates in neurofibrillary tangles, neuropil threads, and 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 Parkinson's disease linked to chromosome 17, Lytico-Bodig disease, Parkinsonism-dementia of Guam complex, tangle-predominant dementia, gangliogliomas and gangliocytomas, 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).

[0062] Tauopathies are classified by the preponderance of the tau subtypes found in pathological tau deposits.Those tauopathies with tau deposits consisting mainly of tau with three MTBRs are referred to as "3R-tauopathies".Pick's disease is a non-limiting example of 3R-tauopathies.For clarification, the pathological tau deposits of some 3R-tauopathies can be a mixture of 3R and 4R tau subtypes, wherein the 3R subtype is dominant.It is generally believed that the intracellular neurofibrillary tangles (i.e. tau deposits) in the brain of the experimenter suffering from Alzheimer's disease contain roughly equal amounts of 3R and 4R subtypes.Those tauopathies with tau deposits consisting mainly 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 the brains of some subjects with genetically confirmed cases of FTLD (e.g., some V334M and R406W mutation carriers) show a mixture of 3R and 4R isoforms.

[0063] The clinical signs of tauopathy can 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., 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]tauPET of JNJ-067, etc.

[0064] As used herein, the terms "treat," "treating," or "treatment" refer to the provision of medical care to a subject in need by a trained and licensed professional. Medical care can be a diagnostic test, therapeutic treatment, and / or preventive or prophylactic measure. The goal of therapeutic and prophylactic treatment is to prevent or slow down (mitigate) undesirable physiological changes or diseases / conditions. The beneficial or desired clinical outcomes of therapeutic or prophylactic treatment include, but are not limited to, relief of symptoms, alleviation of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or total relief), whether detectable or undetectable. "Treatment" can also refer to prolonged survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a disease, condition, or illness, as well as those susceptible to a disease, condition, or illness, or those to be prevented from a disease, condition, or illness. Thus, a subject in need of treatment may or may not have any symptoms or clinical signs of the disease.

[0065] The phrase "tau therapy" generally refers to any imaging agent, therapeutic treatment and / or preventive or prophylactic measure that is considered for use in a subject at risk of developing tau disease or a subject clinically diagnosed with tau disease or used by the subject. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, flubitaban, flubetapir (florbetapir), flumetazol, 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.), γ-secretase inhibitors, β-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 and lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), antagonists of serotonin receptor 6, p38αMAP K inhibitors, recombinant granulocyte macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylsulfoximine chloride, etc.), therapies to improve glycemic 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, beta-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 stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-γ agonists, tubulin modulators, calcium channel blockers, antihypertensive drugs, statins, and any combination thereof.

[0066] The accumulation of aggregated tau in the brain is a hallmark of neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD-tau) (including cortical basal ganglia degeneration (CBD)). Biomarkers in cerebrospinal fluid (CSF) and blood provide accurate performance for identifying AD. However, there are no biomarkers to conclusively identify non-AD tauopathy. Since some FTLD-tau (e.g., CBD) brains accumulate tau species from 4-repeat (4R) tau specific domains in the microtubule binding region (MTBR), the present disclosure examines whether the CSF levels of these MTBR-tau species change in FTLD-tau CSF as a reflection of tau aggregates accumulated in the brain. A method for quantitatively determining the various tau species in CSF was designed and applied to two discovery studies to analyze CSF from individuals (including genetically confirmed and pathologically unconfirmed cases) suffering from AD and frontotemporal lobar dementia (FTD) syndrome. Compared with cases with asymptomatic AD and healthy controls, levels of specific MTBR-tau species relative to total tau (t-tau) were specifically reduced in CSF from individuals with symptomatic AD and FTD syndrome. Notably, pathogenic FTLD-tau mutants (i.e., P301L and R406W) also significantly reduced specific CSF mTBR-tau levels, suggesting that this change may recapitulate tau pathology in FTLD-tau. Finally, all pathologically or genetically confirmed CSF MTBR-tau species were analyzed as a validation study. Levels of specific MTBR-tau species normalized by t-tau distinguished CBD and FTD with certain specific MAPT mutations (P301L and R406W) from other FTLD-taus such as progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), Pick's disease (PiD), and "tau-independent FTLD (e.g., FTLD-TDP43)", suggesting that specific MTBR-tau species in CSF can be used as the first definitive biomarker recapitulating tau pathology in non-AD tauopathies.

[0067] The present disclosure provides methods of measuring MTBR-tau peptide in a biological sample (eg, by mass spectrometry or immunoassay).

[0068] In each of the methods described herein, the present disclosure further contemplates determining the presence / absence of one or more proteins in a biological sample and / or measuring the concentration of one or more other proteins in a biological sample. In some embodiments, one or more proteins may be proteins exhausted from a biological sample before purifying tau. For example, in certain embodiments, N-terminal tau and / or intermediate domain tau species can be separately identified and / or quantified from tau species (e.g., MTBR-tau, C-terminal tau) quantified by the methods disclosed herein. In some embodiments, in addition to endogenous cleavage fragments of tau, post-translational modifications such as phosphorylation of tau at specific residues can also be measured and quantified. Alternatively or additionally, a portion of a biological sample can be processed in parallel, by exhausting target protein from a biological sample before being used in the methods disclosed herein, or by exhausting target protein from a biological sample during the sample processing steps disclosed herein to identify and / or quantify Aβ (e.g., Aβ42 / Aβ40), ApoE, or any other target protein.

[0069] The steps of preparing a biological sample, a suitable internal standard and purified tau, optionally depleting one or more proteins, optionally cleaving the purified tau with a protease, and detecting and measuring endogenous cleaved peptide fragments of tau are described in more detail below. 2. Methods for detecting tau fragments

[0070] The present disclosure provides a method for detecting tau (for example, peptide fragments of tau) in cerebrospinal fluid (CSF) samples. Detection can include confirming the presence of tau, for example, to measure the amount of peptide fragments of tau or the peptide fragments of quantitative tau. The presence, measurement or quantitative amount of the peptide fragments of tau can indicate insoluble tau or tangles (for example, neurofibrillary tangles) of the aggregation relevant to tau disease. Detection and measurement can be carried out by mass spectrometry or immunoassay. Biological sample can be obtained from a subject suffering from or suspected of suffering from tau disease.

[0071] As described herein, peptide fragments of tau (e.g., fragments of tau) may be endogenously cleaved (e.g., cleaved in a cell or tissue of a subject). In some embodiments, the peptide fragments of tau comprise amino acids of the 4R tau splice isoform of tau. In some embodiments, the peptide fragments of tau comprise amino acids of a portion of the R2 region of tau. In some embodiments, as described herein, the peptide fragments of tau comprise the MTBR-tau275 peptide. In some embodiments, the peptide fragments of tau (e.g., MTBR-tau275 peptide) comprise or consist of amino acids 275-280 of tau-441. In some embodiments, the peptide fragments of tau (e.g., MTBR-tau275 peptide) comprise or consist of amino acids 275-280 as shown in SEQ ID NO: 1. In some embodiments, the peptide fragments of tau comprise or consist of the amino acid sequence VQIINK (SEQ ID NO: 2). In some embodiments, as described herein, the peptide fragment of tau comprises the MTBR-tau282 peptide. In some embodiments, the peptide fragment of tau (e.g., the MTBR-tau282 peptide) comprises or consists of amino acids 282-290 of tau-441. In some embodiments, the peptide fragment of tau (e.g., the MTBR-tau282 peptide) comprises or consists of amino acids 282-290 as set forth in SEQ ID NO: 1. In some embodiments, the peptide fragment of tau comprises or consists of the amino acid sequence LDLSNVQSK (SEQ ID NO: 3).

[0072] Detecting tau (e.g., tau peptide fragments) in a CSF sample may include providing a CSF sample and purifying tau peptides from the sample. In some embodiments, the CSF sample contains endogenously cleaved tau. The endogenous cleavage fragments of tau may not be trypsin tau peptides. Purification can be performed without first exposing the sample to additional in vitro proteolytic cleavage. No pre-clearing step (e.g., protein precipitation and / or immunodepletion) may be performed before or after purification of the endogenous cleavage fragments of tau. The endogenous cleavage fragments of tau may be further cleaved in vitro with a protease before or after purification. In some embodiments, the protease is trypsin.

[0073] A sample as described herein (e.g., a CSF sample containing endogenous cleavage fragments of tau) can be subjected to at least one or both steps of immunodepletion and / or immunoprecipitation. Each round of immunodepletion or immunoprecipitation can be performed using one or more anti-tau or anti-MTBR-tau antibodies described herein, or a combination thereof. Following each immunodepletion or immunoprecipitation step, one or more purified tau peptides from the immunoprecipitated or immunodepleted sample can be further cleaved using one or more proteases described herein.

[0074] Liquid chromatography-mass spectrometry (LC / MS) or immunoassays using samples containing purified peptide fragments of tau (e.g., endogenous cleavage fragments of tau) can be used to detect and measure the concentration (relative or absolute) of at least one fragment of tau. One or more tau fragments can be used to detect and measure the amount of tau present in a biological sample, such as the amount of insoluble tau aggregates. The immunoassay can include contacting a sample containing purified peptide fragments of tau with an anti-MTBR-tau antibody disclosed herein. Detection of tau in CSF samples

[0075] As contemplated herein, detecting tau (e.g., tau peptide fragments) in a CSF sample can include one or more of confirming the presence of tau peptides in the CSF sample, measuring tau peptides, and / or quantifying tau peptides (e.g., the amount of tau peptides). In some embodiments, the methods as described herein include detecting and measuring the amount of proteolytic MTBR-tau275 peptides, proteolytic MTBR-tau282 peptides, or both.

[0076] In one aspect, provided herein is a method for detecting tau peptides in a CSF sample. The method comprises the following steps: (a) providing a CSF sample (e.g., a CSF sample containing endogenously cleaved tau fragments); (b) detecting and measuring the amount of one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and MTBR-tau282 peptide). One or more of the MTBR-tau peptides can be detected by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay on the sample to detect and / or measure the amount of one or more of the MTBR-tau peptides.

[0077] In one example, the methods of the present disclosure further include purifying one or more of the MTBR-tau peptides from the CSF sample, wherein the one or more of the MTBR-tau peptides are optionally further cleaved in vitro with one or more proteases before or after purification. In some embodiments, the one or more of the MTBR-tau peptides are not cleaved in vitro (e.g., non-endogenously) prior to purification.

[0078] In another example, the method of the present disclosure further comprises reducing at least one tau peptide in the CSF sample by affinity depletion or immunoprecipitation, wherein the at least one tau peptide that is affinity depleted or immunoprecipitated is not one or more of the MTBR-tau peptides used for detection and / or measurement (e.g., not MTBR-tau275 or MTBR-tau282). In some embodiments, the affinity depletion or immunoprecipitation is affinity depletion or immunoprecipitation of N-terminal tau, middle domain tau, and optionally C-terminal tau, or a combination thereof. Although not required, in each of the methods described herein, additional proteins can be removed from the CSF sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant before or after purification of one or more of the MTBR-tau peptides.

[0079] In another example, the disclosed method of further enriching one or more of the MTBR-tau peptides comprises purifying one or more of the MTBR-tau peptides from an affinity-depleted sample. One or more of the enriched MTBR-tau peptides may be enriched for MTBR-tau275 peptide and / or MTBR-tau282 peptide. MTBR-tau275 peptide and / or MTBR-tau282 peptide may be detected and / or measured using an immunoassay that may include the use of anti-tau antibody 77G7 or an antigen-binding fragment thereof. A sample containing the enriched MTBR-tau275 peptide and / or MTBR-tau282 peptide may be proteolytically cleaved using a protease. The resulting cleavage may be desalted (e.g., by solid phase extraction) to obtain a sample containing one or more of the MTBR-tau peptides (e.g., MTBR-tau275 peptide and / or MTBR-tau282 peptide).

[0080] 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 MTBR-tau275 peptide. In some embodiments, the method comprises detecting and / or measuring the amount of MTBR-tau282 peptide.

[0081] In another aspect, provided herein is a method for detecting 4R tau aggregates in a subject. The method comprises one or more of the following steps: (a) purifying endogenous cleavage fragments of tau from a CSF sample from a subject without exposing the endogenous cleavage fragments of tau to a protease in vitro; (b) exposing the purified endogenous cleavage fragments of tau to an endopeptidase to obtain one or more of a proteolytic MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1 and a proteolytic MTBR-tau282 peptide comprising amino acids 282-290 relative to SEQ ID NO: 1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptide and / or the proteolytic MTBR-tau282 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptide and / or the proteolytic MTBR-tau282 peptide indicates 4R tau aggregates in the subject. In some embodiments, the method further comprises desalting the solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR-Tau282 peptide prior to step (c). In some embodiments, desalting is performed by solid phase extraction. In some embodiments, the purification in step (a) comprises contacting the CSF sample with one or more anti-tau epitope binding agents.

[0082] In some embodiments, the purification in step (a) comprises immunoprecipitation. In some embodiments, the immunoprecipitation of purification step (a) is used to immunoprecipitate N-terminal to middle domain tau (e.g., to deplete N-terminal to middle domain tau from a CSF sample). In some embodiments, immunoprecipitating N-terminal to middle domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal to middle domain tau. In some embodiments, the immunoprecipitation of purification step (a) is used to immunoprecipitate N-terminal tau (e.g., to deplete N-terminal tau from a CSF sample). In some embodiments, immunoprecipitating 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 purification step (a) is used to immunoprecipitate middle domain tau (e.g., to deplete middle domain tau from a CSF sample). In some embodiments, immunoprecipitating middle domain tau comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to middle domain tau. In some embodiments, the immunoprecipitation of the purification step (a) is used to immunoprecipitate one or more MTBR-tau species from the CSF sample (e.g., to capture one or more MTBR-tau species from a biological sample). In some embodiments, immunoprecipitating 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.

