Methods for detecting MTBR-tau and uses thereof

Abstract

Methods and compositions for detecting and measuring MTBR-tau species in CSF, and The use thereof for detecting pathological features and / or clinical symptoms of tauopathies is provided, which can be used to diagnose, stage or select an appropriate treatment for a given disease stage, or to modify 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 to detect pathological features or clinical symptoms of primary tauopathies, which can be used to diagnose, stage, or select appropriate treatments for primary tauopathies. [Background technology]

[0002] 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 is thought to propagate and spread across brain regions by cell-to-cell transmission of specific pathological tau species in a prion-like manner, although the diffusion process and nature of these species (e.g., monomeric, multimeric, and fibrillar species) remain unclear (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 100 potential post-translational modification sites, including phosphorylation sites and multiple truncation sites (Meredith et al., 2013; Sato et al., 2018; Barthelemy et al., 2019; Cicognola et al., 2019; Blennow et al., 2020). Therefore, identifying the specific pathological tau species involved in tau spreading is challenging.

[0003] Mass spectrometry (MS) studies suggest that the microtubule-binding region of tau (MTBR-tau) is abundant in aggregates in Alzheimer's disease brains (Taniguchi-Watanabe et al., 2016; Roberts et al., 2020). Cryo-electron microscopy (cryo-EM) has shown that the core structure of tau aggregates contains a subsegment of MTBR-tau, and specific conformations depend on the tauopathy (Fitzpatrick et al., 2017; Falcon et al., 2018, 2019; Zhang et al., 2020). These findings strongly suggest that MTBR-tau is important for tau aggregation. However, these studies used postmortem brain tissue. Little is known about the pathophysiology of corresponding extracellular MTBR-tau species in biological samples, such as CSF, which could serve as surrogate biomarkers of 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 suggested that MTBR-tau is not present in CSF and have focused on N-terminal tau and central domain tau regions (Meredith et al., 2013; Sato et al., 2018). N-terminal to central domain tau species are thought to be actively secreted from neurons into the extracellular space after truncation between the central and MTBR domains of tau (Sato et al., 2018). Detection of MTBR-tau species has been reported (Barthelemy et al., 2016b, a), but has not been characterized in relation to disease. Recently, a tau species containing a truncation at residue 368 (tau368) within repeat region 4 (R4) was identified in CSF (Blennow et al., 2020). However, considering regions not captured by the antibody, truncations and conformational variations, it is unclear whether tau368 reflects the entire pool of MTBR-tau species. Summary of the Invention

[0005] A method for detecting tau in a cerebrospinal fluid (CSF) sample is provided, which may include: (a) providing a CSF sample; and (b) detecting and measuring the amount of one or more of microtubule-binding region (MTBR)-tau275 peptide and MTBR-tau282 peptide.

[0006] Provided is a method for detecting 4R tau aggregates in a subject, the method may include one or more of: (a) purifying endogenous cleaved tau fragments from a CSF sample from the subject without contacting the endogenous cleaved tau fragments with a protease in vitro; (b) contacting the purified endogenous cleaved tau fragments with 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 the presence of 4R tau aggregates in the subject. The solution containing the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptide may be desalted prior to step (c), optionally 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, e.g., immunoprecipitating the N-terminal to mid-domain of tau, to deplete the N-terminal to mid-domain of tau from the CSF sample. The one or more anti-tau epitope binding agents may specifically bind to an epitope within amino acids 1 to 243 of the sequence set forth in SEQ ID NO: 1. The epitope binding agent may optionally be an anti-tau antibody or an antigen-binding fragment thereof selected from the anti-tau antibodies tau1, HJ8.5, or HJ8.7. The purification in step (a) can include immunoprecipitating one or more MTBR-tau species from the biological sample and capturing one MTBR-tau species from the biological sample. The one or more captured MTBR-tau species can be MTBR-tau275 peptide, MTBR-tau282 peptide, or a combination thereof.Immunoprecipitation of one or more MTBR-tau species may include contacting the CSF 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 or within amino acids 316-335 of the sequence set forth in SEQ ID NO: 1. The epitope binding agent may be anti-tau antibody 77G7 or an antigen-binding fragment thereof. The endopeptidase may be trypsin. The CSF sample may include an internal standard. The method may further include one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample. The method may quantify the amount of mid-domain tau, where optionally, the mid-domain tau comprises tau 212-221 relative to SEQ ID NO: 1 (t-tau). 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 compared to a threshold value can indicate 4R tau aggregates in the subject.

[0007] A method for detecting 4R tau aggregates in a subject, comprising: (a) performing affinity depletion in a CSF sample from the subject by contacting the biological sample with one or more affinity depletion agents, the affinity depletion agents comprising one or more epitope binding agents that each bind to one of N-terminal tau, mid-domain tau, or C-terminal tau but do not bind to an antigen within MTBR-tau, to obtain a depleted sample and an enriched sample, wherein the CSF sample contains endogenous cleaved tau fragments, the depleted sample contains N-terminal tau, mid-domain tau, and / or C-terminal tau, and the enriched sample is enriched for endogenous cleaved tau fragments comprising one or more of endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO:1 and endogenous MTBR-tau282 peptide comprising amino acids 282-290 relative to SEQ ID NO:1; (b) performing immunoprecipitation in the enriched sample to isolate endogenous MTBR-tau275 peptide and endogenous MTBR-tau282 peptide that bind to MTBR-tau. and endogenous MTBR-tau282 peptides in the purified sample with one or more immunoprecipitation agents, including one or more epitope-binding agents that capture one or more of the endogenous MTBR-tau275 peptides and endogenous MTBR-tau282 peptides, to obtain a purified sample; (c) contacting one or more of the endogenous MTBR-tau275 peptides and endogenous MTBR-tau282 peptides in the purified sample with an endopeptidase to obtain a sample containing one or more proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides; and (d) detecting and measuring one or more of the proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides by performing LC / MS or an immunoassay, wherein the amount of one or more of the proteolytic MTBR-tau275 peptides and proteolytic MTBR-tau282 peptides indicates 4R tau aggregates in the subject. One or more of the proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides can be desalted prior to step (d), optionally by solid-phase extraction. The one or more affinity depletors can include one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 1 to 243 of the sequence set forth in SEQ ID NO:1.The epitope-binding agent may optionally be an anti-tau antibody or antigenic fragment thereof selected from the anti-tau antibodies tau1, HJ8.5, or HJ8.7. The one or more immunoprecipitation agents may include one or more anti-tau epitope-binding agents that specifically bind to an epitope within amino acids 244-368 of the sequence set forth in SEQ ID NO:1 or within amino acids 316-335 of the sequence set forth in SEQ ID NO:1. The one or more immunoprecipitation agents may include the anti-tau antibody 77G7 or an antigen-binding fragment thereof. The endopeptidase may be trypsin. The method may further include one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, mid-domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample. The method may quantify the amount of mid-domain tau, where the mid-domain tau optionally comprises tau 212-221 relative to SEQ ID NO:1 (t-tau). 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 compared to a threshold value can indicate 4R tau aggregates in the subject.

[0008] A method for detecting a primary tauopathy in a subject is provided, which may include preparing an amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample from the subject. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the method for detecting tau or 4R tau aggregates described hereinabove. The primary tauopathy may be selected from the group consisting of frontotemporal lobar degeneration (FTLD) (e.g., FTLD-MAPT) and corticobasal degeneration (CBD). A decrease in the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau compared to a threshold value may indicate one or more of FTLD (e.g., FTLD-MAPT) and CBD. FTLD-MAPT can be P301L, R406W, or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0009] A method for detecting primary tauopathy-related deposits in the brain of a subject is provided, which may include preparing an amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample from the subject. One or more of MTBR-tau275 and MTBR-tau282 can be detected according to the method for detecting tau or 4R tau aggregates described hereinabove. Primary tauopathy-related deposits can be from FTLD (e.g., FTLD-MAPT) or CBD. A decrease in the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau compared to a threshold value can indicate FTLD (e.g., FTLD-MAPT) or CBD). FTLD-MAPT can be P301L, R406W, or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0010] Provided is a method for diagnosing FTLD (e.g., FTLD-MAPT) in a subject, the method may include: (a) preparing the amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample from the subject; and (b) diagnosing FTLD-MAPT if the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau detected is decreased compared to a threshold value. FTLD-MAPT can be P301L, R406W, or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0011] A method for measuring the progression of FTLD (e.g., FTLD-MAPT) in a subject is provided, which may include: (a) obtaining the amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample from the subject; and (b) calculating 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 FTLD (e.g., FTLD-MAPT) 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 hereinabove. FTLD-MAPT can be P301L, R406W, or S305I. FTLD-MAPT can be symptomatic or asymptomatic.

[0012] A method for diagnosing CBD in a subject is provided, 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 from the subject; and (b) diagnosing CBD if the detected amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau is decreased compared 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 hereinabove.

[0013] A method for measuring the progression of CBD in a subject is provided, which may include: (a) obtaining an amount of one or more of MTBR-tau275, MTBR-tau275 / t-tau, MTBR-tau282, and MTBR-tau282 / t-tau from a CSF sample from the subject; and (b) calculating a difference between 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 methods for detecting tau or 4R tau aggregates described hereinabove.

[0014] A method of treating a primary tauopathy in a subject in need thereof 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 from the subject; 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 decreased compared 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 hereinabove.

[0015] Provided is a method for treating a primary tauopathy in a subject in need thereof, the method comprising administering to the subject a treatment that alters tau pathology, wherein the subject has been identified as having a decreased amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau compared to a threshold, as measured according to the methods described hereinabove. The treatment may alter or stabilize the amount of one or more of the detected MTBR-tau275 peptide and MTBR-tau282 peptide. Treatments included lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, bepranemab, dasatinib + quercetin, E2814, epothilone D, goslanemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, semolinemab, and TPI. The treatment may be selected from the group consisting of 287, tideglusib, tirabonemab, zagotenemab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors, and tau activation vaccines. The treatment may be selected from the group consisting of 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. The kinase inhibitor may be an inhibitor of thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn. The phosphatase activator may increase the activity of protein phosphatase 2A. The vaccine may be CAD106 or AF20513. The anti-Aβ antibody can be aducanumab or another anti-amyloid antibody that clears plaques. [Brief explanation of the drawings]

[0016] [Figure 1]Figure 1 shows participant demographics and brain MTBR-tau measurements in the primary tauopathy cohort. Abbreviations include: NC: normal controls, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain dementia, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer's disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16), HS: hippocampal sclerosis, MND: motor neuron disease, LBD: dementia with Lewy bodies, SFG: superior frontal gyrus.

[0017] [Figure 2] Figure 2 shows participant demographics and CSF MTBR-tau measurements in the pathologically confirmed primary tauopathy cohort. Abbreviations include: NC: normal controls, FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein, PiD: Pick's disease, AGD: argyrophilic grain dementia, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer's disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations (P301L, S305I, R406W, IVS10+16), HS: hippocampal sclerosis, MND: motor neuron disease, LBD: dementia with Lewy bodies, CDR plus NACC FTLD-SB: Dementia Rating plus National Alzheimer's Coordinating Center FTLD box sum, ALS: amyotrophic lateral sclerosis, MCI: mild cognitive impairment, bvFTD: behavioral frontotemporal dementia, CBS: corticobasal syndrome, DLB: dementia with Lewy bodies, nfvPPA: non-fluent primary progressive aphasia, PPS: pyramidal pallidal syndrome, svPPA: semantic primary progressive aphasia, PAGF: pure akinesia with gait freezing, PSP-RS: progressive supranuclear palsy with Richardson syndrome, TES: traumatic encephalopathy syndrome, EOAD: early-onset Alzheimer's disease, lvPPA: logopenic primary progressive aphasia, PCA: posterior cortical atrophy, CN: cognitively normal. Values ​​in brackets indicate the number of available information within a group when limited.

[0018] [Figure 3] Figure 3 shows the diagnostic accuracy of 4R-specific CSF MTBR-tau for distinguishing 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 dementia, PiD: Pick's disease, AUC: area under the curve, CI: confidence interval.

[0019] [Figure 4]Figures 4A-4C show that 4R-specific insoluble brain MTBR-tau is abundant in CBD, FTLD-MAPT, AD, and PSP. Figure 4A shows a schematic representation of quantified peptides of total tau (t-tau, 181-190) and the 4R isoform-specific microtubule-binding domain of tau (MTBR-tau) in the R2 region (gray 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) were measured in insoluble brain fractions of tauopathy patients from the SFG (circles, n = 54) and insula (triangles, n = 8). Both MTBR-tau species were most abundant in the CBD (n = 12) and FTLD-MAPT (n = 8). PSP (n = 16) and AD (n = 7) had moderate enrichment. AGD (n = 1), PiD (n = 3), and FTLD-TDP (n = 12) did not change in MTBR-tau275 or MTBR-tau282 compared to NC (n = 3). Red (n = 9) and blue (n = 1) filled circles indicate co-pathology of AD and PSP, respectively. Abbreviations include: NC: normal control; FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein; PiD: Pick's disease; AGD: argyrophilic grain dementia; 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. Statistical significance: ****p<0.0001, **p<0.01, *p<0.05. Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. Differences in biomarker values ​​were assessed by one-way ANOVA. A 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%.