[0083] In another aspect, provided herein are methods for detecting 4R tau aggregates in a subject, the methods comprising one or more of the following steps: (a) affinity depleting a cerebrospinal fluid (CSF) sample from the subject by contacting the biological sample with one or more affinity depleting agents to obtain a depleted sample and an enriched sample, the affinity depleting agents comprising one or more epitope binding agents that each bind to one of N-terminal tau, middle domain tau, or C-terminal tau, but not to an antigen within MTBR-tau, wherein the CSF sample comprises endogenous cleavage fragments of tau, wherein the depleted sample comprises N-terminal tau, middle domain tau, and / or C-terminal tau, and wherein the enriched sample is enriched for endogenous cleavage fragments of tau comprising an endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1 and an endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1. (b) immunoprecipitating the enriched sample by contacting the enriched sample with one or more immunoprecipitants to obtain a purified sample, wherein the immunoprecipitant comprises one or more epitope binding agents that bind to MTBR-tau to capture one or more of the endogenous MTBR-tau275 peptide and the endogenous MTBR-tau282 peptide; (c) allowing the endogenous MTBR-tau275 peptide and the endogenous MTBR in the purified sample to be purified. -tau282 peptides are contacted with an endopeptidase to obtain a sample comprising one or more of a proteolytic MTBR-tau275 peptide and a proteolytic MTBR-tau282 peptide; and (d) detecting and measuring one or more of the proteolytic MTBR-tau275 peptide and the proteolytic MTBR-tau282 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein the amount of one or more of the proteolytic MTBR-tau275 peptide and the proteolytic MTBR-tau282 peptide indicates 4R tau aggregates in the subject. In some embodiments, the sample comprising one or more of the proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides is desalted prior to the step. In some embodiments, desalting is performed by solid phase extraction.

[0084] In some embodiments, one or more affinity depletion agents include one or more anti-tau epitope binding agents that specifically bind to N-terminal to middle domain tau. In some embodiments, one or more affinity depletion agents include one or more anti-tau epitope binding agents that specifically bind to N-terminal tau. In some embodiments, one or more affinity depletion agents include one or more anti-tau epitope binding agents that specifically bind to middle domain tau. In some embodiments, one or more immunoprecipitants include one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species (e.g., MTBR-tau275 and / or MTBR-tau282).

[0085] A biological sample containing endogenous cleavage 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. Samples depleted of tau peptides using the above-mentioned antibodies can be proteolytically cleaved in vitro using one or more proteases disclosed herein, and the resulting tau peptides cleaved in vitro (proteolytic fragments) can be contacted with antibodies bound to one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and / or MTBR-tau282 peptide), such as anti-tau antibodies. In some embodiments, the protease is trypsin. 2.2. Epitope Binding Agents

[0086] In some embodiments of the methods described herein, one or more anti-tau epitope binding agents that specifically bind to tau from the N-terminus to the middle domain bind to an epitope within amino acids 1-243 of tau (e.g., tau-441). In some embodiments, one or more anti-tau epitope binding agents that specifically bind to tau from the N-terminus to the middle domain bind to an epitope within amino acids 1-243 relative to SEQ ID NO: 1. In some embodiments, one or more anti-tau epitope binders that specifically bind to tau from the N-terminus to the middle domain bind to an epitope within the following amino acid sequence: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRL (SEQ ID NO: 4). In some embodiments, one or more anti-tau epitope binders that specifically bind to tau from the N-terminus to the middle domain are selected from the anti-tau antibodies HJ8.5, HJ8.7, or Tau1, or antigen-binding fragments thereof.

[0087] In some embodiments of the methods described herein, 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., tau-441). In some embodiments, 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, one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within the following amino acid sequence: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEP GSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTA (SEQ ID NO: 5).

[0088] In some embodiments of the methods described herein, one or more anti-tau epitope binding agents that specifically bind to middle domain tau bind to an epitope within amino acids 104-243 of tau (e.g., tau-441). In some embodiments, one or more anti-tau epitope binding agents that specifically bind to middle domain tau bind to an epitope within amino acids 104-243 relative to SEQ ID NO: 1. In some embodiments, one or more anti-tau epitope binding agents that specifically bind to middle domain tau bind to an epitope within the following amino acid sequence: EEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVA VVRTPPKSPSSAKSRL (SEQ ID NO: 6).

[0089] In some embodiments of the methods described herein, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species specifically bind to an epitope within amino acids 244-368 of tau (e.g., tau-441). In some embodiments, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species bind to an epitope within amino acids 244-368 relative to SEQ ID NO: 1. In some embodiments, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species bind to an epitope of the following amino acid sequence: QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGG SVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPG GGN (SEQ ID NO: 7). In some embodiments, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species specifically bind to an epitope within amino acids 316-335 of tau (e.g., tau-441). In some embodiments, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species bind to an epitope within amino acids 316-335 relative to SEQ ID NO: 1. In some embodiments, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species bind to an epitope of the following amino acid sequence: SKVTSKCGSLGNIHHKPGGG (SEQ ID NO: 8). In some embodiments, one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species are anti-tau antibodies 77G7 or antigen-binding fragments thereof. In some embodiments, immunoprecipitating one or more MTBR-tau species is used to immunoprecipitate MTBR-tau275 peptide and / or MTBR-tau282 peptide. In some embodiments, the one or more captured MTBR-tau species comprises an MTBR-tau275 peptide and / or an MTBR-tau282 peptide.

[0090] A CSF sample containing tau (e.g., an endogenous cleavage fragment of tau) is contacted with one or more, or all, of the following: anti-tau antibody HJ8.5, or an antigen-binding fragment thereof; anti-tau antibody HJ8.7, or an antigen-binding fragment thereof; and / or anti-tau antibody Tau1, or an antigen-binding fragment thereof. In some embodiments, the CSF sample is contacted with anti-tau antibodies Tau1, HJ8.5, and HJ8.7, or antigen-binding fragments thereof. Samples depleted of tau peptides using the above antibodies can be contacted with antibodies that bind to MTBR-tau275 and MTBR-Tau282 (e.g., anti-tau antibody 77G7), or antigen-binding fragments thereof. Biological samples

[0091] Suitable biological samples include cerebrospinal fluid (CSF) samples obtained from experimenter. In some embodiments, experimenter is a human. Human experimenter can be waiting for medical care or treatment, can be in medical care or treatment or can have received medical care or treatment. In various embodiments, human experimenter can be a healthy subject, a subject under the risk of developing neurodegenerative disease, a subject with signs and / or symptoms of neurodegenerative disease or a subject diagnosed with neurodegenerative disease. In further embodiments, neurodegenerative disease can be tauopathy, which can be primary tauopathy. In a particular instance, tauopathy can be frontotemporal lobar degeneration (FTLD-tau) (such as FTLD-MAPT), including cortical basal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), globular glial tauopathy (GGT), chronic traumatic encephalopathy (CTE), Pick's disease (PiD) or Alzheimer's disease. In specific embodiments, primary tauopathy is FTLD-tau or CBD. In certain embodiments, the FTLD-tau is FTLD-MAPT. In certain 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 an experimental animal. In another embodiment, the subject is a laboratory animal genetically engineered to express human tau and, optionally, one or more additional human proteins (e.g., human Aβ, human ApoE, etc.).

[0092] CSF can be obtained by lumbar puncture with or without an indwelling CSF catheter. A plurality of CSF samples collected simultaneously from a subject can be combined. Once collected, the CSF sample can be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cell debris; using additives designed to stabilize and preserve the sample before analysis and testing; etc.). The CSF sample can be used immediately or can be frozen and stored indefinitely. Before being used for the methods disclosed herein, if necessary or desired, the CSF sample can also be modified to include protease inhibitors, isotope-labeled internal standards, detergents and chaotropes, and / or optionally depleted of other analytes (e.g., protein peptides, metabolites).

[0093] The size of the sample used can and will vary depending on the sample type, the health of the subject from whom the sample is obtained, and the analyte (other than tau) to be analyzed. The CSF sample volume can be from about 0.01 mL to about 5 mL, or from about 0.05 mL to about 5 mL. In a specific example, the sample size can be from about 0.05 mL to about 1 mL of CSF.

[0094] In some embodiments, a single sample is obtained from a subject. Alternatively, a sample can be obtained from a subject over time. Therefore, more than one sample can be collected from a subject over time. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more samples can be collected from a subject over time. When more than one sample is collected from a subject over time, samples can be collected every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days or more. In some embodiments, samples are collected at intervals of one month, 3 months, 6 months, 1 year, 2 years, 5 years, 10 years, 20 years or more. Purification of tau

[0095] Another step of the methods disclosed herein includes purifying tau (e.g., peptide fragments of tau), particularly endogenous cleavage and / or proteolytic cleavage fragments of tau. In some instances, the cleavage fragments of tau are N-terminus-independent, mid-domain-independent, and / or C-terminus-independent. The purified tau can be partially purified or completely purified.

[0096] In some embodiments, the methods of the present disclosure include purifying tau peptides (e.g., endogenously cleaved tau fragments) by affinity purification. Affinity purification refers to a method for enriching a target protein by its specific binding properties to a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support such as a bead, resin, tissue culture plate, or the like. Immobilization of the ligand to the solid support can also occur after a ligand-protein interaction occurs. Suitable ligands include antibodies, aptamers, and other epitope binding agents. Purifying tau peptides by affinity purification includes contacting a sample containing endogenous cleavage fragments of tau with a suitable immobilized ligand, one or more washing steps, and eluting the cleavage fragments of tau from the immobilized ligand.

[0097] In some embodiments, the methods of the present disclosure comprise purifying tau peptides (e.g., endogenously cleaved tau fragments) by affinity purification using at least one epitope binding agent that specifically binds to an epitope within amino acids 225-258 (inclusive) of tau-441, or within amino acids 235-258 (inclusive) of tau-441, or within amino acids 235-242 (inclusive) (or within similarly defined regions of other full-length 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. 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.

[0098] In some embodiments, the methods of the present disclosure include purifying tau peptides by solid phase extraction. Purification by solid phase extraction includes contacting a sample containing tau with a solid phase containing an adsorbent to which tau is adsorbed, one or more washing steps, and eluting tau from the adsorbent. Suitable adsorbents include reversed-phase adsorbents. Suitable reversed-phase adsorbents are known in the art and include, but are not limited to, alkyl-bonded silica, aryl-bonded silica, styrene / divinylbenzene materials, and 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, the adsorbent is Oasis HLB (Waters). Prior to contact with the supernatant containing tau, the adsorbent is typically pretreated according to the manufacturer's instructions or as known in the art (e.g., with a water-miscible organic solvent and then with a buffer comprising a mobile phase). In addition, the supernatant may optionally be acidified because some reversed-phase materials retain ionized analytes more strongly than others. The use of volatile components in the mobile phase and for elution is preferred because they promote sample drying. In an exemplary embodiment, the washing step may include using a liquid phase comprising about 0.05% v / v trifluoroacetic acid (TFA) to about 1% v / v TFA or its equivalent. In some instances, the washing can be performed 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 instances, the washing can be performed 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. The bound tau is then eluted with a liquid phase comprising about 20% v / v to about 50% v / v acetonitrile (ACN) or its equivalent. In some examples, tau can 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 can 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 can be dried by methods known in the art, such as vacuum drying (e.g., speed-vac), lyophilization, evaporation under a stream of nitrogen, etc. 2.5. Depletion of one or more proteins

[0099] The methods of the present disclosure may include a step of depleting one or more proteins from a sample. The term "deplete" means a decrease in quantity or number. Thus, a sample depleted of a protein can have any amount of protein that is measurably less than the amount in the original sample, including an amount without the protein.

[0100] It is possible to deplete protein from a sample by a method specifically targeting one or more proteins, for example, by affinity depletion, solid phase extraction, or other methods known in the art. Targeted depletion of a protein or multiple proteins can be used in situations where downstream analysis (e.g., analysis of identification, quantification, post-translational modification, etc.) of the protein is required. For example, Aβ peptides can be identified and quantified by methods known in the art after affinity depletion of Aβ with a suitable epitope binding agent. As another non-limiting example, apolipoprotein E (ApoE) status can be determined by methods known in the art after affinity depletion of ApoE and identification of ApoE subtypes. Targeted depletion can also be used to separate other proteins for subsequent analysis, including but not limited to apolipoprotein J, synuclein, soluble amyloid precursor protein, α-2 macroglobulin, S100B, myelin basic protein, 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 are co-founded in mass spectrometry analysis. For example, in certain embodiments of the present disclosure, N-terminal tau, middle domain tau, and / or C-terminal MTBR-tau are depleted from the sample prior to further sample processing for analysis. Downstream analysis of the depleted tau protein may or may not occur, but the methods of the present disclosure contemplate both options.

[0101] In some embodiments, targeted depletion can occur by affinity depletion. Affinity depletion refers to a method of depleting the target protein from a sample by its specific binding properties to a molecule. Typically, a molecule is a ligand (referred to as an immobilized ligand) attached to a solid support such as a bead, resin, tissue culture plate, or the like. Immobilization of the ligand to the solid support can also occur after a ligand-protein interaction occurs. Suitable ligands include antibodies, aptamers, and other epitope binding agents. The molecule can also be a polymer or other material that selectively adsorbs the target protein. As a non-limiting example, polyhydroxymethylene (e.g., PHM-L LIPOSORB, Sigma Aldrich) substituted with fatty oxyethylated alcohols can be used for selective adsorption of lipoproteins (including ApoE) from serum. Two or more affinity depletion agents can be combined to deplete multiple proteins sequentially or simultaneously.

[0102] In some embodiments, the methods of the present disclosure include affinity depletion of one or more proteins 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 the ON or 1N isoform). 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, the methods of the present disclosure comprise affinity depleting one or more proteins 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 middle domain of tau (e.g., amino acids 104 to 243 of tau-441, inclusive). The epitope-binding agents can be used sequentially or simultaneously.

[0104] In some embodiments, the methods of the present disclosure comprise affinity depleting one or more proteins from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35, inclusive, of tau-441 and an epitope-binding agent that specifically binds to an epitope within amino acids 104 to 243, inclusive, of tau-441 (or within a similarly defined region for the ON or 1N subtype). The epitope-binding agents can be used sequentially or simultaneously.

[0105] In some embodiments, the methods of the present disclosure include affinity depleting one or more proteins 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 the ON or 1N isoform); 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 the ON or 1N isoform); and an epitope-binding agent that specifically binds to an epitope of β-amyloid protein. The epitope-binding agents can be used sequentially or simultaneously.

[0106] In some embodiments, the methods of the present disclosure include affinity depleting one or more proteins 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 the ON or 1N isoform); 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 the ON or 1N isoform); and an epitope-binding agent that specifically binds to an epitope of β-amyloid protein. The epitope-binding agents can be used sequentially or simultaneously.

[0107] In some embodiments, the methods of the present disclosure include affinity depleting one or more proteins 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 the ON or 1N isoform); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents can be used sequentially or simultaneously.

[0108] In some embodiments, the methods of the present disclosure include affinity depleting one or more proteins from a sample using: an epitope binding agent that specifically binds to an epitope within amino acids 1 to 35 (inclusive) of tau-441 (or within a similarly defined region for the ON or 1N isoform); and an epitope binding agent that specifically binds to an epitope of β-amyloid. The epitope binding agents can be used sequentially or simultaneously.

[0109] In some embodiments, the methods of the present disclosure include affinity depleting one or more proteins 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 the ON or 1N isoform); and an epitope binding agent that specifically binds to an epitope of beta amyloid. The epitope binding agents can be used sequentially or simultaneously.

[0110] In some embodiments, the methods of the present disclosure comprise affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 260 to 441, inclusive, of tau-441.

[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 an epitope within amino acids 1 to 103 (inclusive) of tau-441 is HJ8.5, or an epitope binding agent that binds to the same epitope as HJ8.5 and / or competitively inhibits HJ8.5. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 104 to 221 (inclusive) of tau-441 is Tau1, or an epitope binding agent that binds to the same epitope as Tau1 and / or competitively inhibits Tau1.

[0112] In some embodiments, the epitope binder that specifically binds to the middle domain tau is selected from HJ34.8, 77G7, RD3, RD4, UCB1017, PT76, E2815 or 7G6, or is an epitope binder that binds to 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 binders that specifically bind to the same epitope as those antibodies. Methods for identifying the epitope to which an antibody specifically binds and assays for assessing competitive inhibition between two antibodies are known in the art.

[0113] Alternatively, protein can be depleted from the sample by more general methods, such as by ultrafiltration or protein precipitation with acids, organic solvents or salts. In general, these methods are used to reliably reduce high abundance and high molecular weight proteins, and then enrich for low molecular weight and / or low abundance proteins and peptides (e.g., tau, Aβ, etc.).

[0114] In some embodiments, protein can be depleted from a sample by precipitation. In short, precipitation comprises adding a precipitant to the sample and thoroughly mixing, incubating the sample with the precipitant to precipitate the protein, and separating the precipitated protein by centrifugation or filtration. The resulting supernatant can then be used for downstream applications. The amount of the required reagent can be determined experimentally by methods known in the art. Suitable precipitants include perchloric acid, trichloroacetic acid, acetonitrile, methanol, etc. In an exemplary embodiment, protein is depleted from a sample by acid precipitation. In another embodiment, protein is depleted from a sample by acid precipitation using perchloric acid.

[0115] As a non-limiting example, protein can be depleted from a sample by acid precipitation using perchloric acid. Unless otherwise indicated, "perchloric acid" as used herein refers to 70% perchloric acid. 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. After adding perchloric acid, the sample is thoroughly mixed (for example, by a vortex mixer) and remains at low temperatures, usually about 10 minutes or longer, to promote precipitation. For example, the sample can keep about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes or about 30 minutes to about 60 minutes. In other examples, the sample can keep about 15 minutes to about 45 minutes or about 30 minutes to about 45 minutes. In other examples, the sample can keep about 15 minutes to about 30 minutes or about 20 minutes to about 40 minutes. In other examples, the sample keeps about 30 minutes. The sample is then centrifuged at low temperature to pellet the precipitated protein, and the supernatant containing soluble tau (i.e., the acid-soluble fraction) is transferred to a fresh container. As used above, "low temperature" refers to a temperature of 10°C or lower. For example, low temperature can 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 example, from about 3°C to about 5°C, or even about 4°C. In certain embodiments, low temperature can be achieved by placing the sample on ice.

[0116] In one embodiment, the method for the present invention can be combined to deplete multiple proteins in sequence or simultaneously from one or both of two or more methods in the above-mentioned approach.For example, one or more proteins can be selectively depleted (targeted depletion) and then deplete high abundance / molecular weight protein. Alternatively, high abundance / molecular weight protein can be first depleted and then targeted depletion of one or more proteins can be performed. In another alternative, high abundance / molecular weight protein can be first depleted and then first round of targeted depletion of one or more proteins, and then second round of targeted depletion of one or more proteins that are different from the protein targeted in the first round. Other iterations will be apparent to those skilled in the art. 3. Protease Digestion of Purified Tau Fragments

[0117] Another step of the method disclosed herein optionally includes digesting the purified tau fragments (e.g., endogenously cleaved tau) with a protease (e.g., thereby producing proteolytic tau peptides). Digestion can be performed in vitro. Digestion of purified tau with a protease includes contacting a sample containing purified tau with a protease under conditions suitable for digesting tau. When affinity purification is used, digestion can 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 the detection of MTBR-tau275 or MTBR-tau282, the protease includes trypsin. The resulting digestion products (e.g., cleavage products) are compositions comprising proteolytic peptides of tau. When the protease is trypsin, the resulting cleavage products comprise tryptic peptides of tau. After proteolytic cleavage, the resulting digestion products (e.g., cleavage products) can be desalted by solid phase extraction. 3.1. Detection and Quantification of Tau Fragments

[0118] Another step of the method disclosed herein includes detecting the amount of digested tau peptides from the treated biological sample. All suitable methods for detecting the amount of tau protein are considered within the scope of this disclosure. The following describes in detail the method for detecting and quantifying tau peptides. LC-MS

[0119] The steps of detecting and quantifying include performing liquid chromatography-mass spectrometry (LC-MS) with a sample containing tau peptides to detect and measure the concentration of at least one tau peptide. Thus, in practice, the disclosed methods use one or more tau peptides to detect and measure the amount of tau protein present in a biological sample.

[0120] Proteolytic peptides of tau that indicate the presence of endogenous cleavage fragments of tau may comprise MTBR-275 or MTBR-282. When alternative enzymes are used for digestion, the resulting proteolytic peptides may be slightly different, but can be readily determined by one of ordinary skill in the art. Without wishing to be bound by theory, it is believed that the variation in the amount of cleavage fragments of tau peptides between two biological samples of the same type reflects the differences in the cleavage fragments of tau that constitute those biological samples. As disclosed herein, the amounts of certain proteolytic peptides of cleavage fragments of tau and the ratios of certain proteolytic peptides of tau can provide clinically meaningful information to guide treatment decisions. Thus, methods enabling the detection and quantification of cleavage fragments of tau have utility in the diagnosis and treatment of many neurodegenerative diseases.

[0121] The proteolytic peptides of tau can be separated by a liquid chromatography system connected to a high-resolution mass spectrometer. Suitable LC-MS systems may include <1.0 mm ID columns and use a flow rate of less than about 100 μl / min. In a preferred embodiment, a nanoflow LC-MS system (e.g., about 50-100 μm ID columns and <1 μL / min, preferably about 100-800 nL / min, more preferably about 200-600 nL / min flow rate) is used. In an exemplary embodiment, the LC-MS system may include a 0.05 mM ID column and use a flow rate of about 400 nL / min.

[0122] As is known in the art, tandem mass spectrometry can be used to increase resolution, or techniques can be improved 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 mass spectrometers, time-of-flight mass spectrometers, ion trap mass spectrometers, and Orbitrap mass spectrometers, as well as hybrid mass spectrometers (e.g., Orbitrap Fusion Mass Spectrometers) that combine different types of mass analyzers into one architecture. TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TM Mass spectrometer, Q Exactive mass spectrometer, each from ThermoFisher Scientific). In an exemplary embodiment, the LC-MS system may include a mass spectrometer selected from the group consisting of: Orbitrap Fusion TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TM Mass spectrometer or there is similar or improved ion focusing and the mass spectrometer of ion transparency at the quadrupole.Can develop suitable mass spectrometry scheme by optimizing the ion quantity (for example using the AGC setting of orbitrap) and / or the injection time that collects before analysis.In an exemplary embodiment, the mass spectrometry scheme of general introduction in the embodiment is used. Immunoassay

[0123] In some embodiments of the methods described herein, tau peptides (e.g., MTBR-tau peptides) can be measured and quantified by immunoassay. In certain embodiments, ELISA is used to detect and quantify tau fragments.

[0124] Methods for assessing the amount of protein expression using epitope binding agent-based methods are known in the art, and all methods known to those skilled in the art to be suitable for assessing the amount of a protein are considered to be within the scope of the present disclosure.

[0125] Thus, in some embodiments, the method for assessing the amount of tau peptide (e.g., as described herein) is an epitope binding agent-based method. Typically, the epitope binding agent-based method for assessing the amount of protein expression includes contacting a sample comprising the polypeptide with an epitope binding agent that is specific for the polypeptide under conditions that effectively form a complex between the epitope binding agent and the polypeptide. The epitope binding agent-based method can be performed in solution, or the epitope binding agent or sample can be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtiter plates, test tubes, beads, resins, and other polymers.

[0126] As will be appreciated by those skilled in the art, epitope binding agents can be attached to a substrate in a variety of ways. The epitope binding agent can be first synthesized and then attached to the substrate, or it can be synthesized directly on the substrate. The substrate and epitope binding agent can be derivatized with chemical functional groups to allow for subsequent attachment of the two. For example, the substrate can be derivatized with chemical functional groups including, but not limited to, amino, carboxyl, oxo, or thiol groups. Using these functional groups, the epitope binding agent can be attached directly using the functional groups or indirectly using a linker.

[0127] Epitope binding agents can also be non-covalently attached to a substrate. For example, a biotinylated epitope binding agent can be prepared that can bind to a surface covalently coated with streptavidin, resulting in attachment. Alternatively, epitope binding agents can be synthesized on a surface using techniques such as photopolymerization and photolithography. Additional methods for attaching epitope binding agents to solid surfaces and methods for synthesizing biomolecules on substrates are well known in the art, i.e., VLSIPS technology from Affymetrix (e.g., see U.S. Patent No. 6,566,495 and Rockett and Dix, Xenobiotica 30(2): 155-177, both of which are incorporated herein by reference in their entirety).

[0128] Contacting the sample with the epitope binding agent under effective conditions for a period of time sufficient to allow the formation of a complex typically includes adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough to allow the epitope binding agent to bind to any antigen present. Thereafter, the complex will be washed and the complex can be detected by any method known in the art. Methods for detecting epitope binding agent-polypeptide complexes are typically 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, wherein the substance can be detected by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescence quenchers, 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). Methods for detecting epitope binding agent-polypeptide complexes based on the detection of labels or markers are well known in the art.

[0129] In some embodiments, the epitope binding agent-based method is an immunoassay. Immunoassays can be run in many different formats. In general, 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 a labeled analyte for binding to an antibody. The unbound analyte is washed away and the bound analyte is measured. In a non-competitive immunoassay, an antibody is labeled instead of the analyte. A non-competitive immunoassay can use one antibody (e.g., a labeled capture antibody) or more than one antibody (e.g., at least one unlabeled capture antibody and at least one labeled "capping" or detection antibody). Suitable labels are described above.

[0130] In one embodiment, the epitope binding agent method is an immunoassay. In another embodiment, the epitope binding agent method is selected from enzyme-linked immunosorbent assay (ELISA), fluorescence-based assay, dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), radioassay, multiplex immunoassay and flow cytometric microsphere assay (CBA). In some embodiments, the epitope binding agent-based method is an enzyme-linked immunosorbent assay (ELISA). In other embodiments, the epitope binding agent-based method is a radioimmunoassay. In yet other embodiments, the epitope binding agent-based method is an immunoblot or a protein blot. In alternative embodiments, the epitope binding agent-based method is an array. In another embodiment, the epitope binding agent-based method is flow cytometry. In various embodiments, the epitope binding agent-based method is immunohistochemistry (IHC). IHC uses antibodies to detect and quantify antigens in intact tissue samples. Tissue samples can be fresh frozen and / or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC. Methods for preparing tissue blocks for study by IHC and methods for performing IHC are well known in the art. 4. Uses of MTBR-tau peptide measurements

[0131] The present disclosure also encompasses the measurement of the tau peptides (such as endogenous cleavage fragments of tau) in CSF as a biomarker for the pathological characteristics and / or clinical symptoms of tau disease with diagnosis, staging, selection of treatment suitable for a given disease stage and the purposes of revising a given therapeutic regimen (such as changing dosage, converting to different medicines or treatment modalities, etc.). Pathological characteristics can be an aspect of tau pathology (such as the amount of tau deposition, the presence / absence of post-translational modifications, the amount of post-translational modifications, etc.). Alternatively, or except tau deposition, pathological characteristics can be unrelated to tau. For example, when tau disease is Alzheimer's disease, beta amyloid (A β) is deposited in the brain or in the cerebral artery. Clinical symptoms can be the dementia measured by a clinically validated instrument (such as MMSE, CDR-SB, etc.), or any other clinical symptoms relevant to tau disease.