[0020] [Figure 5]Figures 5A-5C show the reduction of 4R-specific CSF MTBR-tau in CBD, FTLD-MAPT, and AD. Figure 5A shows a schematic representation of quantified peptides of total tau (t-tau, 212-221), truncated tau, and 4R isoform-specific microtubule-binding domains of tau in the R2 region (gray 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 reduced in CBD (n=18), AD (n=10), and FTLD-MAPT (n=5) compared to NC (n=29), FTLD-TDP (n=21), and other FTLD-tau peptides. FTLD-MAPT P301L (red, n = 2), R406W (blue, n = 2), and S305I (green, n = 1) showed decreased 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 dementia; PSP: progressive supranuclear palsy; CBD: corticobasal degeneration; AD: Alzheimer's disease; FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation. Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. Differences in biomarker values ​​were assessed by one-way ANOVA. A 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] [Figure 6]Figures 6A-6F show that CSF soluble MTBR-tau correlates with brain insoluble MTBR-tau aggregates. Corresponding CSF and brain MTBR-tau275 / t-tau (Figure 6A) and MTBR-tau282 / t-tau (Figure 6B) were inversely correlated in tauopathy, FTLD-TDP, and controls (N=54, Spearman r=-0.27, p=0.049 and -0.45, p=0.0006, respectively). Corresponding CSF and brain MTBR-tau275 / t-tau (Figure 6C) and MTBR-tau282 / t-tau (Figure 6D) correlated more strongly in 4R tauopathies (CBD, PSP, and AGD, N = 29) (Spearman r = -0.61, p = 0.0004, r = -0.75, p < 0.0001, respectively). Corresponding CSF and brain MTBR-tau275 / t-tau (Figure 6E) and MTBR-tau282 / t-tau (Figure 6F) correlated more strongly in CBD (N = 12, Spearman r = -0.25, p = 0.43, r = -0.31, p = 0.33, respectively). Gray shading represents 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 dementia, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer's disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation.

[0022] [Figure 7]Figure 7 shows that the 4R CSF MTBR-tau assay is reproducible and stable across repeated lumbar punctures. CSF MTBR-tau / t-tau was measured in 25 participants (#01–#25) who underwent three to five repeated lumbar punctures within four months. FTLD-MAPT P301L (red, n=2, #02, #03), symptomatic FTLD-MAPT R406W (blue, n=1, #05), and pathologically confirmed CBD (green, n=2, #10 and #11) had decreased CSF MTBR-tau / t-tau. Data are expressed as mean ± SD. CBS: corticobasal syndrome; PSP-RS: progressive supranuclear palsy-Richardson syndrome; FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation.

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

[0024] [Figure 9] Figure 9 shows participant demographics and CSF MTBR-tau measurements in the repeat lumbar puncture cohort.

[0025] [Figure 10] FIG. 10 shows CSF MTBR-tau275 / t-tau biomarker positivity based on clinical syndrome in the pathologically confirmed cohort.

[0026] [Figure 11] FIG. 11 shows CSF MTBR-tau282 / t-tau biomarker positivity based on clinical syndrome in the pathologically confirmed cohort.

[0027] [Figure 12] Figures 12A-12C show that CSF MTBR-tau and t-tau concentrations do not reflect the pathology of CBD and FTLD-MAPT. CSF MTBR-tau275 (Figure 12A) and MTBR-tau282 (Figure 12B) concentrations are not altered by tauopathy (n = 112 total, including 29 NCs). FTLD-MAPT includes P301L (red, n = 2), R406W (blue, n = 2), and S305I (green, n = 1). CSF t-tau (Figure 12C) was increased in autopsy-confirmed AD (n = 10) compared with NC (n = 29) and PSP (n = 22, p < 0.05). Statistical significance: *P < 0.05. Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum. Differences in biomarker values ​​were assessed by one-way ANOVA. A 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%. Abbreviations include: NC: normal control; FTLD-TDP: frontotemporal lobar degeneration with TAR DNA-binding protein; PiD: Pick's disease; AGD: argyrophilic grain dementia; PSP: progressive supranuclear palsy; CBD: corticobasal degeneration; AD: Alzheimer's disease; FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation.

[0028] [Figure 13]Figures 13A-13B show that CSF MTBR-tau / tau correlates with disease duration in CBD. CSF MTBR-tau275 / t-tau (Figure 13A) and CSF MTBR-tau282 / t-tau (Figure 13B) negatively correlate with disease duration in CBD (i.e., age at onset and interval between CSF draws, n=18, Spearman r=-0.37, p=0.13 and r=-0.39, p=0.11, respectively). Gray shading represents the 95% confidence interval of the linear regression. CBD: Corticobasal degeneration.

[0029] [Figure 14]Figures 14A-14C show that CSF pT217 / T217 distinguishes AD from other tauopathies. Figure 14A shows that CSF pT217 / T217 was 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 (filled purple, n=9) with AD co-pathology had higher CSF pT217 / T217. FTLD-MAPT contains 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) could distinguish AD (n = 10) from FTLD-MAPT (n = 5) with AUCs of 0.794 and 0.987, respectively. Differences in biomarker values ​​were assessed by one-way ANOVA. A two-tailed 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%. Statistical test significance: ****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 dementia, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer's disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation, AUC: area under the curve.

[0030] [Figure 15]Figures 15A-15E show that CSF MTBR-tau275 / t-tau and MTBTR-tau282 / t-tau do not correlate with amyloid pathology in primary tauopathies. CSF pT217 / T217 (Figure 15A) positively correlates with AD Thal phase (Spearman r=0.52, p<0.0001). CSF MTBR-tau275 / t-tau (Figures 15B and 15D) and CSF MTBR-tau282 / t-tau (Figures 15C and 15E) do not correlate with AD Thal phase measured in autopsy brains of the total tauopathy cohort (Figures 15B and 15C, n=79) or CBD-only (Figures 15D and 15E, n=17).

[0031] [Figure 16] Figures 16A-16L show ROC curves for 4R-specific CSF MTBR-tau for distinguishing CBD from controls and other tauopathies. CSF MTBR-tau275 / t-tau (Figures 16A, 16C, 16E, 16G, 16I, and 16K) and MTBR-tau282 / t-tau (Figures 16B, 16D, 16F, 16H, 16J, and 16L) can distinguish CBD from NC (Figures 16A and 16B), FTLD-TDP (Figures 16C and 16D), FTLD-tau (i.e., PSP, PiD, and AGD; Figures 16E and 16F), PiD (Figures 16G and 16H), and PSP (Figures 16I and 16J). The AUC improves when AD co-pathology cases are excluded from CBD and PSP (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 dementia, PSP: progressive supranuclear palsy, CBD: corticobasal degeneration, AD: Alzheimer's disease, FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutations, AUC: area under the curve, ROC: receiver operating characteristic.

[0032] [Figure 17]Figures 17A-17E show retrospective clinical syndrome and CSF MTBR-tau markers in a pathologically confirmed cohort. CSF MTBR-tau275 / t-tau (Figures 17A and 17C) and MTBR-tau282 / t-tau (Figures 16B and 16D) are shown by pathological diagnosis (Figures 16A and 16B) and clinical syndrome (Figures 16C and 16D) (total n = 112). Box plots show the minimum, 25th percentile, median, 75th percentile, and maximum values. Dotted lines indicate cutoffs of 0.00563 and 0.01220 for CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau, respectively. Figure 16E is a schematic diagram showing the relationship between clinical syndrome and pathological diagnosis 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 dementia; PSP: progressive supranuclear palsy; CBD: corticobasal degeneration; FTLD-MAPT: frontotemporal lobar degeneration with MAPT mutation (P301L, S305I); bvFTD: behavioral frontotemporal dementia; CBS: corticobasal syndrome; nfvPPA: non-fluent primary progressive aphasia; PAGF: pure akinesia with freezing of gait; PSP-RS: progressive supranuclear palsy with Richardson syndrome; CN: cognitively normal. DETAILED DESCRIPTION OF THE INVENTION

[0033] Components of aspects of the methods disclosed herein are described below. Where these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that although specific reference to each of the various individual and collective combinations and sequences of these components may not be expressly disclosed, each is specifically contemplated and described herein.

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

[0035] 1. Definition In order that the present invention may be more readily understood, certain terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of embodiments of the present invention without undue experimentation, and the preferred materials and methods are described herein. In describing and claiming embodiments of the present invention, the following terms will be used in accordance with the definitions set forth below.

[0036] The terms "a" or "an" entity refer to one or more of that entity; for example, "a polypeptide subunit" is understood to refer to one or more polypeptide subunits. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0037] Furthermore, "and / or" as used herein should be interpreted as a specific disclosure of each of the specified features or components with or without the others. 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 quantity that may occur with typical measurement techniques and equipment, for example, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Also, given real-world solid and liquid handling procedures, certain inadvertent errors and variations are likely due to differences in the manufacture, source, or purity of ingredients used to make a composition or carry out a method. The term "about" also encompasses these variations, which may be up to ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether modified by the term "about," the claims include equivalents of the quantities.

[0039] "Specifically bind" means that a binding molecule, such as an antibody or its antigen-binding fragment, binds to an epitope via its antigen-binding domain, and that the binding involves 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 if it binds to the epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. Thus, as used herein with respect to an epitope-binding agent, the term "specifically bind" means that the epitope-binding agent does not cross-react to a significant extent with other epitopes on the protein of interest (e.g., tau) or other proteins in general.

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

[0041] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain forms of antibodies and higher or tertiary variants thereof; single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0042] Single-chain forms of antibodies and their higher-order forms may include, but are not limited to, single-domain antibodies, single-chain variant fragments (scFvs), bivalent scFvs (di-scFvs), trivalent scFvs (tri-scFvs), tetravalent scFvs (tetra-scFvs), diabodies, triabodies, and tetrabodies. ScFvs consist of heavy and light chain variable regions connected by a linker. In most, but not all, cases, the linker can be a peptide. The linker peptide is preferably about 5 to 30 amino acids in length, or about 10 to 25 amino acids in length. Typically, the linker stabilizes the variable domains without interfering with proper folding and the generation of an active binding site. In a preferred embodiment, the linker peptide is rich in glycine and serine or threonine. scFvs can be used to facilitate phage display, or can be used for flow cytometry, immunohistochemistry, or as targeting domains. Methods for making and using scFvs are known in the art. The scFv can also be conjugated to a human constant domain (e.g., the heavy chain constant domain is derived from an IgG domain such as IgG1, IgG2, IgG3, or IgG4, or is a heavy chain constant domain derived from IgA, IgM, or IgE). Diabodies, triabodies, and tetrabodies, as well as higher order variants, are typically created by varying the length of the linker peptide from 0 to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be generated using self-assembling units linked to variable domains.

[0043] A "single domain antibody" refers to an antibody fragment consisting of a single monomeric variable antibody domain.

[0044] Multispecific antibodies include bispecific antibodies, trispecific antibodies, or antibodies with four or more specificities. Multispecific antibodies can be made by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. The chains can be covalently linked.

[0045] The antibody of the present disclosure can be a dual affinity retargeting antibody (DART).DART forms are based on a diabody form, which separates the heavy and light chain cognate variable domains of two antigen binding specificities on two separate polypeptide chains.In the diabody form, the two polypeptide chains are non-covalently associated, but the DART form provides additional stabilization through a C-terminal disulfide bridge.DARTs can be produced in high quantity and quality, and show exceptional stability in both formulation buffer and human serum.

[0046] A "monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. A "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies may be produced using hybridoma technology well known in the art, as well as recombinant, phage display, synthetic, or combinations of such technologies with other techniques readily known in the art. Furthermore, monoclonal antibodies may be labeled with a detectable label, 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] "Heavy chain antibody" refers to an antibody consisting of two heavy chains. It can be an IgG-like antibody from camel, llama, alpaca, shark, etc., or an IgNAR from cartilaginous fish.