[0132] Therefore, in one aspect, present disclosure provides a method for measuring tauopathy associated pathology in a subject, the method comprising quantitatively obtaining one or more tau peptides (e.g., as described herein) from a biological sample (e.g., CSF sample) of the subject. The method may comprise providing the amount of one or more tau peptides as described herein. The amount of quantitative tau peptides may represent the tauopathy associated pathology in the subject's brain. Tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. In specific embodiments, tauopathy is 4R-tauopathy. Tauopathy associated pathology may be amyloid plaques in tau deposition, tau post-translational modification, brain and / or cerebral arteries, or other pathological features known in the art. The subject may have or may not have the clinical symptoms of tauopathy.

[0133] On the other hand, present disclosure provides the method for diagnosing the tau disease in experimenter, the method comprises quantitatively obtaining from one or more tau peptides (such as as described herein) in the biological sample (such as CSF sample) of experimenter or provides the amount of one or more tau peptides (such as as described herein) in the biological sample (such as CSF sample) of experimenter, and quantitative tau peptide is about 1.5σ or higher when diagnosing tau disease, wherein σ is the standard deviation defined by the normal distribution recorded in control population, described control population does not have the clinical signs or the symptom of tau disease and as measured by PET imaging and / or the A β42 / A β40 ratio measurement in CSF is amyloid negative.Tau disease can be 3R-tau disease, mixed type 3R / 4R-tau disease or 4R-tau disease.In specific embodiments, tau disease is 4R-tau disease.Experimenter can have or do not have the clinical symptoms of disease.

[0134] On the other hand, present disclosure provides the method for measuring tau disease stability in experimenter, the method comprises quantitatively obtaining from one or more tau peptides (such as described herein) in the first biological sample of experimenter or providing the amount of one or more tau peptides (such as described herein) in the first biological sample of experimenter, and quantitatively obtaining from one or more tau peptides (such as described herein) in the second biological sample of same experimenter in the time later (such as weeks, months or years later) subsequently or providing the amount of one or more tau peptides (such as described herein) in the second biological sample of same experimenter in the time later (such as weeks, months or years later), and calculating the difference between quantitative tau peptides between sample, wherein the statistically significant increase indication disease progression of quantitative tau peptides in the second sample, the statistically significant reduction indication disease improvement of quantitative tau peptides in the second sample, and no change indication stable disease.Tau disease can be 3R-tau disease, mixed type 3R / 4R-tau disease or 4R-tau disease.In specific embodiments, tau disease is 4R-tau disease. The subject may or may not have clinical symptoms of the disease and may or may not be receiving tau therapy. In some examples, the subject is administered tau therapy one or more times during the period between the collection of the first biological sample and the second biological sample, and the measure of disease stability indicates the effectiveness, or lack thereof, of the tau therapy.

[0135] As contemplated by each of the methods described herein, the MTBR-tau peptide species to be measured include MTBR-tau275 / t-tau and / or MTBR-tau282 / t-tau.

[0136] The exemplary uses of the MTBR-tau peptides of tau as described herein can be used to illustrate the various aspects discussed above, but such discussion does not limit the scope of the present invention. Tau peptides (e.g., endogenous cleavage 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 endogenously cleaved at their C-terminal ends. Measuring the amount of tau peptides is a means of measuring the amount of the tau species of this particular group in a given sample. As shown in the Examples, the reduction in the amount of 4R subtype-specific MTBR-tau275 and MTBR-tau282 endogenous cleavage fragments of tau in CSF is relevant to the tau deposition associated with primary tauopathy (especially FTLD (e.g., FTLD-MAPT) and CBD) in the brain. 4R-specific MTBR-tau / t-tau measurements have a negative correlation in CSF and the brain. In addition, the CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau biomarkers with a long interval duration experimenter (such as the experimenter in the later pathological stage) between the age and CSF collection during the onset of the disease have larger reduction. In other words, the amount of CSF MTBR-tau endogenous cleavage fragments (such as MTBR-tau275 and MTBR-tau282) can indicate primary tau disease pathology. In addition, CSF t-tau and pT217 / T217 ratio can be used as the diagnostic marker for distinguishing tau disease (such as AD is distinguished from NC, PSP or other FTLD-tau; Or CBD is distinguished from NC or other FTLD-tau). Therefore the amount of the MTBR-tau peptide detected can be used for measuring primary tau disease pathology, determining the tau state of experimenter, diagnosing the stages of primary tau disease (such as FTLD-MAPT or CBD) in the experimenter and distinguishing primary tau disease etc.

[0137] After the disease is diagnosed and / or staged, treatment can be subsequently provided to the subject to reduce the amount of tau peptides in CSF or prevent any further increase in the amount of tau peptides in CSF and / or reduce another clinical sign or symptom of primary tau disease or prevent any further increase in another clinical sign or symptom of primary tau disease. The selection for the treatment of can further be guided by the knowledge of the specific disease stage known by the amount of tau peptides (such as tau fragments of endogenous cleavage), for example, the therapy designed to prevent A β deposition, reverse A β deposition, prevent tau deposition, reverse tau deposition and improve the clinical signs of disease will be used for the subject with different (although possibly overlapping) tau peptide amounts.

[0138] In a specific embodiment, present disclosure provides a method for detecting the primary tau disease in a subject, the method including providing CSF obtained from the subject, wherein CSF is purified for tau peptides (such as endogenous cleavage fragments of tau);And quantitative tau peptides in the sample. In some embodiments, tau peptides include one or more of peptides with the amino acid sequence of MTBR-tau275, MTBR-tau282 or a combination thereof, wherein the amount of tau peptides or their ratio (such as with each other's ratio or each with CSF t-tau's ratio) indicate the primary tau disease in the brain of the subject. In some embodiments, primary tau disease is selected from frontotemporal lobar degeneration (FTLD-tau) (such as FTLD-MAPT), including cortical basal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), globular glial tau disease (GGT), chronic traumatic encephalopathy (CTE), Pick's disease (PiD) or Alzheimer's disease. In certain embodiments, the primary tauopathy is FTLD-tau or CBD. In certain embodiments, FTLD-tau is FTLD-MAPT. In certain embodiments, 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-associated pathology in a subject, comprising detecting tau or 4R tau aggregates or providing the amount of tau or 4R tau aggregates. In some embodiments, detecting tau or 4R tau aggregates comprises providing CSF obtained from the subject, wherein the CSF is purified for tau peptides; and quantifying tau peptides in the sample. In some embodiments, the tau peptides comprise one or more peptides having the amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of tau peptides or their ratio (e.g., a ratio to each other or a ratio of each to CSF t-tau) indicates FTLD-tau-associated pathology in the brain of the subject. In some embodiments, the method comprises quantifying 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 or providing an amount of 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 from the mean of a control population by about 1.5σ or greater, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and / or is amyloid negative as measured by PET imaging (e.g., by PiB-PET SUVR as described in Ann Neurol 2016;80:379-387) and / or Aβ42 / Aβ40 ratio measurement in CSF (e.g., a cutoff value for the CSF Aβ42 / Aβ40 ratio calculated by PiB-PET SUVR (Ann Neurol 2016;80:379-387) that maximizes % sensitivity + % specificity).

[0140] In another specific embodiment, the present disclosure provides a method for measuring FTLD-tau-related tau deposition in a subject's brain, comprising detecting tau or 4R tau aggregates or providing the amount of tau or 4R tau aggregates. In some embodiments, the method includes providing a CSF sample obtained from a subject, wherein the CSF sample is purified for tau peptides; and quantifying tau peptides in the sample. In some embodiments, the tau peptides comprise one or more peptides having the amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of tau peptides indicates FTLD-tau-related tau deposition in the subject's brain. In specific embodiments, FTLD-tau is FTLD-MAPT. In specific embodiments, 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 for diagnosing a FTLD-tau-related disease, the method comprising providing CSF obtained from a subject, and quantifying tau peptides in the sample. The method may include providing the amount of tau peptides as disclosed herein. In some embodiments, the tau peptide comprises one or more 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 a FTLD-tau-associated disease when the quantified tau peptide differs by about 1.5σ or greater from the mean of a control population, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and / or is amyloid negative as measured by PET imaging (e.g., by PiB-PET SUVR as described in Ann Neurol 2016; 80:379–387) and / or Aβ42 / Aβ40 ratio in CSF (e.g., a cutoff value for the CSF Aβ42 / Aβ40 ratio calculated by PiB-PET SUVR (Ann Neurol 2016; 80:379–387) that maximizes % sensitivity + % specificity). In specific embodiments, FTLD-tau is FTLD-MAPT. In certain 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 for measuring the progression of a FTLD-tau-related disease 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 peptides; and quantifying the tau peptides for each sample. In one example, the amount of tau peptides in the first sample and the second sample is provided. In some embodiments, the tau peptides comprise one or more 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 comprises calculating the difference between the tau peptides quantified in the second sample and the first sample, wherein a statistically significant increase in the tau peptides quantified in the second sample indicates progression of the subject's FTLD-tau-related disease. In specific embodiments, FTLD-tau is FTLD-MAPT. In certain 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 for measuring CBD-associated tau deposition in a subject's brain, comprising detecting tau or 4R tau aggregates or providing an amount of tau or 4R tau aggregates. In some embodiments, the method comprises providing a CSF sample obtained from the subject, wherein the CSF is purified for tau peptides; and quantifying the tau peptides in the sample. In one example, the amount of tau peptides is provided. In some embodiments, the tau peptides comprise one or more peptides having an amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof, wherein the amount of tau peptides is representative of CBD-associated tau deposition in the subject's brain.

[0144] In another specific embodiment, the present disclosure provides a method for diagnosing a CBD-related disease, the method comprising providing CSF obtained from a subject and quantifying tau peptides in the sample. In one example, the amount of tau peptides is provided. In some embodiments, the tau peptide comprises one or more 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, the subject is diagnosed with a CBD-related disease when the quantified tau peptide differs from the mean of a control population by about 1.5σ or greater, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and / or is amyloid negative as measured by PET imaging (e.g., by PiB-PET SUVR as described in Ann Neurol 2016;80:379–387) and / or Aβ42 / Aβ40 ratio in CSF (e.g., a cutoff value for the CSF Aβ42 / Aβ40 ratio calculated by PiB-PET SUVR (Ann Neurol 2016;80:379–387) that maximizes % sensitivity + % specificity).

[0145] In another specific embodiment, the present disclosure provides a method for measuring the progression of a CBD-related disease 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 quantifying tau peptide for each sample. In one example, the amount of tau peptide in the first sample and the second sample is provided. In some embodiments, the tau peptide comprises one or more 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 comprises calculating the difference between the tau peptide quantified in the second sample and the first sample, wherein a statistically significant increase in the tau peptide quantified in the second sample indicates the progression of the CBD-related disease in the subject.

[0146] In each of the aforementioned methods, additional biomarkers may be detected and measured to help stage and / or determine the subject's pathology and / or the subject's disease progression. For example, the method may include detecting and measuring the amount of Aβ. In some embodiments, Aβ42 / Aβ40 values are determined to help measure amyloid plaque-specific abnormalities. Deviations from the Aβ42 / Aβ40 values of a normal control population without amyloid pathology begin to appear about 20 years before the onset of symptoms and have been shown to be a highly sensitive measure of these changes. In addition to or in place of Aβ measurements, each of the aforementioned methods may include detecting and measuring the amount of tau phosphorylation at specific residues. In some embodiments, tau phosphorylation at 217, 181, 231, 205, 153, 111, 208, and / or any combination thereof is determined to help measure amyloid plaque-specific abnormalities. Aβ42 / Aβ40 changes first, followed by ptau217 / 181 / 231 shortly thereafter, and these are associated with amyloid plaques. Then, after about 10 years, ptau205 increased and was associated with both Aβ plaques and tau tangles, similar to total tau (N-terminal to middle domain tau). In some embodiments, the composite value of ptau phosphorylation and Aβ42 / Aβ40 values can be measured and determined. For example, pT217×Aβ42 / Aβ40 values are determined to help measure amyloid plaque-specific abnormalities, and it is found to be highly sensitive and specific. Finally, about 20 years after Aβ42 / Aβ40 abnormalities, endogenous cleavage fragments of tau as disclosed herein increase and are only associated with tau pathology (not associated with amyloid pathology). Thus, when measured and combined in the methods disclosed herein, each marker provides the ability to more accurately determine the subject's stage, pathology, and / or disease progression, which was not available before the present disclosure. Aβ42 / Aβ40 is a measure of pre-abnormal amyloid plaques; Aβ42 / Aβ40 and ptau217% abnormal are measures of amyloid plaque abnormalities, ptau205 is a measure of amyloid plaques plus neurodegeneration; and endogenous cleavage fragments of tau as disclosed herein are a measure of tau tangles and clinical onset of dementia.

[0147] Alternatively or in addition to the above, in any of the above embodiments, other markers can be used, such as the measurement of total tau, the ratio calculated from the phosphorylation level measured, or the ratio calculated from the phosphorylation level measured and total tau. Mathematical operations other than ratios can also be used. For example, the exemplary use of endogenous cleavage fragments, A β values and / or site-specific tau phosphorylation values of tau as disclosed herein can be combined with other known biomarkers (such as MAPT state, ApoEε4 state, age, sex, cognitive test score, functional test score, etc.) for various statistical models (such as linear regression, LME curve, LOESS curve, etc.). The selection of measurement and the selection of mathematical operations can be optimized to maximize the specificity of the method. For example, diagnostic accuracy can be assessed by area under the ROC curve, and in some embodiments, 0.7 or greater ROCAUC values are set to threshold values (such as 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.).