[0048] A "humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of eliciting an immune response in humans after administration, while retaining the same binding specificity and affinity as the starting non-human antibody. A humanized antibody binds to the same or similar epitope as the non-human antibody. The term "humanized antibody" includes antibodies that are partially or completely composed of amino acid sequences derived from human antibody germline sequences by altering the sequence of an antibody with non-human hypervariable regions ("HVRs"). The simplest such alteration may consist of simply replacing the mouse constant regions with those of a human antibody, thus resulting in a human / mouse chimera that may have sufficiently low immunogenicity to be acceptable for pharmaceutical use. Preferably, the variable regions of the antibody are also humanized using techniques now well known in the art. For example, the framework regions of the variable regions may be replaced with corresponding human framework regions while retaining one, some, or all six non-human HVRs. Some framework residues may be substituted with corresponding residues from a non-human VL or VH domain (e.g., the non-human antibody from which the HVR residues are derived) to, for example, restore or improve the specificity or affinity of the humanized antibody. A substantially human framework region has at least about 75% homology (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) with a known human framework sequence. HVRs may also be randomly mutated such that binding activity and affinity for the antigen are maintained or enhanced in the context of fully human germline or substantially human framework regions. As noted above, antibody fragments are sufficient for use in the methods of the present invention. Furthermore, the term "humanized antibody," as used herein, refers to an antibody comprising substantially human framework regions, at least one HVR from a non-human antibody, and in which any constant regions present are substantially human. A substantially human constant region has at least about 90% (ie, about 90%, about 95% or about 99% sequence identity) with a known human constant sequence.Hence, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to corresponding pairs of one or more germline human immunoglobulin sequences.

[0049] The term "Aβ" refers to a peptide derived from the carboxy-terminal region of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. Many forms of Aβ exist that can have toxic effects: Aβ peptides are typically 37–43 amino acids in length but can have truncations and modifications that alter their overall size. They can be found intracellularly or extracellularly in monomeric, multimeric, and aggregated forms, in soluble and insoluble compartments, and complexed with other proteins or molecules. The harmful or toxic effects of Aβ can result from any or all of the above forms, as well as others not specifically described. For example, two such Aβ isoforms include Aβ40 and Aβ42; the Aβ42 isoform is particularly fibrillogenic or insoluble and is associated with disease states. The term "Aβ" typically refers to multiple Aβ species without distinguishing between individual Aβ species. Specific Aβ species are identified by the size of the peptide (eg, 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, pets, etc. A subject may be awaiting medical care or treatment, undergoing medical care or treatment, or receiving medical care or treatment.

[0052] As used herein, the terms "control population," "normal population," or samples from "healthy" subjects refer to a subject or group of subjects who have been 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 from alternative splicing of the mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein, genetic mutation, and somatic recombination. The terms "isoform" and "variant" are used interchangeably.

[0054] The term "tau" refers to multiple isoforms encoded by the gene MAPT (or its homologue), and its species that are C-terminally truncated in vivo, N-terminally truncated in vivo, post-translationally modified in vivo, or any combination thereof.As used herein, the terms "tau", "tau protein" and "tau species" can be used interchangeably.In many animals, including but not limited to humans, non-human primates, rodents, fish, cows, frogs, goats and chickens, tau is encoded by the gene MAPT.In animals where the gene is not identified as MAPT, homologues can be identified by methods well known in the art.

[0055] In humans, six isoforms of tau exist, generated by alternative splicing of exons 2, 3, and 10 of MAPT. These isoforms range in length from 352 to 441 amino acids. Exons 2 and 3 each encode a 29-amino acid insert at the N-terminus (referred to as N), and full-length human tau isoforms may have both inserts (2N), one insert (1N), or no inserts (0N). All full-length human tau isoforms also have three repeats of the microtubule-binding domain (referred to as R). Inclusion of exon 10 at the C-terminus includes the fourth microtubule-binding domain encoded by exon 10. Thus, full-length human tau isoforms may consist of four repeats of the microtubule-binding domain (including exon 10: R1, R2, R3, and R4) or three repeats of the microtubule-binding domain (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 glycated.Human tau may also be proteolytically processed or not at C-terminus, N-terminus, or C-terminus and N-terminus in vivo.Therefore, the term "human tau" includes 2N3R, 2N4R, 1N3R, 1N4R, 0N3R and 0N4R isoforms, and those species that are C-terminus truncated in vivo, N-terminus truncated in vivo, post-translationally modified in vivo, or any combination thereof.Alternative splicing of the gene encoding tau also 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 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPP The amino acid sequence of Tau-441 is shown as TREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 1). N-terminal tau, intermediate domain tau, MTBR-tau and C-terminal tau are described below for this isoform. These regions vary in predictable ways for other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R and 0N3R). Thus, when an amino acid position is identified for Tau-441, those skilled in the art can determine the corresponding amino acid position for other isoforms.

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

[0058] As used herein, the term "mid-domain tau" refers to a tau peptide or multiple tau proteins that include two or more or all of the amino acids of the mid-domain of tau (e.g., 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 that contain 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 that include two or more or all of the C-terminal amino acids of tau (such as, for example, amino acids 369-441 of tau-441).

[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 parkinsonism linked to chromosome 17, Richie-Bodig disease, Parkinson's disease dementia complex of Guam, tangle-predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis complex, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration (CBD), argyrophilic grain dementia (AGD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and frontotemporal dementia (FTD).

[0062] Tauopathies are classified according to the predominance of tau isoforms found in pathological tau deposits. Tauopathies with tau deposits primarily composed of tau with three MTBCs are referred to as "3R-tauopathies." Pick's disease is a non-limiting example of a 3R-tauopathies. For clarity, the pathological tau deposits of some 3R-tauopathies may be a mixture of 3R and 4R tau isoforms, with the 3R isoform predominating. Intracellular neurofibrillary tangles (i.e., tau deposits) in the brains of subjects with Alzheimer's disease are generally thought to contain approximately equal amounts of both 3R and 4R isoforms. Tauopathies with tau deposits primarily composed of tau with four MTBCs are referred to as "4R-tauopathies." PSP, CBD, and AGD, as well as some forms of FTLD, are non-limiting examples of 4R-tauopathies. In particular, pathological tau deposits in the brains of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, display a mixture of 3R and 4R isoforms.

[0063] A clinical manifestation of tauopathy may be tau aggregates 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., [ 18 F]THK5317,[ 18 F]THK5351,[ 18 F]AV1451,[ 11 C]PBB3, 18 F]MK-6240,[ 18 F]RO-948,[ 18 F]PI-2620,[ 18 F]GTP1, 18 F]PM-PBB3, and [ 18 F]JNJ64349311,[ 18 tau PET using tau-specific ligands such as [F]JNJ-067).

[0064] As used herein, the terms "treat," "treating," or "treatment" refer to the provision of medical care by a trained and licensed professional to a subject in need thereof. Medical care can be a diagnostic test, therapeutic treatment, and / or a prophylactic or preventative measure. The purpose of therapeutic and prophylactic treatment is to prevent or slow (alleviate) undesirable physiological changes or diseases / disorders. Beneficial or desired clinical results of therapeutic or prophylactic treatment include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliation of disease state, and remission (partial or total). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder, as well as those prone to have the disease, condition, or disorder, or those in whom the disease, condition, or disorder is to be prevented. Thus, a subject in need of treatment may or may not have any symptoms or clinical signs of disease.

[0065] The term "tau therapy" collectively refers to any imaging agent, therapeutic treatment, and / or preventive or preventative measures that are intended for or used with subjects at risk of developing tauopathy or clinically diagnosed with tauopathy. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.).Non-limiting examples of therapeutic agents include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-Ab antibodies (including antigen-binding fragments, variants, or derivatives thereof), anti-tau antibodies (including antigen-binding fragments, variants, or derivatives thereof), stem cells, and dietary supplements. (e.g., lithium water, omega-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract, etc.), serotonin receptor 6 antagonists, 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, methylthionium chloride, etc.), glycemic control improvement therapies (e.g., insulin, exenatide, Liraglutide, pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma 1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, b-2 adrenoceptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenoceptor antagonists, 5-HT1A and 1D receptor agonists 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, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof.

[0066] 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 corticobasal degeneration (CBD). Biomarkers in cerebrospinal fluid (CSF) and blood provide accurate characteristics for identifying AD. However, no biomarkers exist for positively identifying non-AD tauopathies. Because some FTLD-tau, such as CBD brains, accumulate tau species from the 4-repeat (4R) tau-specific domain in the microtubule-binding region (MTBR), this disclosure investigated whether CSF levels of these MTBR-tau species change in FTLD-tau CSF, reflecting the accumulation of tau aggregates in the brain. A method for quantifying various tau species in CSF was designed and applied in two discovery studies to analyze CSF from individuals with AD and frontotemporal dementia (FTD) syndromes, including genetically confirmed and pathologically unconfirmed cases. The 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 compared with those from asymptomatic AD and healthy controls. Notably, pathogenic FTLD-tau variants (i.e., P301L and R406W) also significantly reduced specific CSF MTBR-tau levels, suggesting that this alteration 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 distinguish CBD and FTD with several specific MAPT mutations (P301L and R406W) from other FTLD-tau disorders such as progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), Pick's disease (PiD), and tau-unrelated FTLD (e.g., FTLD-TDP43), suggesting that specific MTBR-tau species in CSF may serve as the first positive biomarker for recapitulating tau pathology in non-AD tauopathies.

[0067] The present disclosure provides methods for measuring MTBR-tau peptides in biological samples (eg, by mass spectrometry or immunoassay).

[0068] 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 additional proteins in the biological sample in each of the methods described herein. In some embodiments, the one or more proteins may be proteins depleted from the biological sample before tau purification. For example, in certain embodiments, N-terminal tau and / or mid-domain tau species can be identified and / or quantified separately from the tau species (e.g., MTBR-tau, C-terminal tau) quantified by the methods disclosed herein. In some embodiments, in addition to endogenous truncated tau fragments, post-translational modifications such as tau phosphorylation at specific residues can be measured and quantified. Alternatively or additionally, Aβ (e.g., Aβ42 / Aβ40), ApoE, or any other protein of interest can be identified and / or quantified by processing a portion of the biological sample in parallel, by depleting the protein of interest from the biological sample before use in the methods disclosed herein, or by depleting the protein of interest from the biological sample during the sample processing steps disclosed herein.

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

[0070] 2. Cleavage method of tau fragments The present disclosure provides a method for detecting tau (e.g., tau peptide fragments) in a cerebrospinal fluid (CSF) sample. Detection can include confirming the presence of tau, such as measuring or quantifying the amount of tau peptide fragments. The presence, measured, or quantified amount of tau peptide fragments can indicate aggregated insoluble tau or tangles (e.g., neurofibrillary tangles) associated with tauopathy. Detection and measurement can be performed by mass spectrometry or immunoassay. The biological sample can be obtained from a subject having or suspected of having a tauopathy.

[0071] The tau peptide fragments (e.g., tau fragments) described herein can be endogenously cleaved (e.g., cleaved in a cell or tissue of a subject). In some embodiments, the tau peptide fragment comprises amino acids of the 4R tau splicing isoform of tau. In some embodiments, the tau peptide fragment comprises an R2 region fragment of tau. In some embodiments, the tau peptide fragment comprises a portion of amino acids of the R2 region of tau. In some embodiments, the tau peptide fragment comprises the MTBR-tau275 peptide described herein. In some embodiments, the tau peptide fragment (e.g., the MTBR-tau275 peptide) comprises or consists of amino acids 275-280 of tau-441. In some embodiments, the tau peptide fragment (e.g., the MTBR-tau275 peptide) comprises or consists of amino acids 275-280 set forth in SEQ ID NO: 1. In some embodiments, the tau peptide fragment comprises or consists of the amino acid sequence VQIINK (SEQ ID NO: 2). In some embodiments, the peptide fragment of tau comprises the MTBR-tau282 peptide described herein. In some embodiments, the peptide fragment of tau (e.g., the MTBR-tau282 peptide) comprises or consists of amino acids 282-290 of tau441. In some embodiments, the peptide fragment of tau (e.g., the MTBR-tau282 peptide) comprises or consists of amino acids 282-290 set forth in SEQ ID NO: 1. In some embodiments, the peptide fragment of tau comprises or consists of amino acids LDLSNVQSK (SEQ ID NO: 3).

[0072] Detecting tau (e.g., tau peptide fragments) in a CSF sample can include preparing a CSF sample and purifying tau peptides from the sample. In some embodiments, the CSF sample contains endogenously cleaved tau. The endogenously cleaved tau fragments may not be tryptic tau peptides. Purification can be performed without first exposing the sample to further in vitro proteolytic cleavage. A prior removal step (e.g., protein precipitation and / or immunodepletion) before or after purifying the endogenously cleaved tau fragments may not be performed. The endogenously cleaved tau fragments can be further cleaved in vitro using a protease before or after purification. In some embodiments, the protease is trypsin.

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

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

[0075] 2.1. Detection of tau in CSF samples 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 described herein include detecting and measuring the amount of proteolytic MTBR-tau275 peptide, proteolytic MTBR-tau282 peptide, or both.