[0148] Brain amyloid plaques are routinely measured in humans by amyloid positron emission tomography (PET). 11 C-Pittsburgh compound B (PiB) PET imaging 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). Therefore, in the above-mentioned embodiments, a control population without brain amyloid plaques as measured by PET imaging can refer to a population of subjects with cortical PiB-PET SUVR<1.25. Other values of PiB binding (e.g., mean cortical binding potential) or analysis of target areas other than cortical areas can also be used to classify subjects as PIB positive or negative. Other PET imaging agents can also be used.

[0149] On the other hand, present disclosure provides the method for treating the experimenter with tau disease, the method includes the tau in the biological sample such as CSF sample quantitatively obtained from the experimenter; And tau therapy is provided to the experimenter to improve the measurement of tau disease related pathology or clinical symptoms, wherein the experimenter has at least 1 standard deviation of mean value higher than or lower than control population, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations or even more preferably at least 2 standard deviations (that is, differ respectively by 1σ, 1.3σ, 1.5σ or 2σ, wherein σ is the standard deviation defined by the normal distribution measured in control population, described control population does not have the clinical signs or symptoms of tau disease and as measured by PET imaging and / or the Aβ42 / Aβ40 ratio in CSF for amyloid negative) quantitative tau endogenous cleavage peptide fragment.Except using threshold value (for example, higher than or lower than at least 1 standard deviation of mean value), in some embodiments, the degree of variation higher than or lower than mean value can be used as the standard for treating experimenter. Protein tau disease can be 3R-tau disease, mixed type 3R / 4R-tau disease or 4R-tau disease.In specific embodiments, protein tau disease is 4R-tau disease.The measurement of protein tau disease associated pathology can be as the tau deposition recorded by PET imaging, as the tau post-translational modification recorded by mass spectrum or other suitable methods, as the amyloid plaque in the brain or cerebral artery recorded by PET imaging, as the amyloid plaque recorded by the A β42 / A β40 ratio in CSF or other pathological features known in the art.Clinical symptoms can be as the dementia measured by the instrument (such as MMSE, CDR-SB etc.) of clinical validation, or other clinical symptoms of 3R-tau disease and 4R-tau disease known in the art.In other embodiments, quantitatively two or more tau species.The specific pathophysiological changes of many tau therapy targets. For example, Aβ targeted therapies are generally designed to reduce Aβ production, antagonize Aβ aggregation, or increase brain Aβ clearance; tau targeted therapies are generally designed to change tau phosphorylation patterns, antagonize tau aggregation (general antagonism of tau or antagonism of specific tau subtypes), or increase NFT clearance; various therapies are designed to reduce CNS inflammation or brain insulin resistance; and so on. 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 who are correctly identified as having tau pathology, including determining the disease stage in which the subject is located based on the subject's specific pathological state, thereby more effectively changing tau phosphorylation patterns, antagonizing tau aggregation, or increasing NFT clearance.

[0150] Treatment may include one or more of the following: lencaneluzumab, donetumab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, berenuzumab, dasatinib + quercetin, E2814, epothilone D, gonelizumab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember™, sirenuzumab, TPI 287, Tideglusib, tipranezumab, zagteneizumab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors, and tau active vaccines.

[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 reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, etc.), a γ-secretase inhibitor, a β-secretase inhibitor, an anti-Aβ antibody (including an antigen-binding fragment, variant, or derivative thereof), an anti-tau antibody (including an antigen-binding fragment, variant, or derivative thereof), an anti-TREM2 antibody (including an antigen-binding fragment, variant, or derivative thereof), a TREM2 antagonist, stem cells, a dietary supplement (e.g., lithium water, omega-3 fatty acids and lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract etc.), antagonists of serotonin receptor 6, p38α MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylsulfoximine chloride, etc.), therapies to improve glycemic 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, beta-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 stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-γ agonists, tubulin regulators, calcium channel blockers, antihypertensive drugs, statins and any combination thereof. In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors may inhibit tau plus one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. In another exemplary embodiment, the pharmaceutical composition may include 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 targeted therapy (including but not limited to an active pharmaceutical ingredient that changes the tau phosphorylation pattern, antagonizes tau aggregation, or increases the 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 antagonist, a γ-secretase inhibitor, a β-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau aggregation inhibitor.

[0152] In some embodiments, when the detected Aβ42 / Aβ40 value deviates from the average value of the healthy control population and optionally pTau217 / tau217, ptau205 / tau205, pTau181, pTau231 and / or the cleavage peptide fragments of the present invention do not significantly deviate from the average value of the healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent that prevents an increase in amyloid deposition is selected. In an exemplary embodiment, the detected Aβ42 / Aβ40 value is significantly lower than the average value of the healthy control population. In some embodiments, when the detected Aβ42 / Aβ40 value and pTau217 / tau217 (or pTau217×Aβ42 / Aβ40 composite value) deviate significantly from the average value of the healthy control population and optionally ptau205 / tau205 and / or the cleavage peptide fragments of the present invention do not significantly deviate from the average value of the healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent that prevents an increase in amyloid deposition and / or reduces the subject's existing plaque load is selected. In an exemplary embodiment, the detected Aβ42 / Aβ40 value is significantly lower than the average of the healthy control population, the detected pTau217 / tau217 is higher than the average of the healthy control population and / or the pTau217×Aβ42 / Aβ40 composite value is higher than the average of the healthy control population. In some embodiments, when the detected Aβ42 / Aβ40 value, pTau217 / tau217 (or pTau217×Aβ42 / Aβ40 composite value) and pTau205 / tau205 value deviate significantly from the average of the healthy control population and optionally the cleavage peptide fragment of the present disclosure does not deviate significantly from the average of the healthy control population, the subject is administered a therapeutic agent or a therapeutic agent is selected to prevent the increase in amyloid deposition and / or reduce the subject's existing plaque load and / or treat or prevent neurodegeneration and / or prevent Tau tangles or related pathologies. In an exemplary embodiment, the detected Aβ42 / Aβ40 value is significantly lower than the average of the healthy control population, the detected pTau217 / tau217 is higher than the average of the healthy control population and / or the pTau217×Aβ42 / Aβ40 composite value is higher than the average of the healthy control population and / or the pTau205 / tau205 value is higher than the average of the healthy control population. In some embodiments, when the detected Aβ42 / Aβ40 value, pTau217 / tau217 (or pTau217×Aβ42 / Aβ40 composite value), pTau205 / tau205 and the cleavage peptide fragments of the present disclosure deviate significantly from the average of the healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent is selected to prevent an increase in amyloid deposition and / or reduce the subject's existing plaque load and / or treat or prevent neurodegeneration and / or prevent an increase in tau tangles and / or reduce the subject's existing tangles or related pathology.In an exemplary embodiment, the detected Aβ42 / Aβ40 value is significantly lower than the average value of a healthy control population, the detected pTau217 / tau217 is higher than the average value of a healthy control population and / or the pTau217×Aβ42 / Aβ40 composite value is higher than the average value of a healthy control population and / or the pTau205 / tau205 value is higher than the average value of a healthy control population and / or the cleavage peptide fragment value of the present disclosure is higher than the average value of a healthy control population.

[0153] In each of the above embodiments, the pharmaceutical composition administered to the 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 compound B, flubetaban, flubetapyr, flumetazol, radionuclide-labeled antibodies, etc.).

[0154] Another aspect of the present disclosure is a method for selecting a subject to enter a clinical trial, particularly a clinical trial for Aβ or tau therapy, provided that all other criteria for the clinical trial have been met. In one embodiment, the method for selecting a subject to enter a clinical trial may include (a) providing a CSF sample obtained from the subject, wherein the CSF sample is purified for cleavage peptide fragments of tau; (b) quantifying the cleavage peptide fragments of tau in the sample, and (c) when the cleavage peptide fragment value of tau is approximately the same as that of a healthy control group and the subject's Aβ42 / Aβ40 value is lower than the mean value (man) of the healthy control group, selecting the subject to enter the clinical trial for Aβ therapy. In some embodiments, the cleavage peptide fragment of tau comprises one or more of the peptides having the amino acid sequence in Table 1 (wherein 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 cleavage peptide fragment of tau or its ratio represents tau deposition in the subject's brain.

[0155] In another embodiment, a method for selecting a subject for a clinical trial may include (a) providing a CSF sample obtained from the subject, wherein the CSF sample is purified for cleavage peptide fragments of tau; (b) quantifying the cleavage peptide fragments of tau in the sample, and (c) excluding the subject from the clinical trial for Aβ therapy when the cleavage peptide fragment value of tau is higher than the average value of a healthy control population and the subject's Aβ42 / Aβ40 value is approximately the same as or lower than the average value of a healthy control population. In some embodiments, the cleavage peptide fragment of tau comprises one or more of the peptides having the amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid at the C-terminus of the peptide) or a combination thereof, wherein the amount of the cleavage peptide fragment of tau or the ratio thereof indicates tau deposition in the brain of the subject.

[0156] In another embodiment, a method for selecting a subject to enter a clinical trial, particularly a clinical trial for tau therapy, is provided that all other criteria for the clinical trial have been met. In one embodiment, the method for selecting a subject to enter a clinical trial may include (a) providing a CSF sample obtained from the subject, wherein the CSF sample is purified for cleavage peptide fragments of tau; (b) quantifying cleavage peptide fragments of tau in the sample, and (c) selecting the subject to enter a clinical trial for tau therapy when the cleavage peptide fragment value of tau is higher than the average value of the healthy control and optionally when the subject's Aβ42 / Aβ40 value is approximately the same as or lower than the average value of the healthy control group. In some embodiments, the cleavage peptide fragment of tau comprises one or more of a peptide containing the amino acid sequence in Table 1 (wherein 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 cleavage peptide fragment of tau or its ratio represents tau deposition in the subject's brain.

[0157] In another embodiment, a method for selecting a subject to enter a clinical trial, particularly a clinical trial for tau therapy, is provided that all other criteria for the clinical trial have been met. In one embodiment, the method for selecting a subject to enter a clinical trial may include (a) providing a CSF sample obtained from the subject, wherein the CSF sample is purified for cleavage peptide fragments of tau; (b) quantifying the cleavage peptide fragments of tau in the sample, and (c) excluding the subject from the clinical trial for tau therapy when the cleavage peptide fragment value of tau is approximately the same as the average value of the healthy control group. In some embodiments, the cleavage peptide fragment of tau comprises one or more of the peptides containing the amino acid sequence in Table 1 (wherein 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 cleavage peptide fragment of tau or its ratio represents tau deposition in the subject's brain. The phrase "a control group without brain amyloid plaques as measured by PET imaging" is defined above.

[0158] Alternatively or in addition to using a cleavage peptide fragment of tau as disclosed herein, in any of the above embodiments, site-specific tau phosphorylation (optionally together with the measurement of total tau), a ratio calculated by the measured phosphorylation level, or a ratio calculated by the measured phosphorylation level and total tau can be used. The ratio calculated by the measured phosphorylation level can be a ratio between pT181 and pT205, between pT217 and pT205, or between pT181 and pT217. The ratio calculated by the measured phosphorylation level and total tau can be a ratio between pT181 and total tau, between pT205 and total tau, or between pT217 and total tau. Mathematical operations other than ratios can also be used. For example, examples use site-specific tau phosphorylation values combined with other known biomarkers (e.g., APOEε4 status, age, sex, cognitive test scores, functional test scores, etc.) in various statistical models (e.g., linear regression, LME curves, LOESS curves, etc.).

[0159] Can greatly help the design of the clinical trial of tau disease (such as FTLD-MAPT, CBD) and tau therapy by method disclosed herein.Many clinical trials are designed to test the imaging agent or the effect of the therapeutic agent of the specific pathophysiological change that targeting occurs before the onset of symptoms of tau disease.As described herein, can improve the effect of these various medicaments by administering medicament to the experimenter with some site-specific tau phosphorylation level as recorded by the method disclosed and illustrated herein.Similarly, the clinical trial of the experimenter of the symptom of selecting to have A β pathology or only tau pathology will also benefit from the pathology that can accurately distinguish the person that recruits so that determine whether effect is relevant to specific morbid state.Therefore, before selecting the experimenter to enter clinical trial, particularly entering the treatment arm of clinical trial, measuring tau phosphorylation level as described herein can cause test less and / or result improvement.In some cases, can develop method as described herein and use it as the companion diagnosis of therapeutic agent.

[0160] In each of the above embodiments, subjects can be recruited into the treatment arm of a clinical trial. "Treatment" is defined above. The pharmaceutical composition can be administered to subjects recruited into the treatment arm of a clinical trial. In some embodiments, the pharmaceutical composition may include 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, flubetaban, flubetapyr, flumetazol, radionuclide-labeled antibodies, etc.). Alternatively, the pharmaceutical composition may include 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.), γ-secretase inhibitors, β-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 and lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), antagonists of serotonin receptor 6, p38αMA PK inhibitors, recombinant granulocyte macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylsulfoximine 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, beta-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 stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-γ agonists, tubulin regulators, calcium channel blockers, antihypertensive drugs, statins and any combination thereof. In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors can inhibit thousand-plus-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, a phosphatase activator can increase the activity of protein phosphatase 2A.

[0161] In each of the above-mentioned embodiments, the subject may or may not be symptomatic. " Asymptomatic subject " as used herein refers to a subject that does not show any signs or symptoms of tauopathy. Alternatively, the subject may show signs or symptoms (such as memory loss, misplaced items, changes in mood or behavior, etc.), but does not show enough cognitive or functional impairment for clinical diagnosis. Symptomatic or asymptomatic subjects may have Aβ amyloidosis; However, the prior knowledge of Aβ amyloidosis is not necessary for treatment. In further embodiments, the subject may suffer from AD. In any of the above-mentioned embodiments, the subject may carry one of the genetic mutations known to cause hereditary tauopathy. In alternative embodiments, the subject may not carry the genetic mutations known to cause hereditary tauopathy. 5. Dosage box

[0162] Also provided is a kit. Such a kit may include the agents or compositions described herein, and in certain embodiments, instructions for administering or measuring tau peptides (e.g., endogenous cleavage fragments of tau). Such a kit can facilitate the execution of the methods described herein. When provided as a kit, the different components of the composition can be packaged in separate containers and mixed just before use. Components include, but are not limited to, systems, assays, epitope binding agents, reagents, internal standards, or software. If desired, such separate packaging of components can be present in a package or dispenser device that can include one or more unit dosage forms containing the composition. Packaging can, for example, include metal or plastic foil, such as blister packs. In some cases, such separate packaging of components can also allow long-term storage without losing the activity of the components.