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

[0077] In one example, the method of the present disclosure further includes purifying one or more MTBR-tau peptides from the CSF sample, and optionally, one or more of the MTBR-tau peptides are further cleaved in vitro using one or more proteases before or after purification. In some embodiments, one or more of the MTBR-tau peptides are not cleaved in vitro (e.g., are not endogenously cleaved) before purification.

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

[0079] In another example, the method of the present disclosure further includes enriching one or more MTBR-tau peptides, including purifying one or more MTBR-tau peptides from the affinity-depleted sample. The enriched one or more MTBR-tau peptides may be enriched for MTBR-tau275 peptide and / or MTBR-tau282 peptide. The MTBR-tau275 peptide and / or MTBR-tau282 peptide may be detected and / or measured using an immunoassay, which may include the use of anti-tau antibody 77G7 or an antigen-binding fragment thereof. The sample containing the enriched MTBR-tau275 peptide and / or MTBR-tau282 peptide may be proteolytically cleaved using a protease. The resulting cleavage product may be desalted (e.g., by solid-phase extraction) to obtain a sample containing one or more 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) prior to purification to obtain a sample containing proteolytic tau peptides (e.g., endogenous cleaved tau). 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 embodiment, a method for detecting 4R tau aggregates in a subject is provided. The method may include one or more of the following steps: (a) purifying endogenous cleaved tau fragments from a CSF sample from the subject without contacting the endogenous cleaved tau fragments with a protease in vitro; (b) contacting the purified endogenous cleaved tau fragments with an endopeptidase to obtain one or more of a proteolytic MTBR-tau275 peptide comprising amino acids 275 to 280 relative to SEQ ID NO: 1 and a proteolytic MTBR-tau282 peptide comprising amino acids 282 to 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 containing the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptide prior to step (c). In some embodiments, the desalting is accomplished 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 in purification step (a) is for immunoprecipitation of N-terminal to mid-domain tau (e.g., for depleting N-terminal to mid-domain tau from a CSF sample). In some embodiments, the immunoprecipitation of N-terminal to mid-domain tau comprises contacting a CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau. In some embodiments, the immunoprecipitation in purification step (a) is for immunoprecipitation of N-terminal tau (e.g., for depleting N-terminal tau from a CSF sample). In some embodiments, the immunoprecipitation of N-terminal tau comprises contacting a CSF sample with one or more anti-tau epitope binding agents that specifically bind to N-terminal tau. In some embodiments, the immunoprecipitation in purification step (a) is for immunoprecipitation of mid-domain tau (e.g., for depleting mid-domain tau from a CSF sample). In some embodiments, immunoprecipitation of mid-domain tau comprises contacting a CSF sample with one or more anti-tau epitope binding agents that specifically bind to mid-domain tau. In some embodiments, the immunoprecipitation in purification step (a) is for immunoprecipitation of one or more MTBR-tau species from a CSF sample (e.g., capturing one or more MTBR-tau species from a biological sample). In some embodiments, immunoprecipitation of one or more MTBR-tau species comprises contacting a CSF sample with one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species.

[0083] In another embodiment, there is provided a method for detecting 4R tau aggregates in a subject, comprising one or more of the following steps: (a) performing affinity depletion in cerebrospinal fluid (a CSF sample from the subject) by contacting the biological sample with one or more affinity depletion agents comprising one or more epitope binding agents that each bind to one of N-terminal tau, central domain tau, or C-terminal tau but do not bind to an antigen within MTBR-tau, to obtain a depleted sample and an enriched sample, wherein the CSF sample is enriched for endogenous cleaved tau fragments; (b) immunoprecipitation of the enriched sample, wherein the enriched sample contains endogenous cleaved tau fragments, the depleted sample contains N-terminal tau, central domain tau, and / or C-terminal tau, and the enriched sample is enriched for endogenous cleaved tau fragments, the endogenous MTBR-tau 275 peptide containing amino acids 275-280 relative to SEQ ID NO: 1, and the endogenous MTBR-tau 282 peptide containing amino acids 282-290 relative to SEQ ID NO: 1; (b) immunoprecipitation of the enriched sample, wherein the enriched sample is enriched for endogenous MTBR-tau 275 peptide containing amino acids 275-280 relative to SEQ ID NO: 1, and the endogenous MTBR-tau 282 peptide containing amino acids 282-290 relative to SEQ ID NO: 1; and (d) detecting and measuring one or more of the proteolytic MTBR-tau275 peptides and the proteolytic MTBR-tau282 peptides by performing liquid chromatography-mass spectrometry (LC / MS) or an 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. In some embodiments, the sample containing one or more of the proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides is desalted prior to the step. In some embodiments, the desalting is done by solid phase extraction.

[0084] In some embodiments, the one or more affinity depletors comprise one or more anti-tau epitope binding agents that specifically bind to tau from the N-terminus to the mid-domain. In some embodiments, the one or more affinity depletors comprise one or more anti-tau epitope binding agents that specifically bind to N-terminal tau. In some embodiments, the one or more affinity depletors comprise one or more anti-tau epitope binding agents that specifically bind to mid-domain tau. In some embodiments, the one or more immunoprecipitation agents comprise 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 cleaved tau fragments 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. The sample depleted of tau peptides using the above-mentioned antibody may be proteolytically cleaved in vitro using one or more proteases disclosed herein, and the resulting in vitro cleaved tau peptides (proteolytic fragments) may be contacted with an antibody that binds to one or more MTBR-tau peptides (e.g., MTBR-tau275 peptide and / or MTBR-tau282 peptide, e.g., anti-tau). In some embodiments, the protease is trypsin.

[0086] 2.2. Epitope-binding agents In some embodiments of the methods described herein, the one or more anti-tau epitope binding agents that specifically bind to tau from the N-terminal to the mid-domain bind to an epitope within amino acids 1-243 of tau (e.g., tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to tau from the N-terminal to the mid-domain bind to an epitope within amino acids 1-243 relative to SEQ ID NO:1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau bind to an epitope within the amino acid sequence of MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRL (SEQ ID NO: 4). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal to mid-domain tau are selected from anti-tau antibodies HJ8.5, HJ8.7, or tau1, or antigen-binding fragments thereof.

[0087] In some embodiments of the methods described herein, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within amino acids 1-103 of tau (e.g., tau-441). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within amino acids 1-103 relative to SEQ ID NO: 1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to N-terminal tau bind to an epitope within the amino acid sequence of MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTA (SEQ ID NO: 5).

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

[0089] In some embodiments of the methods described herein, the 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, the 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, the one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species bind to an epitope of the amino acid sequence QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGN (SEQ ID NO: 7). In some embodiments, the 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, the 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 SEQ ID NO: 1. In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species specifically bind to an epitope of the amino acid sequence SKVTSKCGSLGNIHHKPGGG (SEQ ID NO: 8). In some embodiments, the one or more anti-tau epitope binding agents that specifically bind to one or more MTBR-tau species is the anti-tau antibody 77G7 or an antigen-binding fragment thereof. In some embodiments, the immunoprecipitation of one or more MTBR-tau species is immunoprecipitation of the MTBR-tau275 peptide and / or the 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., endogenously cleaved tau fragments) is contacted with one or more or all of the following: anti-tau antibody HJ8.5 or its antigen-binding fragment; anti-tau antibody HJ8.7 or its antigen-binding fragment; and / or anti-tau antibody tau1 or its antigen-binding fragment. In some embodiments, the CSF sample is contacted with the anti-tau antibodies tau1, HJ8.5, and HJ8.7, or their antigen-binding fragments. A sample depleted of tau peptides using the above antibodies may be contacted with an antibody that binds to MTBR-tau275 and MTBR-tau282, such as the anti-tau antibody 77G7 or its antigen-binding fragment.

[0091] 2.3. Biological samples Suitable biological samples include cerebrospinal fluid (CSF) samples obtained from a subject. In some embodiments, the subject is a human. The subject may be awaiting medical treatment or treatment, undergoing medical treatment or treatment, or receiving medical treatment or treatment. In various embodiments, the human subject may be a healthy subject, a subject at risk of developing a neurodegenerative disease, a subject with signs and / or symptoms of a neurodegenerative disease, or a subject diagnosed with a neurodegenerative disease. In further embodiments, the neurodegenerative disease may be a primary tauopathy or a tauopathy. In certain examples, the tauopathy may be corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), globular glial tauopathy (GGT), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), or frontotemporal lobar degeneration (FTLD-tau) (e.g., FTLD-MAPT), including Alzheimer's disease. In certain embodiments, 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.In other embodiments, the subject is an experimental animal.In further embodiments, the subject is an experimental animal that is genetically engineered to express human tau and optionally one or more additional human proteins (for example, human Aβ, human ApoE, etc.).

[0092] CSF can be obtained by lumbar puncture, with or without an indwelling CSF catheter. Multiple CSF samples collected simultaneously from a subject can be pooled. Once collected, the CSF sample can be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cellular debris; use of additives designed to stabilize and preserve specimens prior to analytical testing, etc.). CSF samples can be used immediately or frozen and stored indefinitely. Prior to use in the methods disclosed herein, CSF samples can also be modified, if necessary or desired, to include protease inhibitors, isotope-labeled internal standards, detergents and chaotropic agents, and / or to deplete other analytes (e.g., proteins, peptides, metabolites), as desired.

[0093] The size of the sample used can vary depending on the type of sample, the health status of the subject from whom the sample was obtained, and the analytes (in addition to tau) being analyzed. The volume of the CSF sample can be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In a specific example, the sample size can be about 0.05 mL to about 1 mL of CSF.

[0094] In some embodiments, a single sample is obtained from a subject. Alternatively, samples can be obtained from a subject over time. Thus, two or more samples 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 two or more samples are collected from a subject over time, the samples can be collected every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days. In some embodiments, the samples are collected at intervals of 1 month, 3 months, 6 months, 1 year, 2 years, 5 years, 10 years, 20 years or more.

[0095] 2.4. Tau purification Another step of the methods disclosed herein includes purifying tau (e.g., peptide fragments of tau), particularly endogenously cleaved and / or proteolytically cleaved fragments of tau. In some examples, the cleavage fragments of tau are N-terminus-independent, central domain-independent, and / or C-terminus-independent. The purified tau may be partially purified or completely purified.

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

[0097] In some embodiments, the methods of the present disclosure include purifying tau peptides (e.g., endogenous cleavage 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) of tau-441 (or within a similarly defined region for 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 method of the present disclosure comprises purifying tau peptides by solid-phase extraction. Purification by solid-phase extraction involves contacting a sample containing tau with a solid phase containing an adsorbent that adsorbs tau, 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 containing N-vinylpyrrolidone and divinylbenzene, or a polymer containing styrene and divinylbenzene. In an exemplary embodiment, the adsorbent is Oasis HLB (Waters). Before contacting the supernatant containing tau, the adsorbent is typically preconditioned according to the manufacturer's instructions or as known in the art (e.g., with a water-miscible organic solvent, followed by a buffer containing the mobile phase). Additionally, because some reversed-phase materials retain ionized analytes more strongly than others, the supernatant can be acidified if desired. The use of volatile components in the mobile phase and for elution is preferred to facilitate sample drying. In exemplary embodiments, the wash step can involve the use of a liquid phase containing about 0.05% v / v trifluoroacetic acid (TFA) to about 1% v / v TFA, or its equivalent. In some instances, the wash can involve the use of a liquid phase containing 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 wash can involve the use of a liquid phase containing about 0.1% v / v to about 1.0% v / v TFA or about 0.1% v / v to about 0.5% v / v TFA. Bound tau is then eluted with a liquid phase containing 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 containing about 20% v / v to about 40% v / v ACN, or about 20% v / v to about 30% v / v ACN, or about 30% v / v to about 50% v / v ACN, or about 30% v / v to about 40% v / v ACN.The eluate may be dried by methods known in the art, such as vacuum drying (eg, speed-vac), freeze-drying, evaporation under a stream of nitrogen, and the like.

[0099] 2.5. Depletion of One or More Proteins The method of the present disclosure can include the step of depleting one or more proteins from a sample. The term "deplete" means to reduce the amount or number. Thus, a protein-depleted sample can have any amount of protein that is measurably less than the amount in the original sample, including no amount of protein.

[0100] Proteins can be depleted from samples by methods that specifically target one or more proteins, such as affinity depletion, solid-phase extraction, or other methods known in the art. Targeted depletion of one or more proteins can be used in situations where downstream analysis of the protein is desired (for example, identification, quantification, analysis of post-translational modification, etc.). For example, Aβ peptides can be identified and quantified by methods known in the art after affinity depletion of Aβ using 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 isoforms. Targeted depletion can also be used to isolate other proteins for subsequent analysis, including, but not limited to, apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, interleukins, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, NFL, prion protein, pNFH, and DJ-1. Targeted depletion of specific tau peptide fragments is also used herein to enrich for other tau peptides and / or to eliminate proteins that co-detect mass spectrometry. For example, in certain embodiments of the present disclosure, N-terminal tau, mid-domain tau, and / or C-terminal MTBR-tau are depleted from the sample before further sample processing for analysis. Downstream analysis of the depleted tau proteins may or may not be performed, although both options are contemplated by the methods of the present disclosure.