[0163] Test kit can also include the reagent in a separate container, such as sterile water or saline to be added to the freeze-dried active component of a separate package. For example, a sealed glass ampoule can contain freeze-dried components, and in a separate ampoule, contains sterile water, sterile saline or sterile material (sterile), which is packaged separately under a neutral non-reactive gas (such as nitrogen). Ampoules can be made up of any suitable material (such as glass, organic polymers (such as polycarbonate, polystyrene), ceramics, metal or any other material generally used to hold reagents). Other examples of suitable containers include bottles that can be made of materials similar to ampoules, and medicine bags that can be made up of the inside (such as aluminum or alloy) of a foil lining. Other containers include test tubes, vials, flasks, bottles, syringes, etc. Containers can have sterile access ports, such as bottles with a stopper that can be pierced by a hypodermic needle. Other containers can have two compartments, which are separated by a film that is easy to remove, and the film allows component mixing after removal. Removable film can be glass, plastics, rubber, etc.

[0164] In certain embodiments, the kit may be provided with instructional materials. The instructions may be printed on paper or other substrates, and / or may be provided as an electronically readable medium or video. The detailed instructions may not be physically associated with the kit; instead, the user may be directed to an internet website designated by the manufacturer or distributor of the kit.

[0165] As described herein, a control sample or reference sample can be a sample from a healthy subject or from a random group of subjects. A reference value can be used instead of a control or reference sample previously obtained from a healthy subject or group of healthy subjects. A control sample or reference sample can also be a sample or spiked sample with a known amount of a detectable compound.

[0166] The methods and algorithms of the present invention can be encapsulated in a controller or processor. In addition, the methods and algorithms of the present invention can be embodied as one or more computer-implemented methods for executing one or more such computer-implemented 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 ("computer program" herein), wherein when the computer program is loaded into a computer or other processor ("computer" herein) and / or executed by the computer, the computer becomes a device for practicing the one or more methods. Storage media for containing such computer programs include, for example, floppy disks and magnetic disks, compact disks (CD)-ROMs (whether writable or not), DVD digital disks, RAM and ROM memories, computer hard drives and backup drives, external hard drives, "thumb" drives, and any other storage media readable by a computer. The one or more methods can also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted through a transmission medium such as an electrical conductor, optical fiber or other optical conductor, or transmitted by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or executed by a computer, the computer becomes a device for practicing the one or more methods. The one or more methods can be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice one or more processes. When a general-purpose microprocessor is employed, the computer program code configures the microprocessor's circuitry to create a specific logic circuit arrangement. Computer-readable storage media include media that can be read by the computer itself or by another machine that reads computer instructions to provide those instructions to the computer for controlling its operation. Such machines may include, for example, machines for reading the storage media mentioned above. Example

[0167] The present invention has various aspects illustrated by the following non-limiting examples. Example 1: Identification of pathological changes in primary tauopathies by the microtubule-binding domain of CSFtau summary:

[0168] In the fluid biomarker research of Alzheimer's disease (AD), progress has been made, but there is no fluid biomarker or the imaging tracer with the effectiveness for diagnosis and / or therapeutic diagnosis (theragnosis) that can be used for other tau diseases.Use immunoprecipitation and mass spectrometry, show and contrast and other FTLD-tau (i.e. Pick's disease) compared, 4 repeat (4R) subtype specific tau species (MTBR-tau275 and MTBR-tau282) from microtubule binding zone increase in the brain of corticobasal ganglia degeneration (CBD), progressive supranuclear palsy (PSP), frontotemporal lobar degeneration (FTLD)-MAPT and AD, but on the contrary reduce in the CSF of CBD, FTLD-MAPT and AD.CSF MTBR-tau measurement is reproducible in repeated lumbar punctures, and can distinguish CBD from contrast (area under the curve of 0.889) and other FTLD-tau for example PSP (area under the curve of 0.886). CSF MTBR-Tau275 and MTBR-tau282 may represent the first definitive biomarkers to aid in the diagnosis of primary tauopathies and facilitate the design of clinical trials. introduction:

[0169] Tauopathies are a heterogeneous group of neurodegenerative diseases, all of which involve aggregated tau protein. The symptomatic phase of these fatal diseases involves neurological impairment, which typically progresses over years to decades, resulting in substantial medical, social, and economic burdens for patients and their families. For Alzheimer's disease (AD), the most common tauopathy, cerebrospinal fluid (CSF) biomarkers beta-amyloid (Aβ) as well as total tau and phosphorylated tau have been used to aid diagnosis. 1-8 , and these biomarkers have become useful in evaluating treatment outcomes in clinical trials. 9-11 Furthermore, recent advances in positron emission tomography (PET) imaging now enable the measurement of aggregated Aβ and tau in the brains of living AD patients. 12In recent years, the research progress of the present invention has been made in the field of tauopathy.In recent years, the research progress of tauopathy is the most important progress of tauopathy.In contrast, for being classified as frontotemporal lobar degeneration (FTLD-tau), other tauopathy including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), globular glial tauopathy (GGT), chronic traumatic encephalopathy (CTE) and Pick's disease (PiD) lacks this type of progress. In the case of not having the fluid biomarker for described tauopathy, accurate diagnosis is challenging because these diseases fall into the pedigree comprising multiple and overlapping clinical phenotypes. Most of tauopathy can only be clearly diagnosed by brain autopsy. It will be necessary to use antemortem fluid biomarker for these tauopathy to improve the accuracy of clinical diagnosis and promote the clinical trial of tauopathy therapy.

[0170] Recent structural and biochemical analyses have shown that different tau species constitute brain tau aggregates in subtypes of tauopathies. Six tau splice isoforms are expressed in the adult brain, including isoforms containing R1, R3, R4 repeat domains (3R) and R1, R2, R3, and R4 repeat domains (4R) in the MTBR. 13 Based on the dominant isoforms found in tau aggregates, tauopathies can be classified as 3R, 4R, and mixed 3R / 4R tauopathies. Cryo-electron microscopy (CryoEM) studies have shown that in AD (3R / 4R) 14 、PiD(3R) 15 、CBD(4R) 16 、PSP(4R) 17 and CTE(3R / 4R) 18 Different tau fibril structures exist in tau aggregates. R3 and R4 repeat domains are commonly found in tau aggregates from AD and other tauopathies. 14 In contrast, the 4R subtype-specific R2 repeat domain, in addition to R3 and R4, was found in tau aggregates from CBD and PSP. 16,17 Biochemical extraction and mass spectrometry approaches recently revealed that specific tau peptides, such as residues 243–254 (MTBR-tau243; R1), 299–317 (MTBR-tau299; R2–R3), and 354–369 (MTBR-tau354; R4), are differentially enriched in AD brains with disease progression. 19 Furthermore, truncated tau containing MTBR can be detected and quantified in CSF, and CSF-soluble concentrations of MTBR-tau peptide reflect AD clinical severity and strongly correlate with tau PET measurements. 19 Others have also reported a correlation between CSF soluble truncated MTBR-tau and insoluble tau aggregates measured by tau PET.20 , suggesting that measurement of MTBR-tau peptide may serve as a fluid biomarker of tau aggregation in AD.

[0171] As a solution, the inventors hypothesized that 4R subtype-specific MTBR-tau species accumulate in the brain of the specific subtype of 4R tau disease. Studied whether these changes are reflected in CSF, and whether these changes can distinguish the different subtypes of primary tau disease. Particularly monitored tau peptides 275-280 (MTBR-tau275) and 282-290 (MTBR-tau282), which are in the R2 region and specific to the 4R tau splicing subtype. Tested the differential diagnostic ability of MTBR-tau275 and MTBR-tau282 normalized with respect to total tau, to distinguish the effect in FTLD-tau, FTLD-TdP, controls and in the different subtypes of tau disease. method: Human studies

[0172] This retrospective cohort of pathologically confirmed cases included participants seen at UCSF or participating study sites under the following programs: the Hillblom Healthy Aging Study, the UCSF Alzheimer's Disease Research Center Program Project Grant (P30AG062422), the ARTFL-LEFFTDS Longitudinal Frontotemporal Lobar Degeneration (ALLFTD, U19AG063911), and the Four Repeat Tauopathy Neuroimaging Initiative (4RTNI, R01AG038791). The collection and use of biospecimens were approved by the institutional review board of ALLFTD or each recruiting individual's study center, and the study was approved by the UCSF Biospecimen Resource Committee. Participants provided written informed consent at the time of recruitment. Participants underwent a standardized clinical assessment, which included the collection of demographic data, a structured participant / informant interview, functional assessment, neurologic examination, and neuropsychological testing. CSF extraction was performed at the same visit as the clinical evaluation; the primary clinical syndrome was determined at the time of the clinical evaluation by an experienced neurologist or team of neurologists based on all available data, following established diagnostic criteria. The patient or their surrogate consented to the autopsy, in accordance with the principles outlined in the Declaration of Helsinki.

[0173] The Repeat Lumbar Puncture (LP) Study (NCT03545126) was approved by the Institutional Review Boards of Washington University in St. Louis, MO, USA, and University College London (UCL), UK. All participants or their surrogates consented to the collection and sharing of biofluid samples and brain autopsies and were compensated. Exclusion criteria included any contraindications to LP or lumbar catheterization, including bleeding disorders, active anticoagulation, and active infection.

[0174] The study of the clinical diagnostic cohort was approved by the Ethics Committee of Montpellier University Hospital, France (CSF-Neurobank #DC-2008-417, certification number NFS 96-900, CHU Resource Center BB-0033-00031 (http: / / www.biobanques.eu)). Authorization for the processing of personal data was granted by the French Data Protection Authority (CNIL) under number 1709743v0. Human brain samples

[0175] Neuropathological diagnosis of human brain donations was performed at Washington University and UCSF according to established diagnostic criteria. Frozen human brain tissue samples selected for this study were processed as previously described. 19,36 Briefly, frozen brain tissue was 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 at 11,000 g for 20 min at 4°C and stored at −80°C as whole brain extracts. The demographic characteristics of the brain donors included in this study are described in Figure 1 middle. Human CSF samples

[0176] CSF collection methods were similar in all cohorts examined in this study. CSF collection methods from pathologically confirmed cases were as previously described in the Alzheimer's Disease Neuroimaging Initiative (ADNI) procedure manual.

[0177] Demographic characteristics of participants in the pathology-confirmed cohort were Figure 2 CSF collection methods from replicate LP studies were collected using the same human tau stable isotope labeling kinetic protocol as previously described.37 The demographic characteristics of the participants in the repeat lumbar puncture study are described in Figure 9 The CSF collection methods and demographic characteristics of the clinical diagnosis cohort were previously described. 24 . Mass spectrometry analysis of MTBR-tau

[0178] Brain-insoluble MTBR-tau was analyzed using filter-assisted sample preparation as previously described. 19,36 Briefly, whole brain extracts were incubated with 1% sarkosyl for 60 min on ice, followed by ultracentrifugation at 100,000 g for 60 min at 4°C to obtain an insoluble pellet. The insoluble brain fraction was filtered, digested, desalted, and injected into a mass spectrometer for analysis.

[0179] CSF MTBR-tau was analyzed as previously described. 19 , with the following modifications: Before adding CSF, a premix containing detergent and chaotropic reagent (final 1% NP-40, 5 mM guanidine, protease inhibitor cocktail) and an internal standard for tau (15N-labeled 2N4R recombinant tau) was prepared in a polypropylene tube. 0.5 mL of CSF was added, and immunoprecipitation was performed using Tau1, HJ8.5, and HJ8.7 anti-tau antibodies with epitopes present in the N-terminal to middle domains of tau. 24,37 . The immunoprecipitated samples depleted of the N-terminus of tau to the middle domain were further immunoprecipitated with the 77G7 anti-tau antibody against MTBR (residues 316-335) to measure MTBR-Tau species. After washing, the samples were digested with trypsin, desalted, and analyzed by Orbitrap Eclipse mass spectrometer (ThermoScientific). The mass spectrometry method for measuring MTBR-tau was as described above with some modifications. To account for individual variability in t-tau concentrations, the ratio of MTBR-tau275 or MTBR-tau282 normalized to the middle domain tau peptide (181-190 and 212-221 for brain and CSF analysis, respectively) was used, which was common to all isoforms. statistics

[0180] All statistical analyses were performed using GraphPad Prism software (v9.3.1). Unless otherwise stated, differences in biomarker values were assessed using one-way ANOVA. Two-sided p < 0.05 was considered statistically significant and corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) method, with the FDR set at 5%. 38P values reported in tables and figures were corrected by the Benjamini-Hochberg method (FDR=5%). Spearman correlation was used to assess the association between CSF tau biomarkers and neuropathological changes. result: 4R-specific brain MTBR-tau is increased in primary tauopathies

[0181] First, frozen brain tissue from 59 individuals with autopsy-confirmed AD, FTLD-tau, or FTLD with TAR DNA-binding protein aggregates (FTLD-TDP) and 3 normal controls (NC) (p < 0.0001, p < 0.01, respectively), 4R tauopathy, 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 was moderately increased (approximately 2-fold) in PSP compared with FTLD-TDP (p < 0.01). Brain MTBR-tau282 / t-tau had a similar increase profile as MTBR-tau275 / t-tau ( Figure 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 indicate that 4R-specific MTBR-tau species are enriched in the insoluble fraction of SFG / insular cortex brain tissue in subgroups of 4R tauopathies (e.g., CBD and FTLD-MAPT) and are moderately increased in different PSPs (4R tauopathies) and AD (3R / 4R mixed tauopathies). 4R-specific CSFMTBR-tau is reduced in primary tauopathies

[0182] Next, CSF from 29 NC, -FTLD-MAPT and 78 autopsy-confirmed AD, primary tauopathy, and FTLD-TDP cases were analyzed for MTBR-tau275 and MTBR-tau282. Figure 2 CSF MTBR-tau275 and MTBR-tau282 concentrations are affected by individual variability in t-tau concentrations ( Figure 12C ) but failed to separate different tauopathies ( Figure 12A and 12BThus, CSF MTBR-tau275 and MTBR-tau282 derived from truncated tau are expressed by the intermediate domain tau 212-221 ( Figure 5A ) was normalized to t-tau, similar to the normalization method previously reported in the measurement of truncated tau and Aβ isoforms. 20,22 CSF MTBR-tau275 / t-tau was decreased in CBD (0.00525±0.00117), AD (0.00472±0.00085), and FTLD-MAPT (0.00491±0.00207) compared with 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). Figure 5B ). This reduction is particularly significant in FTLD-MAPT P301L, which has more typical FTLD pathology than R406W with many features of AD. Compared with other 4R tauopathy AGD (0.00759 ± 0.00013) and PSP (0.00669 ± 0.00091, p < 0.001, p < 0.0001, p < 0.01) and 3R tauopathy PiD (0.00676 ± 0.00138, p < 0.05, p < 0.01, p < 0.05), CSF MTBR-tau275 also reduces in CBD, AD and FTLD-MAPT. CSF MTBR-tau282 / t-tau has a reduction profile similar to CSF MTBR-tau275 / t-tau ( Figure 5C Interestingly, CSF MTBR-tau275 / t-tau were unchanged in PSP compared with controls or FTLD-TDP, although these ratios were moderately elevated in the brain.