[0101] In some embodiments, targeted depletion can be achieved by affinity depletion. Affinity depletion refers to a method of depleting a protein of interest from a sample based on 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, or tissue culture plate. Ligand immobilization to a solid support can also occur after ligand-protein interaction has occurred. Suitable ligands include antibodies, aptamers, and other epitope-binding agents. The molecule can also be a polymer or other material that selectively absorbs the protein of interest. As a non-limiting example, polyhydroxymethylene substituted with fatty oxyethylated alcohol (e.g., P PHM-L LIPOSORB, Sigma-Aldrich) can be used to selectively absorb lipoproteins (including ApoE) from serum. Two or more affinity depletion agents can be combined to sequentially or simultaneously deplete multiple proteins.

[0102] In some embodiments, the methods of the disclosure comprise affinity depleting one or more proteins from a sample using at least one epitope binding agent that specifically binds to an epitope within amino acids 1-243 (inclusive) of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms). In various embodiments, one, two, three, or more epitope binding agents may be used. When two or more epitope binding agents are used, they may be used sequentially or simultaneously.

[0103] In some embodiments, the methods of the 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 1-103, inclusive, of tau-441) and an epitope binding agent that specifically binds to an epitope within the central domain of tau (e.g., amino acids 104-243, inclusive, of tau-441). The epitope binding agents may be used sequentially or simultaneously.

[0104] In some embodiments, the methods of the 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 1-35 (inclusive) of Tau-441 and an epitope binding agent that specifically binds to an epitope within amino acids 104-243 (inclusive) of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms). The epitope binding agents may be used sequentially or simultaneously.

[0105] In some embodiments, the methods of the 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 1-103, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); an epitope binding agent that specifically binds to an epitope within amino acids 104-243, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0106] In some embodiments, 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 1-35, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); an epitope binding agent that specifically binds to an epitope within amino acids 104-243, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0107] In some embodiments, 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 1-103 (inclusive) of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0108] 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 1-35 (inclusive) of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0109] 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 104-243 (inclusive) of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0110] In some embodiments, the methods of the 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 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 1-103 (inclusive) of Tau-441 is HJ8.5 or is 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 104-221 (inclusive) of Tau-441 is Tau1 or is an epitope binding agent that binds to the same epitope as Tau1 and / or competitively inhibits Tau1.

[0112] In some embodiments, the epitope binding agent that specifically binds to mid-domain tau is selected from HJ34.8, 77G7, RD3, RD4, UCB1017, PT76, E2815, or 7G6, or an epitope binding agent 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., those described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, and Roberts et al., Acta Neuropathol Commun, 2020, 8: 13) and other epitope-binding agents 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, proteins can be depleted from samples by more conventional methods, such as ultrafiltration or protein precipitation with acids, organic solvents, or salts. Generally speaking, these methods are used to ensure the reduction of high-abundance and high-molecular-weight proteins, thereby enriching for low-molecular-weight and / or low-abundance proteins and peptides (e.g., tau, Aβ, etc.).

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

[0115] As a non-limiting example, proteins can be depleted from samples by acid precipitation using perchloric acid. As used herein, "perchloric acid" refers to 70% perchloric acid unless otherwise specified. 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 the addition of perchloric acid, the sample is mixed well (e.g., using a vortex mixer) and typically maintained at a low temperature for about 10 minutes or longer to promote precipitation. For example, the sample may be maintained for about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes. In other examples, the sample may be maintained for about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes. In other examples, the sample may be maintained for about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes. In other examples, the sample is maintained for about 30 minutes. The sample is then centrifuged at a low temperature to pellet precipitated proteins, and the supernatant containing soluble tau (i.e., the acid-soluble fraction) is transferred to a new container. As used in the above context, "low temperature" refers to a temperature of 10°C or below. 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, e.g., about 3°C ​​to about 5°C, or even about 4°C, may be preferred. In certain embodiments, low temperature can be achieved by placing the sample on ice.

[0116] Two or more methods from one or both of the above approaches can be combined to sequentially or simultaneously deplete multiple proteins.For example, one or more proteins can be selectively depleted (targeted depletion), and then high abundance / molecular weight proteins can be depleted.Alternatively, high abundance / molecular weight proteins can be depleted first, and then one or more proteins can be targeted depleted.In yet another method, high abundance / molecular weight proteins can be depleted first, and then one or more proteins can be depleted in a first round of targeted depletion, and then one or more proteins that are different from the proteins targeted in the first round can be depleted in a second round of targeted depletion.Other iterations are readily apparent to those skilled in the art.

[0117] 3. Protease digestion of purified tau fragments Another step of the methods disclosed herein optionally includes digesting purified tau fragments (e.g., endogenously cleaved tau) with a protease (e.g., thereby generating proteolytic tau peptides). Digestion can be in vitro. Digesting purified tau with a protease includes contacting a sample containing purified tau with the 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. To detect MTBR-tau275 or MTBR-tau282, the protease includes trypsin. The resulting digestion product (e.g., cleavage product) is a composition containing 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.

[0118] 3.1. Detection and quantification of tau fragments Another step of the method disclosed herein involves detecting the amount of digested tau peptides from the processed biological sample. Any suitable method for detecting the amount of tau protein is contemplated within the scope of the present disclosure. Methods for detecting and quantifying tau peptides are described in detail below.

[0119] LC-MS The detecting and quantifying step includes performing liquid chromatography-mass spectrometry (LC-MS) on a sample containing tau peptides to detect and measure the concentration of at least one peptide of tau. Thus, in effect, the disclosed method uses one or more tau peptides to detect and measure the amount of tau protein present in a biological sample.

[0120] Proteolytic tau peptides that indicate the presence of endogenous cleaved tau fragments may include MTBR-275 or MTBR-282. When using different enzymes for digestion, the resulting proteolytic peptides may be slightly different, but this can be easily determined by those skilled in the art. Without wishing to be bound by theory, it is believed that the variation in the amount of cleaved tau peptide fragments between two biological samples of the same type reflects the difference in the cleaved tau fragments that make up those biological samples. As disclosed herein, the amount of specific proteolytic peptides of cleaved tau fragments and the ratio of specific proteolytic peptides of tau can provide clinically meaningful information for guiding treatment decisions. Therefore, methods that enable the detection and quantification of cleaved tau fragments are useful in the diagnosis and treatment of many neurodegenerative diseases.

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

[0122] Tandem mass spectrometry can be used to improve resolution, as is known in the art, or techniques can be improved to achieve the resolution of tandem mass spectrometry using a single mass analyzer. Suitable types of mass analyzers are known in the art. These include quadrupoles, time-of-flight, ion traps and orbitraps, as well as hybrid mass analyzers that combine different types of mass analyzers into one architecture (e.g., Orbitrap Fusion). TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TM In an exemplary embodiment, the LC-MS system includes, but is not limited to, an Orbitrap Fusion LC-MS system, a Q Exactive mass spectrometer, and a Q Exactive mass spectrometer, each manufactured by ThermoFisher Scientific. TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TMThe mass spectrometer can include a mass spectrometer selected from mass spectrometers, or a mass spectrometer with similar or improved ion focusing and ion transmission in quadrupole.Suitable mass analysis protocol can be developed by optimizing the number of ions collected before analysis (for example, AGC setting using Orbitrap) and / or injection time.In exemplary embodiments, the mass analysis protocol outlined in Examples is used.

[0123] 3.1.2. Immunoassays 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, tau fragments are detected and quantified using ELISA.

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

[0125] Thus, in some embodiments, the method for assessing the amount of tau peptide (e.g., those described herein) is an epitope-binding agent-based method. Generally, an epitope-binding agent-based method for assessing the amount of protein expression comprises contacting a sample containing a polypeptide with an epitope-binding agent specific for the polypeptide under conditions effective to allow the formation of a complex between the epitope-binding agent and the polypeptide. The epitope-binding agent-based method may be performed in solution, or the epitope-binding agent or the sample may be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtiter plates, test tubes, beads, resins, and other polymers.

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

[0127] Epitope binding agents can also be non-covalently bound to a substrate. For example, biotinylated epitope binding agents can be prepared, which can bind to a surface covalently coated with streptavidin to effect attachment. Alternatively, epitope binding agents can be synthesized on a surface using techniques such as photopolymerization and photolithography. Additional methods for binding epitope binding agents to solid surfaces and synthesizing biomolecules on a substrate are well known in the art, such as Affymetrix's VLSIPS technology (see, e.g., U.S. Pat. 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 a sample with an epitope binding agent under conditions effective for a time sufficient to allow complex formation generally involves adding an epitope binding agent composition to the sample and incubating the mixture for a time sufficient for the epitope binding agent to bind to any antigen present. After this time, the complexes are washed and may be detected by any method known in the art. Methods for detecting epitope binding agent-polypeptide complexes generally rely on the detection of a label or marker. As used herein, the term "label" refers to any substance that binds to an epitope binding agent or other substrate material and that is detectable 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, and the like. 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 a label or marker are well known in the art.

[0129] In some embodiments, the method based on the epitope-binding agent is an immunoassay. Immunoassays can be performed in several different formats. Generally speaking, immunoassays can be divided into two categories: competitive immunoassays and non-competitive immunoassays. In competitive immunoassays, unlabeled analytes in the sample compete with labeled analytes to bind to antibodies. Unbound analytes are washed away, and bound analytes are measured. In non-competitive immunoassays, the antibodies are labeled, not the analytes. Non-competitive immunoassays can use one antibody (e.g., a labeled capture antibody) or two or more antibodies (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 the group consisting of enzyme-linked immunoassay (ELISA), fluorescence-based assay, dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), radiometric assay, multiplex immunoassay, and cytometric bead assay (CBA). In some embodiments, the epitope-binding agent-based method is an enzyme-linked immunoassay (ELISA). In other embodiments, the epitope-binding agent-based method is a radioimmunoassay. In still other embodiments, the epitope-binding agent-based method is an immunoblot or Western blot. In another embodiment, the epitope-binding agent-based method is an array. In another embodiment, the epitope-binding agent-based method is flow cytometry. In a different embodiment, the epitope-binding agent-based method is immunohistochemistry (IHC). IHC uses antibodies to detect and quantify antigens in intact tissue samples. The tissue sample may be a fresh-frozen and / or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue block prepared for testing by IHC. Methods for preparing tissue blocks for testing by IHC and for performing IHC are well known in the art.

[0131] 4. Use of MTBR-tau peptide measurements The present disclosure also encompasses the use of measuring tau peptides (e.g., endogenous cleaved tau fragments) in CSF as a biomarker of pathological features and / or clinical symptoms of tauopathy to diagnose, stage, and select appropriate treatments for a given disease stage, or to modify a given treatment regimen (e.g., changing the dose, switching to a different drug or treatment modality, etc.). Pathological features can be aspects of tau pathology (e.g., the amount of tau deposits, the presence / absence of post-translational modifications, the amount of post-translational modifications, etc.). Instead of or in addition to tau deposits, pathological features can be tau-independent. For example, if the tauopathy is Alzheimer's disease, amyloid beta (Aβ) deposits in the brain or cerebral arteries. Clinical symptoms can be dementia measured by a clinically validated instrument (e.g., MMSE, CDR-SB, etc.), or any other clinical symptoms associated with tauopathy.

[0132] Thus, in one aspect, the present disclosure provides a method for measuring tauopathy-related pathology in a subject, the method comprising quantifying one or more tau peptides (e.g., those described herein) in a biological sample obtained from the subject, such as a CSF sample. The method may include providing an amount of one or more tau peptides described herein. The quantified amount of tau peptides may be representative of tauopathy-related pathology in the brain of the subject. The tauopathy may be 3R-tauopathy, 3R / 4R mixed-tauopathy, or 4R-tauopathy. In certain embodiments, the tauopathy is 4R-tauopathy. The tauopathy-related pathology may be tau deposition in the brain and / or cerebral arteries, tau post-translational modifications, amyloid plaques, or other pathological features known in the art. The subject may or may not have clinical symptoms of tauopathy.

[0133] In another aspect, the present disclosure provides a method for diagnosing tauopathy in a subject, the method comprising: quantifying or preparing the amount of one or more tau peptides (for example, those described herein) in a biological sample obtained from the subject, such as a CSF sample; and diagnosing tauopathy if the quantified tau peptide is greater than or equal to about 1.5σ, where σ is the target deviation defined by the normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as measured by PET imaging and / or CSF Aβ42 / Aβ40 ratio measurement.Tauopathy can be 3R-tauopathy, 3R / 4R mixed-tauopathy or 4R-tauopathy.In certain embodiments, tauopathy is 4R-tauopathy.Subject can have or not have clinical symptoms of disease.

[0134] In another aspect, the present disclosure provides a method for measuring the stability of tauopathy disease in a subject, the method comprising: quantifying or preparing the amount of one or more tau peptides (e.g., those described herein) in a first biological sample obtained from the subject, and then in a second biological sample obtained from the same subject at a later time point (e.g., weeks, months, or years later); and calculating the difference between the quantified tau peptides between the samples; wherein a statistically significant increase in the quantified tau peptides in the second sample indicates disease progression; a statistically significant decrease in the quantified tau peptides in the second sample indicates disease improvement; and no change indicates stable disease. The tauopathy can be 3R-tauopathy, 3R / 4R mixed-tauopathy, or 4R-tauopathy. In certain embodiments, the tauopathy is 4R-tauopathy. The subject may or may not have clinical symptoms of the disease, and may or may not be receiving tau therapy. In some instances, the tau treatment is administered to the subject one or more times during the period between collection of the first and second biological samples, and the measure of disease stability is indicative of the effectiveness, or lack thereof, of the tau treatment.

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

[0136] The exemplary use of the MTBR-tau peptide of tau described herein may serve to illustrate various aspects described above, but such description does not limit the scope of the present invention. Tau peptides (e.g., endogenously cleaved tau fragments, e.g., MTBR-tau) and their detection are described in detail in the Examples. Generally speaking, these tau peptides include MTBR-tau275 and / or MTBR-tau282, which are endogenously cleaved at their C-terminus. Measuring the amount of tau peptides is one way to measure the amount of this specific group of tau species in a given sample. As shown in the Examples, a decrease in the amount of 4R isoform-specific MTBR-tau275 and MTBR-tau282 endogenous cleavage fragments of tau in CSF correlates with tau deposition in the brain associated with primary tauopathy, particularly FTLD (e.g., FTLD-MAPT) and CBD. 4R-specific MTBR-tau / t-tau measurements have an inverse correlation in CSF and brain. Furthermore, subjects with a longer age at disease onset and a longer interval between CSF sampling (e.g., subjects at a later pathological stage) show a greater decrease in CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau biomarkers.In other words, the amount of CSF MTBR-tau endogenous cleavage fragments (e.g., MTBR-tau275 and MTBR-tau282) can indicate primary tauopathy pathology.Furthermore, CSF t-tau and pT217 / T217 ratios are useful as diagnostic markers for distinguishing between tauopathies (e.g., AD, which is differentiated from NC, PSP, or other FTLD-tau; or CBD, which is differentiated from NC or other FTLD-tau). Thus, the amount of MTBR-tau peptide detected can be used to measure primary tauopathy pathology, determine a subject's tau status, diagnose the stage of primary tauopathy (e.g., FTLD-MAPT or CBD) in a subject, and, inter alia, distinguish between primary tauopathies.

[0137] After diagnosing and / or staging the disease, a subject may be provided with a treatment to reduce or prevent further increase in the amount of tau peptide in the CSF and / or to reduce or prevent further increase in another clinical sign or symptom of the primary tauopathy. The selection of treatment may be further guided by knowledge of the particular disease stage, as informed by the amount of tau peptide (e.g., endogenous cleaved tau fragments), e.g., therapies designed to prevent Aβ deposition, reverse Aβ deposition, prevent tau deposition, reverse tau deposition, and improve clinical symptoms of the disease may be used in subjects with potentially overlapping but different amounts of tau peptide.

[0138] In certain embodiments, the present disclosure provides a method for detecting primary tauopathy in a subject, the method comprising: preparing CSF obtained from a subject, the CSF being purified for tau peptides (for example, endogenous cleaved tau fragments); quantifying the 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 combinations thereof, wherein the amount of tau peptides or their ratio (for example, relative to each other, or each relative to CSF ​​t-tau) indicates primary tauopathy in the brain of the subject. In some embodiments, the primary tauopathy is selected from corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), glioglobular tauopathy (GGT), chronic traumatic encephalopathy (CTE), Pick's disease (PiD) or frontotemporal lobar degeneration (FTLD-tau) (for example, FTLD-MAPT), including Alzheimer's disease.In certain embodiments, the 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.

[0139] In certain embodiments, the present disclosure provides a method for measuring FTLD-tau-related pathology in subject, comprising detecting or preparing the amount of tau or 4R tau aggregates.In some embodiments, detecting tau or 4R tau aggregates comprises: preparing CSF obtained from subject, and the CSF is purified for tau peptide; and quantifying the tau peptide in the sample.In some embodiments, the tau peptide comprises one or more peptides with the amino acid sequence of MTBR-tau275, MTBR-tau282 or combinations thereof, wherein the amount of tau peptide or their ratio (for example, relative to each other, or each relative to CSF ​​t-tau) indicates the FTLD-tau-related pathology in the brain of subject. In some embodiments, the method includes quantifying and providing the 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 if the quantified tau peptide differs from the mean of a control population by about 1.5σ or more, where σ is the target 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 CSF Aβ42 / Aβ40 ratio measurement (e.g., a cutoff value for the CSF Aβ42 / Aβ40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379-387) that maximizes sensitivity % + specificity %).

[0140] In another specific embodiment, the present disclosure provides a method for measuring FTLD-tau-associated tau deposition in the brain of a subject, comprising detecting or preparing tau or 4R tau aggregates.In some embodiments, the method comprises preparing CSF obtained from a subject, wherein the CSF is purified for tau peptides; and quantifying the 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-associated tau deposition in the brain of the subject.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.

[0141] In another specific embodiment, the present disclosure provides a method for diagnosing FTLD-tau-related disease, the method comprising: preparing CSF obtained from a subject; and quantifying tau peptide in the sample. The method may comprise preparing the amount of tau peptide disclosed herein. In some embodiments, the tau peptide comprises one or more peptides having the 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, if the quantified tau peptide differs from the mean of the control population by about 1.5σ or more, the subject is diagnosed with FTLD-tau-related disease, where σ is the target deviation defined by the normal distribution measured in the control population, and the subject does not have the clinical signs or symptoms of tauopathy and / or is amyloid-negative, as measured by PET imaging (for example, by PiB-PET SUVR as described in Ann Neurol 2016; 80:379-387) and / or CSF Aβ42 / Aβ40 ratio measurement (for example, the cutoff value of the CSF Aβ42 / Aβ40 ratio calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379-387) that maximizes sensitivity%+specificity%).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, or FTLD-MAPT IVS10+16.

[0142] In another specific embodiment, the present disclosure provides a method for measuring the progression of FTLD-tau-related disease in a subject, the method comprising: preparing a first CSF sample and a second CSF sample, each sample obtained from a single subject, each sample purified for tau peptide; and quantifying the tau peptide for each sample. In one example, the amount of tau peptide in the first and second samples is provided. In some embodiments, the tau peptide comprises one or more peptides having the amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof. In some embodiments, the method further comprises measuring p-tau or Aβ, or a combination thereof. The method further comprises calculating the difference between the quantified tau peptide in the second sample and the first sample, and a statistically significant increase in the quantified tau peptide in the second sample indicates the progression of the FTLD-tau-related disease in the subject. 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, or FTLD-MAPT IVS10+16.

[0143] In another specific embodiment, the present disclosure provides a method for measuring CBD-related tau deposition in a subject, comprising detecting or preparing the amount of tau or 4R tau aggregates.In some embodiments, the method comprises preparing CSF obtained from a subject, wherein the CSF is purified for tau peptides; and quantifying the amount of tau peptides in the sample.In one example, the amount of tau peptides is provided.In some embodiments, the tau peptides include 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 CBD-related tau deposition in the brain of the subject.

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

[0145] In another specific embodiment, the present disclosure provides a method 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, each sample obtained from a single subject, each sample purified for tau peptide; and quantifying the tau peptide for each sample. In one example, the amount of tau peptide in the first and second samples is provided. In some embodiments, the tau peptide comprises one or more peptides having the amino acid sequence of MTBR-tau275, MTBR-tau282, or a combination thereof. In some embodiments, the method further comprises measuring p-tau or Aβ, or a combination thereof. The method further comprises calculating the difference between the quantified tau peptide in the second sample and the first sample, and a statistically significant increase in the quantified tau peptide in the second sample indicates the progression of the CBD-related disease in the subject.

[0146] In each of the above methods, additional biomarkers may be detected and measured to assist in staging and / or determining a subject's pathology and / or disease progression. For example, the method may include detecting and measuring the amount of Ab. In some embodiments, Aβ42 / Aβ40 levels are determined to assist in measuring amyloid plaque-specific abnormalities. Aβ42 / Aβ40 levels that deviate from normal control populations without amyloid pathology begin to occur approximately 20 years before the onset of symptoms, demonstrating a highly sensitive measure of these changes. In addition to or instead of Ab measurement, each of the above 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 assist in measuring amyloid plaque-specific abnormalities. Aβ42 / Aβ40 changes first, followed shortly by pTau217 / 181 / 231, which are associated with amyloid plaques. Then, about 10 years later, pTau205 increases and is associated with both Ab plaques and tau tangles, as is total tau (n-terminal to central domain tau). In some embodiments, the combined value of phosphorylated pTau and Aβ42 / Aβ40 can be measured and determined. For example, determining pT217×Aβ42 / Aβ40 can help measure amyloid plaque-specific abnormalities and proves to be highly sensitive and specific. Finally, about 20 years after Aβ42 / Aβ40 abnormalities, the endogenous cleaved tau fragments disclosed herein increase and are correlated only with tau pathology (not with amyloid pathology). Thus, each marker, when measured and combined in the methods disclosed herein, provides a more accurate ability to determine a subject's stage, pathology, and / or disease progression that was not available prior to this disclosure. Aβ42 / Aβ40 is a measure of pre-amyloid plaque abnormalities; Aβ42 / Aβ40 and pTau217% abnormalities are measures of amyloid plaque abnormalities, pTau205 is a measure of amyloid plaques and neurodegeneration; and the endogenous cleaved tau fragments disclosed herein are a measure of the clinical onset of tau tangles and dementia.

[0147] Alternatively or additionally, other markers can be used, such as the measurement of total tau in any of the above embodiments, the ratio calculated from the measured phosphorylation level, or the ratio calculated from the measured phosphorylation level and total tau.Mathematical operations other than ratios can also be used.For example, the use of endogenous cleaved tau fragments, Ab values ​​and / or site-specific tau phosphorylation values ​​disclosed herein can be used in various statistical models (for example, linear regression, LME curve, LOESS curve, etc.) in conjunction with other known biomarkers (for example, MAPT status, APOEε4 status, age, sex, cognitive test score, functional test score, 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 evaluated by the area under the ROC curve, and in some embodiments, the ROC AUC value of 0.7 or more is set as the threshold (for example, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.).

[0148] Cerebral amyloid plaques in humans are routinely measured by amyloid-positron emission tomography (PET). For example, cortical Aβ plaques 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 is used to reliably identify significant cortical Aβ plaques and classify subjects as PIB positive (SUVR≧1.25) or negative (SUVR<1.25). Thus, in the above embodiment, a control population without brain amyloid plaques measured by PET imaging may 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 regions of interest other than the cortical region can also be used to classify subjects as PiB positive or negative. Other PET contrast agents can also be used.

[0149] In another embodiment, the present disclosure provides a method for treating a subject with tauopathy, the method comprises: quantifying tau in biological samples obtained from subject, such as CSF samples; and providing tau treatment to improve the measurement of tauopathy-related pathology or clinical symptoms, wherein subject has the endogenous cleaved tau peptide fragments that are quantified above or below the mean of control population by at least 1 standard deviation, preferably at least 1.3 standard deviation, more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ respectively), where σ is the target deviation defined by the normal distribution measured in the control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative by PET imaging and / or measuring the Aβ42 / Aβ40 ratio in CSF.In addition to using a threshold (for example, above or below the mean by at least 1 standard deviation), in some embodiments, the degree of change above or below the mean can be used as the criterion for treating subject. Tauopathy can be 3R-tauopathy, 3R / 4R mixed-tauopathy or 4R-tauopathy. In certain embodiments, tauopathy is 4R-tauopathy. Measurement of tauopathy-related pathology can be tau deposition measured by PET imaging, tau post-translational modification measured by mass spectrometry or other suitable methods, amyloid plaques in the brain or cerebral arteries measured by PET imaging, amyloid plaques measured by the Aβ42 / Aβ40 ratio in CSF, or other pathological features known in the art. Clinical symptoms can be dementia measured by clinically validated instruments (e.g., MMSE, CDR-SB, etc.), or other clinical symptoms known in the art for 3R- and 4R-tauopathy. In further embodiments, two or more tau species are quantified. Many tau treatments target specific pathophysiological changes.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 tau antagonism or antagonism of specific tau isoforms), or increase NFT clearance; various therapies are designed to reduce CNS inflammation or brain insulin resistance, etc. However, not all tauopathies share the same pathophysiological changes. Therefore, the effectiveness of these various tau therapies can be improved by administering them to subjects accurately identified as having tau pathology, including determining the subject's disease stage and thereby more effectively changing tau phosphorylation patterns, antagonizing tau aggregation, or increasing NFT clearance based on the subject's specific pathological condition.