[0183] To assess whether soluble CSF MTBR-tau / t-tau measurements reflect brain tau pathology as measured by paired insoluble brain MTBR-tau / t-tau measurements, we analyzed the correlation of MTBR-tau / t-tau between antemortem CSF and brain from the same individuals (N = 54, Figure 6A and Figure 6BMTBR-tau275 / t-tau and MTBR-tau282 / t-tau from CSF and brain were moderately correlated in all disease groups (N=54) (r=-0.27, p=0.049, and r=-0.45, p=0.0006, respectively) and strongly correlated in the 4R tauopathies (PSP, CBD, and AGD, N=29) (r=-0.61, p=0.0004, and r=0.75, p<0.0001, respectively). Figure 6C and Figure 6D This suggests that 4R-specific MTBR-tau species are negatively correlated in CSF and brain of 4R tauopathies. In CBD, MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from CSF and brain were moderately correlated, but did not achieve statistical significance (N=12, r=-0.25, p=0.43 and r=-0.31, p=0.33, respectively). Figure 3 E, F). A CBD participant without cognitive impairment (sum of Clinical Dementia Rating Scale plus National Alzheimer's Disease Coordinating Center FTLD score) 23 (CDR plus NACC FTLD-SB) = 0) changes in MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in both brain and CSF were small (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 indicate that changes in these biomarkers depend on the severity of the disease.

[0184] To assess whether CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau decrease with disease stage, the correlation between these biomarkers and duration (i.e., the interval between age at onset (AAO) and CSF collection) was investigated. Figure 2 , Figure 13B). The average duration of these diseases was 5±4 years (N=81), and only 4±1 years (N=18) for CBD. In CBD, there was a negative correlation between duration and CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau (r=-0.37 and -0.39, respectively), although no statistical significance was observed (p=0.13 and 0.11, respectively). This result suggests that participants with a longer duration at a later stage of pathology had a greater reduction in CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau biomarkers. CSFMTBR-tau is reproducible in repeat lumbar punctures

[0185] To evaluate the reproducibility and stability of 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 (LP, 3-5 times) over approximately 4 months as part of an ongoing study examining protein turnover kinetics. 37 ( Figure 9 , Figure 7 ). These participants included individuals clinically diagnosed with PSP-Richardson syndrome (PSP-RS, N=7) or corticobasal syndrome (CBS, a clinical syndrome associated with a heterogeneous neuropathological substrate (including AD, CBD, PSP and FTLD-TDP, N=9), of which 2 participants had autopsy-confirmed CBD. They also included 7 symptomatic or asymptomatic carriers of MAPT mutations (P301L, R406W and IVS10+16) and 2 non-carrier family members who served as normal controls (NC). The average coefficient of variation (CV) of CSF MTBR-tau275 / t-tau in repeated LP was 12±7%, which established the high reproducibility and stability of CSF MTBR-tau measurements over 4 months.

[0186] Consistent with the FTLD-MAPT cases analyzed in the pathology-confirmed cohort, CSF MTBR-tau275 / t-tau was reduced in FTLD-MAPT mutation carriers in the repeat LP cohort. Interestingly, CSF MTBR-tau275 / t-tau was reduced in two symptomatic FTLD-MAPT P301L mutation carriers (participants #02, #03, 0.00381 ± 0.00021) and one symptomatic FTLD-MAPT R406W mutation carrier (#05, 0.00508) compared with NC (#01, #04, 0.00666 ± 0.00027). However, CSF MTBR-tau275 / t-tau did not change in FTLD-MAPT R406W mutation carriers (#06, #07) who were asymptomatic at the time of LP. The FTLD-MAPT variant IVS10+16 promotes splicing of tau exon 10, resulting in higher production of the 4R than the 3R isoform. 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 with NC, suggesting an increase in the 4R isoform reflected in the CSF.

[0187] CSF MTBR-tau275 / t-tau was reduced in two participants (#10 (0.00396), #11 (0.00535)) who were clinically diagnosed with PSP-RS but subsequently confirmed to have CBD at autopsy, consistent with the results of the pathologically confirmed CSF cohort. 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 had not yet been autopsied during the repeated measurement study. The average duration of these diseases was 5 ± 3 years (N = 21), and only CBS was 4 ± 2 years (N = 9), which was similar to the pathologically confirmed cohort. CSFMTBR-tau in clinically diagnosed primary tauopathies

[0188] To evaluate the performance of the CSF MTBR-tau biomarker in clinically diagnosed primary tauopathies, CSF MTBR-tau275 / t-tau ( Figure 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 the CBS-PSP continuum 25 Individuals with the CBS-PSP continuum were defined as patients who initially presented with CBS but subsequently developed clinical features of PSP-RS as the disease progressed. Compared with cognitive NC, CSF MTBR-tau275 / t-tau was reduced in the CBS-PSP continuum and FTLD-MAPT (p < 0.05). However, CSF MTBR-tau275 / t-tau was not statistically different in AD or clinically diagnosed CBS compared with controls or other tauopathies. Diagnostic accuracy of CSFMTBR-tau in primary tauopathies

[0189] Finally, the diagnostic accuracy of CSF MTBR-tau 275 / t-tau and MTBR-tau 282 / t-tau was examined in a cohort of pathologically confirmed primary tauopathies. First, CSF t-tau (middle domain peptide 212-221) and phosphorylated tau (pT217 / T217) were examined for comparison in primary tauopathies ( Figure 12C and Figure 14A ). CSFt-tau was increased in AD compared with NC and PSP (p < 0.05) and could distinguish AD from FTLD-tau (PSP, CBD, AGD, PiD, FTLD-MAPT) with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.794 ( Figure 14B However, CSF t-tau did not discriminate FTLD-tau. CSF pT217 / T217 was increased in AD compared with NC, FTLD-TDP, and FTLD-tau (p < 0.0001) and could discriminate AD from FTLD-tau with an AUC of 0.987 ( Figure 14C AD co-pathology in other neurodegenerative diseases (i.e., FTLD-TDP, CBD, PSP) also increases CSF pT217 / T217. These results suggest that CSF pT217 / T217 can be used to accurately identify individuals with AD pathology, regardless of co-pathology.

[0190] The effect of amyloid on CSF MTBR-tau 275 / t-tau and MTBR-tau 282 / t-tau in primary tauopathies was further evaluated using the AD Thal stage. CSF pT217 / T217 was strongly correlated with AD Thal stage (r = 0.52, p < 0.0001, Figure 15A However, CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau were significantly higher in the entire cohort (r = -0.22, p = 0.06 and r = -0.24, p = 0.04, respectively). Figure 15B and Figure 15C ) or in CBD (r = -0.14, p = 0.60 and r = -0.07, p = 0.78, respectively, Figure 15D and Figure 15E ) was not associated with AD Thal stage. These results suggest that CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau are reduced in CBD independently of AD co-pathology.

[0191] The diagnostic accuracy of CSF MTBR-tau 275 / t-tau and MTBR-tau 282 / t-tau was examined to determine whether they differentiate CBD from controls, FTLD-TDP, FTLD-tau as a group, and individual tauopathies ( Figure 3 , Figures 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 AUCs of 0.800 to 0.889. CBD can be distinguished from FTLD-TDP, with AUCs of 0.701 to 0.770. When AD co-pathology cases are excluded, CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau can distinguish CBD from PSP, with AUCs of 0.859 and 0.886, respectively ( Figure 3 , Figure 16K and Figure 16L ).

[0192] Finally, CSF MTBR-tau 275 / t-tau and MTBR-tau 282 / t-tau were retrospectively evaluated in a neuropathologically confirmed cohort by final clinical syndrome to determine whether these biomarkers could facilitate antemortem diagnosis of primary tauopathies. Figure 3The number of individuals with CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau (defined in the cutoff to differentiate CBD from PSP) was included to differentiate CBD from PSP in PSP-RS, as 0 / 11 (0%) and 2 / 11 (18%) PSP had CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau below the cutoff values. discuss:

[0193] Despite the long-standing search for antemortem biomarkers of FTLD-tau pathology, no fluid-derived biomarkers have been identified to date that can distinguish subgroups of tauopathies other than AD. Previous studies have shown that CSF tau phosphorylated at T181 is reduced in PSP and FTLD-TDP relative to controls. 26–30 Recently, CSF pT217 / T217 was shown to be increased in FTLD-MAPT R406W compared with controls without Aβ pathology. 24 However, phosphorylated tau is most significantly altered in AD, and thus these studies may not have captured the key pathological changes in primary tauopathies. In this study, we focused on MTBR-tau, which constitutes the core region of tau aggregates in the brain and is also present as a truncated C-terminal tau peptide in CSF. 19 . Using biochemical purification and quantitative mass spectrometry methods, it was shown that 4R isoform-specific MTBR-tau275 and MTBR-tau282, normalized to t-tau, decreased in CSF soluble tau as they increased in brain insoluble tau in primary tauopathies (especially CBD and FTLD-MAPT P301L). MTBR-tau / t-tau measurements were negatively correlated in CSF and brain, suggesting that there may be an equilibrium or unidirectional transfer between soluble CSF MTBR-tau and insoluble brain MTBR-tau in these primary tauopathies. This study provides the first possibility of a fluid biomarker reflecting brain pathology in primary tauopathies.

[0194] One of the findings that attract people's attention of this research is that the change of 4R subtype specificity MTBR-tau 275 and MTBR-tau 282 is only observed in the subgroup of 4R and 3R / 4R mixed type tau disease.As expected, these 4R subtype specific measurements do not change in 3R tau disease (PiD) or non-tau disease FTLD (FTLD-TDP).However, CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau specifically reduce in CBD and FTLD-MAPT, but do not reduce in PSP and AGD, even if it is all classified as 4R tau disease.This may be due to compared with CBD and most FTLD-MAPT cases, and the variability of neocortex pathological degree is higher in PSP and AGD cases.

[0195] Many PSP cases have 4R tau aggregates primarily in subcortical regions such as the thalamus and brainstem. 31 , and AGD pathology is most severe in the medial temporal lobe, even in advanced stages 32 In contrast, CBD was associated with more abundant and widespread tau pathology in the brain. 33 FTLD-MAPT (e.g., P301L) leads to very high deposition of 4R tau aggregates in neurons and glia in multiple brain regions, including the hippocampus, neocortex, and substantia nigra. 34 An alternative explanation is that CBD has delicate filamentous inclusions in neuronal cell bodies, whereas PSP neurons tend to carry a greater proportion of more compact tau aggregates. 35 CBD is generally associated with abundant cortical astrocyte plaque pathology and neuroinflammatory tau pathology in both gray and white matter, while PSP neuronal and astroglial pathology (i.e., clustered astrocytes) is generally limited to the motor cortex and premotor cortex and subcortical nuclei. Tau aggregates in PSP may have different physicochemical properties from CBD, and the equilibrium state between insoluble and soluble forms may be different. In summary, it is speculated that the amount or total load of 4R tau pathology in the whole brain may reflect changes in CSF 4R-specific MTBR-tau.

[0196] CSF MTBR-tau 275 / t-tau and MTBR-tau 282 / t-tau have the potential to positively identify subgroups of primary tauopathies and aid in antemortem differential diagnosis. In our repeated lumbar puncture study, CSF MTBR-tau / t-tau measurements were reproducible and stable over 4 months, which would reliably provide biomarker values in clinical or clinical trial settings. In a cohort of patients with clinical diagnoses without autopsy confirmation, CSF MTBR-tau 275 / t-tau did not change in CBS, and there was a high overlap between CBS and PSP-RS, which may be attributed to the lack of a one-to-one relationship between clinical syndromes and neuropathological diagnoses in FTLD. However, a retrospective clinical syndrome analysis from a pathologically confirmed cohort showed that CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau biomarkers could identify individuals with CBD regardless of clinical syndrome (ie, CBS, bvFTD, and PSP-RS) with an accuracy of up to 83%, which is approximately 25 to 50% higher than the diagnostic accuracy of CBD in the absence of these biomarkers.