[0150] Treatments included lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, bepranemab, dasatinib + quercetin, E2814, epothilone D, goslanemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, semolinemab, and TPI. 287, tideglusib, tirabonemab, zagotenemab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors, and tau-activating vaccines.

[0151] In some embodiments, treatment includes the administration of a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., selective serotonin reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, etc.), a gamma-secretase inhibitor, a beta-secretase inhibitor, an anti-Aβ antibody (including 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), an anti-T ... variants or derivatives), TREM2 agonists, stem cells, nutritional supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract), serotonin receptor 6 antagonists, p38α MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthionium chloride), and improvement of glycemic control Therapies (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 adrenoceptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenoceptor antagonists, 5-HT1A and 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, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof. In exemplary embodiments, the pharmaceutical composition may comprise a kinase inhibitor.Suitable kinase inhibitors may inhibit thousand-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, the 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 alters tau phosphorylation patterns, 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 agonist, a γ-secretase inhibitor, a β-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor.

[0152] In some embodiments, if the detected Aβ42 / Aβ40 value significantly deviates from the mean of a healthy control population, and optionally, if pTau217 / Tau217, pTau205 / Tau205, pTau181, pTau231, and / or the truncated peptide fragments of the present disclosure do not significantly deviate from the mean of a healthy control population, a therapeutic agent that prevents an increase in amyloid deposition is administered to the subject or a therapeutic agent is selected.In exemplary embodiments, the detected Aβ42 / Aβ40 value significantly deviates from the mean of a healthy control population. In some embodiments, if the detected Aβ42 / Aβ40 value and pTau217 / Tau217 (or pTau217×Aβ42 / Aβ40 composite value) significantly deviate from the mean of a healthy control population, and optionally, if pTau205 / Tau205 and / or the truncated peptide fragments of the present disclosure do not significantly deviate from the mean of a healthy control population, a therapeutic agent is administered to the subject or selected to prevent an increase in amyloid deposition and / or reduce the subject's existing plaque burden. In exemplary embodiments, the detected Aβ42 / Aβ40 value significantly deviates below the mean of a healthy control population, the detected pTau217 / Tau217 is above the mean of a healthy control population, and / or the pTau217×Aβ42 / Aβ40 composite value is above the mean of a healthy control population. In some embodiments, if the detected Aβ42 / Aβ40 values, pTau217 / Tau217 (or pTau217×Aβ42 / Aβ40 composite value) and pTau205 / Tau205 values ​​significantly deviate from the average of a healthy control population, and optionally, if the truncated peptide fragments of the present disclosure do not significantly deviate from the average of a healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent is selected that prevents an increase in amyloid deposition and / or reduces the subject's existing plaque burden and / or treats or prevents neurodegeneration and / or prevents tau tangles or related pathologies. In exemplary embodiments, the detected Aβ42 / Aβ40 values ​​deviate significantly below the mean of a healthy control population, the detected pTau217 / Tau217 values ​​are above the mean of a healthy control population, and / or the pTau217×Aβ42 / Aβ40 combined value is above the mean of a healthy control population, and / or the pTau205 / Tau205 values ​​are above the mean of a healthy control population.In some embodiments, if the detected Aβ42 / Aβ40 values, pTau217 / Tau217 (or pTau217×Aβ42 / Aβ40 composite value), pTau205 / Tau205 and the truncated peptide fragments disclosed herein significantly deviate from the average of a healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent is selected that prevents an increase in amyloid deposits and / or reduces the subject's existing plaque burden and / or treats or prevents neurodegeneration and / or prevents an increase in tau tangles and / or reduces existing tangles or related pathologies in the subject. In exemplary embodiments, the detected Aβ42 / Aβ40 values ​​deviate significantly below the mean of a healthy control population, the detected pTau217 / Tau217 values ​​are above the mean of a healthy control population, and / or the pTau217×Aβ42 / Aβ40 combined values ​​are above the mean of a healthy control population, and / or the pTau205 / Tau205 values ​​are above the mean of a healthy control population, and / or the cleaved peptide fragment values ​​of the present disclosure are above the mean of a healthy control population.

[0153] In each of the above embodiments, the pharmaceutical composition administered to the subject may contain an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radionuclide-labeled antibodies, etc.).

[0154] Another aspect of the present disclosure is a method for selecting subjects for clinical trials, particularly clinical trials for Aβ or tau treatment, provided that all other criteria for clinical trials are met. In one embodiment, the method for selecting subjects for clinical trials can include: (a) preparing a CSF sample from a subject, wherein the CSF sample is purified for tau cleavage peptide fragments; (b) quantifying the amount of tau cleavage peptide fragments in the sample; and (c) selecting the subject for clinical trials for Aβ treatment if the tau cleavage peptide fragment value is approximately the same as the average of a healthy control population and the subject's Aβ42 / Aβ40 value is lower than the average of a healthy control population. In some embodiments, the tau cleavage peptide fragments include one or more peptides with the amino acid sequence of Table 1, wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, and the amount of tau cleavage peptide fragments or their ratio is an indication of tau deposition in the brain of the subject.

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

[0156] In another embodiment, provided is a method for selecting subjects for clinical trials, particularly for clinical trials for tau treatment, provided that all other criteria for clinical trials are met.In one embodiment, the method for selecting subjects for clinical trials can include: (a) obtaining a CSF sample from a subject, and the CSF sample is purified for tau cleavage peptide fragments; (b) quantifying the tau cleavage peptide fragments in the sample; and (c) selecting the subject for clinical trials for tau treatment if the tau cleavage peptide fragment value is higher than the average of a healthy control population, and optionally the subject's Aβ42 / Aβ40 value is approximately the same as or lower than the average of a healthy control population.In some embodiments, the tau cleavage peptide fragment comprises one or more peptides comprising the amino acid sequence of Table 1, wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, and the amount of tau cleavage peptide fragments or their ratio is an indication of tau deposition in the brain of a subject.

[0157] In another embodiment, provided is a method for selecting subjects for clinical trials, particularly for tau treatment, provided that all other criteria for clinical trials are met.In one embodiment, the method for selecting subjects for clinical trials can include: (a) preparing a CSF sample from a subject, and the CSF sample is purified for tau cleavage peptide fragments; (b) quantifying the amount of tau cleavage peptide fragments in the sample; and (c) excluding the subject from the clinical trial for tau treatment if the value of tau cleavage peptide fragments is approximately the same as the average of a healthy control population.In some embodiments, the tau cleavage peptide fragments include one or more peptides comprising the amino acid sequence of Table 1, wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide, or a combination thereof, and the amount of tau cleavage peptide fragments or their ratio is an indication of tau deposition in the brain of the subject.The phrase "control population without brain amyloid plaques measured by PET imaging" is defined above.

[0158] Instead of or in addition to measuring the cleaved peptide fragments of tau disclosed herein, site-specific tau phosphorylation, optionally with the measurement of total tau, can be used in any of the above embodiments, such as a ratio calculated from the measured phosphorylation levels, or a ratio calculated from the measured phosphorylation levels and total tau. The ratio calculated from the measured phosphorylation levels can be the ratio between pT181 and pT205, pT217 and pT205, or pT181 and pT217. The ratio calculated from the measured phosphorylation levels and total tau can be the ratio between pT181 and total tau, pT205 and total tau, or pT217 and total tau. Mathematical calculations other than ratios can also be used. For example, site-specific tau phosphorylation values ​​can be used in various statistical models (e.g., linear regression, LME curve, LOESS curve, etc.) in conjunction with other known biomarkers (e.g., APOE ε4 status, age, sex, cognitive test score, functional test score, etc.).

[0159] The design of clinical trials for tauopathies (e.g., FTLD-MAPT, CBD) and tau treatments can be greatly aided by the methods disclosed herein. Many clinical trials are designed to test the effectiveness of imaging or therapeutic agents targeting specific pathophysiological changes that occur before the onset of tau pathology. As described herein, the efficacy of these various drugs can be improved by administering the drug to subjects with specific site-specific tau phosphorylation levels, as measured by the methods disclosed and exemplified herein. Similarly, clinical trials that select subjects with symptoms of Aβ pathology or tau-only pathology also benefit from being able to accurately distinguish between enrollee conditions to determine whether efficacy is associated with a specific disease state. Therefore, measuring tau phosphorylation levels as described herein in clinical trials, particularly before selecting subjects for treatment arms of the clinical trial, can result in smaller trials and / or improved results. In some instances, the methods described herein can be developed and used as companion diagnostics for therapeutic agents.

[0160] In each of the above embodiments, a subject may be enrolled in a treatment group of a clinical trial. "Treatment" is defined above. A pharmaceutical composition may be administered to a subject enrolled in a treatment group 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, florbetaben, florbetapir, flutemetamol, 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.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-Abeta antibodies (including antigen-binding fragments, variants, or derivatives thereof), anti-tau antibodies (including antigen-binding fragments, variants, or derivatives thereof), stem cells, nutritional supplements, Foods (e.g., lithium water, omega-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract), serotonin receptor 6 antagonists, p38α MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthionium chloride), glycemic control improvement therapies (e.g., insulin, exenatide, etc.) , 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 adrenoceptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenoceptor antagonists, 5-HT1A and 1D receptor agonists 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, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof. In an exemplary embodiment, the pharmaceutical composition may comprise a kinase inhibitor.Suitable kinase inhibitors may inhibit thousand and 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, the phosphatase activator may increase the activity of protein phosphatase 2A.

[0161] In each of the above embodiments, the subject may or may not be symptomatic. As used herein, an "asymptomatic subject" refers to a subject that does not exhibit any signs or symptoms of tauopathy. Alternatively, the subject may exhibit signs or symptoms (e.g., memory loss, misplacing objects, changes in mood or behavior, etc.), but may not exhibit sufficient cognitive or functional impairment for clinical diagnosis. A symptomatic or asymptomatic subject may have Aβ amyloidosis, but prior recognition of Aβ amyloidosis is not required for treatment. In a further embodiment, the subject may have AD. In any of the above embodiments, the subject may have one of the genetic mutations known to cause hereditary tauopathy. In another embodiment, the subject may not have a genetic mutation known to cause hereditary tauopathy.

[0162] 5. Kit Kits are also provided. Such kits may include a drug or composition described herein and, in certain embodiments, instructions for administering or measuring tau peptides (e.g., endogenously cleaved tau fragments). Such kits may facilitate the implementation of the methods described herein. When provided as a kit, different components of the composition may be packaged in separate containers and mixed immediately before use. Components include, but are not limited to, systems, assays, epitope binding agents, reagents, internal standards, or software. Such separate packaging of components may optionally be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil, such as a blister pack. Such separate packaging of components may, in certain instances, allow for long-term storage without loss of activity of the components.

[0163] The kit may also contain reagents in separate containers, such as sterile water or saline, to be added to the separately packaged lyophilized active ingredient. For example, a sealed glass ampoule may contain the lyophilized ingredient, and a separate ampoule may contain sterile water, sterile saline, or other sterile materials, each packaged under a neutral, non-reactive gas, such as nitrogen. The ampoules may be made of any suitable material, such as glass, organic polymers such as polycarbonate, polystyrene, ceramic, metal, or any other material typically used to hold reagents. Other examples of suitable containers include bottles made from materials similar to ampoules, and envelopes, which may be made from foil-lined interiors such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile access port, such as a bottle with a stopper pierceable by a hypodermic needle. Other containers may have two compartments separated by a readily removable membrane, which, when removed, allows the components to mix. The removable membrane can be glass, plastic, rubber, or the like.