[0197] The other limitation of this research includes the following. Tauopathies with shorter duration or during the asymptomatic phase may not yet show the minimizing of these biomarkers. Future research for larger cohorts with different degrees of severity and longitudinal samples with clinical measurements will help solve whether CSF MTBR-tau can capture disease in the earlier stages of primary taupathies. Lack of orthogonal measurements identifies the brain tau pathology in living patients (that is, using primary taupathies with specific tracers to carry out tau PET imaging) has limited the ability of the correlation between CSF and brain MTBR-tau in our assessment before death. For subgroups (such as AGD and PiD), the medium-sized cohorts with small sample sizes may limit explanation. The 77G7 MTBR-tau antibody for sequential immunoprecipitation in research can also target the specific pool of tau of brachymemma, and there may be future technological advancements and analytical method developments that can disclose other or new tau species colonies in the biofluid, which reflect the qualitative and quantitative aspects of tau pathology in primary taupathies. Together, these findings advance our understanding of the heterogeneous pathophysiology in primary tauopathies and open avenues for therapeutic development and clinical trials targeting primary tauopathies. References: 1. Ovod, V et al., Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis. Alzheimers Dement. J. Alzheimers Assoc. 13, 841–849 (2017). 2. Nakamura, A et al., High performance plasma amyloid-β biomarkers for Alzheimer’s disease. Nature 554, 249–254 (2018). 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). 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). 5. Janelidze, S et al., Cerebrospinal fluid p-tau217 performs better than p-tau181 as a biomarker of Alzheimer’s disease. Nat. Commun. 11, 1–12 (2020). 6. Barthélemy, N.R. et al., Cerebrospinal fluid phospho-tau T217 outperforms T181 as a biomarker for the differential diagnosis of Alzheimer’s disease and PET amyloid-positive patient identification. Alzheimers Res. Ther. 12, 26 (2020). 7. Palmqvist, S. et al., Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders. JAMA 324, 772–781 (2020). 8. Barthélemy, N.R., Horie, K., Sato, C. and Bateman, R.J. Blood plasma phosphorylated-tau isoforms track CNS change in Alzheimer’s disease. J. Exp. Med. 217, (2020). 9. Mintun, M.A. et al., Donanemab in Early Alzheimer’s Disease. N. Engl. J. Med. 384, 1691–1704 (2021). 10. Salloway, S. et al., Atrial of gantenerumab or solanezumab in dominantly inherited Alzheimer’s disease. Nat. Med. 27, 1187–1196 (2021). 11. Swanson, C.J. et al., A randomized, double-blind, phase 2b proof-of-concept clinical trial in early Alzheimer’s disease with lecanemab, an anti-Aβ protofibril antibody. Alzheimers Res. Ther. 13, 80 (2021). 12. Villemagne, V.L., Doré, V., Burnham, S.C., Masters, C.L. and Rowe, C.C. Imaging tau and amyloid-β proteinopathies in Alzheimer disease and other conditions. Nat. Rev. Neurol. 14, 225–236 (2018). 13. Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D. and Crowther, R.A. Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer’s disease. Neuron 3, 519–526 (1989). 14. Fitzpatrick, A.W.P et al., Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 547, 185–190 (2017). 15. Falcon, B et al., Structures of filaments from Pick’s disease reveal a novel tau protein fold. Nature 561, 137–140 (2018). 16. Zhang, W et al., Novel tau filament fold in corticobasal degeneration. Nature 580, 283–287 (2020). 17. Shi, Y et al., Structure-based classification of tauopathies. Nature 598, 359–363 (2021). 18. Falcon, B et al., Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules. Nature 568, 420–423 (2019). 19. Horie, K., Barthélemy, N.R., Sato, C. and Bateman, R.J. CSF tau microtubule binding region identifies tau tangle and clinical stages of Alzheimer’s disease. Brain 144, 515–527 (2021). 20. Blennow, K et al., Cerebrospinal fluid tau fragment correlates with tau PET: a candidate biomarker for tangle pathology. Brain 143, 650–660 (2020). 21. Bang, J., Spina, S. and Miller, B.L. Frontotemporal dementia. The Lancet 386, 1672–1682 (2015). 22. Wiltfang, J et al., Amyloid β peptide ratio 42 / 40 but not Aβ42 correlates with phospho-Tau in patients with low- and high-CSF Aβ40 load. J. Neurochem. 101, 1053–1059 (2007). 23. Miyagawa, T et al., Utility of the global plus NACC FTLD rating and development of scoring rules: Data from the ARTFL / LEFFTDS Consortium. Alzheimers Dement. 16, 106–117 (2020). 24. Sato, C et al., MAPT R406W increases tau T217 phosphorylation in absence of amyloid pathology. Ann. Clin. Transl. Neurol. 8, 1817–1830 (2021). 25. Ling, H. Clinical Approach to Progressive Supranuclear Palsy. J. Mov. Disord. 9, 3–13 (2016). 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). 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). 28. Hu, W. T et al., Reduced CSF p-Tau181 to Tau ratio is a biomarker for FTLD-TDP. Neurology 81, 1945–1952 (2013). 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). 30. M et al., Biomarkers for tau pathology. Mol. Cell. Neurosci. 97, 18–33 (2019). 31. Kovacs, G. G. et al., Distribution patterns of tau pathology in progressive supranuclear palsy. Acta Neuropathol. (Berl.) 140, 99–119 (2020). 32. Togo, T. et al., Argyrophilic Grain Disease Is a Sporadic 4-Repeat Tauopathy. J. Neuropathol. Exp. Neurol. 61, 547–556 (2002). 33. Kouri, N., Whitwell, J. L., Josephs, K. A., Rademakers, R. and Dickson, D. W. Corticobasal degeneration: a pathologically distinct 4R tauopathy. Nat. Rev. Neurol. 7, 263–272 (2011). 34. Spillantini, M. G., Crowther, R. A., Kamphorst, W., Heutink, P. and van Swieten, J. C. 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Claims

1. A method for 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 the microtubule binding region (MTBR)-tau275 peptide and the MTBR-tau282 peptide.

2. A method for detecting 4R tau aggregates in a subject, the method comprising: (a) purifying endogenous cleavage fragments of tau from a CSF sample from the subject without exposing the endogenous cleavage fragments of tau to a protease in vitro; (b) contacting the purified endogenous cleavage fragment of tau with an endopeptidase to obtain one or more of a proteolytic microtubule binding region (MTBR)-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1 and a proteolytic MTBR-tau282 peptide comprising amino acids 282-290 relative to SEQ ID NO: 1; and (c) detecting and measuring the proteolytic MTBR-tau275 peptide and / or the proteolytic MTBR-tau282 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptide and / or the proteolytic MTBR-tau282 peptide is indicative of 4R tau aggregates in the subject.

3. The method of claim 2, wherein the solution comprising the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptide is desalted prior to step (c), optionally wherein the desalting is performed 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 an antigen-binding fragment thereof.

6. The method of any one of claims 2 to 5, wherein the purification in step (a) comprises immunoprecipitation.

7. The method of any one of claims 2 to 6, wherein the purification in step (a) comprises immunoprecipitation of N-terminal to middle domain tau to deplete N-terminal to middle domain tau from the biological sample.

8. The method of claim 7, wherein the immunoprecipitation of N-terminal to middle 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 shown 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 immunoprecipitating 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 an MTBR-tau275 peptide and / or an MTBR-tau282 peptide.

12. The method of claim 10 or 11, wherein immunoprecipitation of the 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 immunoprecipitation of the 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 shown in SEQ ID NO:

1.

14. The method of any one of claims 10 to 13, wherein the epitope binding agent is the 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.

17. The method of any one of claims 1 to 16, further comprising one or more of detection and quantification of one or more of beta-amyloid, N-terminal tau, middle domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample.

18. The method of claim 17, wherein the mid-domain tau is quantified, wherein the mid-domain tau comprises tau212-221 (t-tau) relative to SEQ ID NO:

1.

19. The method of claim 18, wherein the amount of one or more of MTBR-tau275 peptide and MTBR-tau282 peptide is normalized to the amount of t-tau, and the amount of 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 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) affinity-depleting a cerebrospinal fluid (CSF) sample from the subject by contacting the biological sample with one or more affinity-depleting agents comprising one or more epitope binding agents that each bind to one of N-terminal tau, middle domain tau, or C-terminal tau, but do not bind to an antigen within the microtubule-binding region (MTBR) tau, to obtain a depleted sample and an enriched sample, wherein the CSF sample comprises endogenous cleavage fragments of tau, wherein the depleted sample comprises N-terminal tau, middle domain tau, and / or C-terminal tau, and wherein the enriched sample is enriched for endogenous cleavage fragments of tau comprising one or more of an endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO: 1 and an endogenous MTBR-tau282 peptide comprising amino acids 282-290 relative to SEQ ID NO: 1; (b) immunoprecipitating the enriched sample by contacting the enriched sample with one or more immunoprecipitants to obtain a purified sample, wherein the immunoprecipitants comprise one or more epitope binding agents that bind to MTBR-tau to capture one or more of endogenous MTBR-tau275 peptide and endogenous MTBR-tau282 peptide; (c) contacting one or more of the endogenous MTBR-tau275 peptide and the endogenous MTBR-tau282 peptide in the purified sample with an endopeptidase to obtain a sample comprising one or more of the proteolytic MTBR-tau275 peptide and the proteolytic MTBR-tau282 peptide; and d) detecting and measuring one or more of a proteolytic MTBR-tau275 peptide and a proteolytic MTBR-tau282 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein the amount of the one or more of the proteolytic MTBR-tau275 peptide and the proteolytic MTBR-tau282 peptide is indicative of 4R tau aggregates in the subject.

22. The method of claim 21, wherein the sample comprising one or more of the proteolytic MTBR-tau275 peptide and the proteolytic MTBR-tau282 peptide is desalted prior to step (d), optionally wherein the desalting is performed by solid phase extraction.

23. The method of claim 21 or 22, wherein the one or more affinity depleting agents comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 1-243 of the sequence shown 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 antigenic 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 immunoprecipitating agents comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244-368 of the sequence shown in SEQ ID NO:

1.

27. The method of claim 26, wherein the one or more immunoprecipitating agents comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 316-335 of the sequence shown in SEQ ID NO:

1.

28. The method of any one of claims 21 to 27, wherein the one or more immunoprecipitating 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.

30. The method of any one of claims 21 to 29, further comprising one or more of detection and quantification of one or more of beta-amyloid, N-terminal tau, middle domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample.

31. The method of claim 30, wherein the mid-domain tau is quantified, wherein the mid-domain tau comprises tau212-221 (t-tau) relative to SEQ ID NO:

1.

32. The method of claim 31, wherein the amount of one or more of the MTBR-tau275 peptide and the MTBR-tau282 peptide is normalized to the amount of t-tau, and wherein the amount of 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 indicates 4R tau aggregates in the subject.

34. A method of detecting a primary tauopathy in a subject, the method comprising detecting tau or 4R tau aggregates according to any one of claims 1 to 33.

35. The method of claim 34, wherein the primary tauopathy is selected from 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 indicates one or more of frontotemporal lobar degeneration (FTLD)-MAPT and corticobasal degeneration (CBD).

37. A method for detecting primary tauopathy-associated deposits in the brain of a subject, the method comprising detecting tau or 4R tau aggregates according to any one of claims 1 to 33.

38. The method of claim 37, wherein the primary tauopathy-associated deposits are from frontotemporal lobar degeneration (FTLD)-MAPT and 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).

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) 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, FTLD-MAPT is diagnosed.

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, or providing the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from the CSF samples according to the method of any one of claims 1 to 33; and (b) calculating a difference between the amount 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 one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in the second sample compared to the first sample indicates progression of FTLD-MAPT in the subject.

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) CBD is diagnosed when the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau detected is reduced relative to a threshold value.

43. A method of measuring the progression of CBD in a subject, comprising: (a) detecting tau or 4R tau aggregates in a first CSF sample and a second CSF sample from the subject, or providing the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from the CSF samples according to the method of any one of claims 1 to 33; and (b) calculating a difference between the amount 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 one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in the second sample compared to the first sample indicates progression of CBD in the subject.

44. A method of treating primary tauopathy in a subject in need thereof, 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) administering to the subject a treatment that alters tau pathology if the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau is reduced relative to a threshold value.

45. A method of treating a primary tauopathy in a subject in need thereof, comprising administering to the subject a therapy that alters tau pathology, wherein the subject has been identified as having an amount of one or more of MTBR-tau 275 / t-tau and MTBR-tau 282 / t-tau that is reduced relative to a threshold, optionally as measured according to a method according to any one of claims 1 to 33.

46. The method of claim 44 or 45, wherein the treatment alters or stabilizes the amount of one or more of the MTBR-tau275 peptide and the MTBR-tau282 peptide detected.

47. The method of any one of claims 44 to 46, wherein the treatment is selected from lencaneluzumab, donetumab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, berenuzumab, dasatinib + quercetin, E2814, epothilone D, gonelizumab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember™, cirenuzumab, TPI 287, Tideglusib, tipranezumab, zagteneizumab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors, and tau active vaccines.

48. The method of any one of claims 44 to 47, wherein the treatment is selected from anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, γ-secretase inhibitors, β-secretase inhibitors, kinase inhibitors, phosphatase activators, vaccines, and tau protein aggregation inhibitors.

49. The method of claim 48, wherein the kinase inhibitor is an inhibitor of thousand-plus-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.

51. The method of claim 48, wherein the vaccine is CAD106 or AF20513.

52. The method of claim 48, wherein the anti-A[beta] antibody is aducanumab or another anti-amyloid antibody that removes plaques.

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