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

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

[0166] The methods and algorithms of the present invention may be included in a controller or processor. Furthermore, the methods and algorithms of the present invention may be embodied as one or more computer-implemented methods for performing such methods, and may also be embodied in the form of a tangible or non-transitory computer-readable storage medium containing a computer program or other machine-readable instructions (herein "computer program"), which, when loaded into and / or executed by a computer or other processor (herein "computer"), causes the computer to become an apparatus for performing one or more methods. Storage media containing such computer programs include, for example, floppy disks and diskettes, compact disks (CD)-ROMs (whether or not writable), DVD digital discs, RAM and ROM memory, computer hard drives and backup drives, external hard drives, "thumb" drives, and any other storage medium readable by a computer. One or more methods may be embodied in the form of a computer program, whether stored on a storage medium or transmitted via a transmission medium such as electrical conductors, optical fibers or other optical conductors, or by electromagnetic radiation; when the computer program is loaded into and / or executed by a computer, the computer becomes an apparatus for performing one or more methods. One or more methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to perform one or more processes. If a general-purpose microprocessor is used, the computer program code configures the microprocessor's circuitry to create specific logic circuitry. A computer-readable storage medium includes a medium readable by the computer itself or by another machine that reads computer instructions to provide the computer with these instructions to control its operation. Such a machine may include, for example, a machine for reading the storage medium described above. [Example]

[0167] The present invention has multiple aspects, illustrated by the following non-limiting examples.

[0168] Example 1: CSF tau microtubule-binding domain identifies pathological changes in primary tauopathies (overview) Despite recent advances in fluid biomarker research in Alzheimer's disease (AD), no fluid biomarkers or imaging tracers useful for diagnosis and / or theragnostic analysis are available for other tauopathies. Using immunoprecipitation and mass spectrometry, we demonstrate that microtubule-binding region-derived 4-repeat (4R) isoform-specific tau species (MTBR-tau275 and MTBR-tau282) are elevated in the brains of patients with corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), frontotemporal lobar degeneration (FTLD)-MAPT, and AD compared with controls and other FTLD-tau species (i.e., Pick's disease). However, they are conversely decreased in the CSF of patients with CBD, FTLD-MAPT, and AD. CSF MTBR-tau measurements are reproducible in repeated lumbar punctures and can distinguish CBD from controls (area under the curve of 0.889) and other FTLD-tau species, such as PSP (area under the curve of 0.886). CSF MTBR-tau275 and MTBR-tau282 may represent the first positive biomarkers to aid in the diagnosis of primary tauopathies and facilitate clinical trial design.

[0169] (Introduction) Tauopathies are a heterogeneous group of neurodegenerative disorders that all involve aggregated tau protein. The symptomatic phase of these fatal diseases typically involves progressive neurological impairment over years to decades, resulting in substantial medical, social, and financial burdens for patients and their families. For the most common tauopathies, Alzheimer's disease (AD), cerebrospinal fluid (CSF) biomarkers amyloid beta (Aβ) as well as total and phosphorylated tau are used to aid diagnosis. 1~8 These biomarkers have been useful in assessing the efficacy of treatments in clinical trials.9~11 Furthermore, recent advances in positron emission tomography (PET) imaging now allow for the measurement of aggregated Aβ and tau in the brains of living AD patients. 12 In contrast, such progress has not been seen for other tauopathies classified as frontotemporal lobar degeneration (FTLD-tau), including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain disease (AGD), glioglobular tauopathy (GGT), chronic traumatic encephalopathy (CTE), and Pick's disease (PiD). Accurate diagnosis of tauopathies is difficult without biologic biomarkers for tauopathies because these disorders fall into a spectrum that includes multiple overlapping clinical phenotypes. Most tauopathies can only be conclusively diagnosed by brain autopsy. Premortem biologic biomarkers for these tauopathies are needed to improve the accuracy of clinical diagnosis and facilitate clinical trials for tauopathy treatments.

[0170] Recent structural and biochemical analyses suggest that different tau species constitute brain tau aggregates in subtypes of tauopathies. Six tau splicing isoforms are expressed in the adult brain, including isoforms containing the R1, R3, and R4 repeat domains (3R) and the R1, R2, R3, and R4 repeat domains (4R) in the MTBR. 13 Tauopathies can be classified into 3R, 4R, and mixed 3R / 4R tauopathies based on the predominant isoform found in tau aggregates. Cryo-electron microscopy (CryoEM) studies have shown that AD(3R / 4R) 14 , PiD(3R) 15 , CBD(4R) 16 , PSP(4R) 17 , and CTE(3R / 4R) 18 We demonstrated that tau aggregates from AD and other tauopathies have a distinct tau filament structure. The R3 and R4 repeat domains are commonly present in tau aggregates from AD and other tauopathies. 14 In contrast, the 4R isoform-specific R2 repeat domain, in addition to R3 and R4, is found in CBD- and PSP-derived tau aggregates. 16、17Biochemical extraction and mass spectrometry have recently demonstrated 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 as the disease progresses. 19 Furthermore, cleaved tau-containing MTBR could be detected and quantified in CSF, and the CSF soluble concentration of MTBR-tau peptide reflected the clinical severity of AD and correlated strongly with tau PET measurements. 19 Other investigators have also reported a correlation between CSF soluble cleaved MTBR-tau and insoluble tau aggregates measured by tau PET. 20 , suggesting that measurement of MTBR-tau peptides may serve as a fluid biomarker of tau aggregation in AD.

[0171] As a solution, we hypothesized that 4R isoform-specific MTBR-tau species accumulate in the brains of specific subtypes of 4R tauopathies. We investigated whether these changes are reflected in CSF and whether they can distinguish different subtypes of primary tauopathies. We specifically monitored tau peptides 275-280 (MTBR-tau275) and 282-290 (MTBR-tau282), which are located in the R2 region and specific for 4R tau splicing isoforms. We tested the differential diagnostic ability of MTBR-tau275 and MTBR-tau282 normalized to total tau to distinguish between effects in FTLD-tau, FTLD-TDP, and controls, as well as within different subtypes of tauopathies.

[0172] (method) Human trials This retrospective study of pathologically confirmed cohorts included participants seen at UCSF or participating research sites under the following projects: the Hillblom Healthy Aging Study, 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). Biospecimen collection and use were approved by the Institutional Review Boards of ALLFTD and each research center where individuals were recruited, and this study was approved by UCSF's Biospecimen Resource Committee. Participants provided written informed consent at the time of recruitment. Participants underwent a standardized clinical evaluation, including collection of demographic data, a structured participant / informant interview, functional assessment, neurological examination, and neuropsychological testing. CSF collection was performed at the same visit as the clinical evaluation; the primary clinical syndrome was determined based on all data available at the time of clinical evaluation by an experienced neurologist or panel of neurologists according to established diagnostic criteria. Consent to undergo autopsy was provided by the patient or their representative 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 (St. Louis, MO, USA) and University College London (UCL) (UK). All participants or their representatives consented to and were compensated for the collection and sharing of biofluid samples and brain autopsy. Exclusion criteria included any contraindication to LP or lumbar catheterization, including bleeding disorders, active anticoagulation, and active infection.

[0174] The clinically diagnosed cohort study was approved by the Ethics Committee of the Montpellier University Hospital (Centre des Sciences ...

[0175] human brain samples Neuropathological diagnosis of human brain donations was performed according to established diagnostic criteria at Washington University and UCSF. 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-HCl (pH 7.4), 150 mM sodium chloride, 10 mM EDTA, 10 mM EGTA, a phosphatase inhibitor cocktail, and a 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 extract. The demographics of the brain donors included in this study are described in Figure 1.

[0176] Human CSF samples CSF collection methods were similar across 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] Demographics of participants in the pathologically confirmed cohort are shown in Figure 2. CSF collection methods from the repeat LP study have been previously described. 37CSF was collected using the same human tau stable isotope labeling kinetic protocol as described previously. Demographics of participants in the repeat lumbar puncture study are described in Figure 9. CSF collection methods and demographics for the clinically diagnosed cohort have been described previously. 24 .

[0178] Mass spectrometry of MTBR-tau Brain insoluble MTBR-tau was analyzed using a filter-aided sample preparation method as previously described 19、36 Briefly, whole brain extracts were incubated with 1% sarkosyl on ice for 60 min, 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 with the following modifications. 19 A master mix containing detergent and chaotropic reagents (final 1% NP-40, 5 mM guanidine, protease inhibitor cocktail) and an internal tau standard (15N-labeled 2N4R recombinant tau) was prepared in a polypropylene tube before the addition of CSF. 0.5 mL of CSF was added, and immunoprecipitation was performed with tau1, HJ8.5, and HJ8.7 anti-tau antibodies, which possess epitopes present in the N-terminus to central domain of tau. 24、37 To measure MTBR-tau species, post-immunoprecipitation samples depleted from the N-terminus to the central domain of tau were sequentially immunoprecipitated with the 77G7 anti-tau antibody against MTBR (residues 316–335). After washing, samples were digested with trypsin, desalted, and analyzed by an Orbitrap Eclipse mass spectrometer (Thermo Scientific). The mass spectrometry method used to measure MTBR-tau was as previously described, with some modifications. To account for individual variability in t-tau concentrations, we used the ratio of MTBR-tau275 or MTBR-tau282 normalized to the mid-domain tau peptide (181–190 and 212–221 for brain and CSF analyses, respectively), which is common to all isoforms.

[0180] statistics All statistical analyses were performed using GraphPad Prism software (v9.3.1). Differences in biomarker values ​​were assessed by one-way ANOVA unless otherwise specified. A 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%. 38 p-values ​​reported in tables and figures are corrected by the Benjamini-Hochberg method (FDR=5%). Spearman correlation was used to assess associations between CSF tau biomarkers and neuropathological changes.

[0181] (result) Increased 4R-specific brain MTBR-tau in primary tauopathies We first analyzed frozen brain tissue from 59 individuals with autopsy-confirmed AD, FTLD-tau, or FTLD with TAR DNA-binding protein aggregates (FTLD-TDP), and three normal controls (NC) (p<0.0001 and p<0.01, respectively), 4R tauopathies, AGD (0.200) and PSP (0.489±0.229, p<0.0001 and p<0.05, respectively), and mixed 3R / 4R tauopathies, AD (0.319±0.047, p<0.0001 and p<0.01, respectively). MTBR-tau275 / t-tau was moderately (approximately 2-fold) increased in PSP compared with FTLD-TDP (p<0.01). Brain MTBR-tau282 / t-tau had a similar increase profile to MTBR-tau275 / t-tau (Figure 4C), but was moderately (2.6-fold) increased in AD (0.915 ± 0.180) compared with FTLD-TDP (0.348 ± 0.133, p < 0.05), which was not observed in MTBR-tau275 / t-tau. These results suggest that 4R-specific MTBR-tau species are enriched in the insoluble fraction of SFG / insular cortex brain tissue in a subset of 4R tauopathies, such as CBD and FTLD-MAPT, and are moderately increased in distinct PSPs (4R tauopathies) and AD (mixed 3R / 4R tauopathies).

[0182] Reduction of 4R-specific CSFMTBR-tau in primary tauopathies Next, CSF from 29 NC, -FTLD-MAPT, and 78 autopsy-confirmed AD, primary tauopathy, and FTLD-TDP cases was analyzed for MTBR-tau275 and MTBR-tau282 (Figure 2). CSF MTBR-tau275 and MTBR-tau282 concentrations did not distinguish between different tauopathies (Figures 12A and 12B) due to individual variability in t-tau concentrations (Figure 12C). Therefore, CSF MTBR-tau275 and MTBR-tau282 from truncated tau were consistent with previously reported measurements of truncated tau and Aβ isoforms. 20、22Similar to the previously described normalization method, normalization was performed by t-tau measured by the mid-domain tau (Figure 5A). CSF MTBR-tau / t-tau was decreased in CBD (0.00525 ± 0.00117), AD (0.00472 ± 0.00085), and FTLD-MAPT (0.00491 ± 0.00207) compared to NC (0.00657 ± 0.00078, p<0.001, p<0.0001, and p<0.01, respectively) and non-tauopathy controls, FTLD-TDP (0.00611 ± 0.00115, p<0.05, p<0.01, and p<0.05, respectively) (Figure 5B). This decrease was particularly pronounced in FTLD-MAPT P301L, which has typical FTLD pathology, compared with R406W, which has many features of AD. CSF MTBR-tau275 was also decreased in CBD, AD, and FTLD-MAPT compared with other 4R tauopathies, AGD (0.00759 ± 0.00013) and PSP (0.00669 ± 0.00091, p < 0.001, p < 0.0001, and p < 0.01, respectively), and the 3R tauopathies, PiD (0.00676 ± 0.00138, p < 0.05, p < 0.01, and p < 0.05, respectively). CSF MTBR-tau282 / t-tau had a similar decrease profile to CSF ​​MTBR-tau275 / t-tau (Figure 5C). Interestingly, CSF MTBR-tau275 / t-tau was unchanged in PSP compared with controls or FTLD-TDP, even though these ratios were moderately increased in the brain.

[0183] To assess whether soluble CSF MTBR-tau / t-tau measurements reflected brain tau pathology measured by paired insoluble brain MTBR-tau / t-tau measurements, antemortem CSF and brain MTBR-tau / t-tau from the same individuals were analyzed for correlation (N=54, Figures 6A and 6B). MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from CSF and brain were moderately correlated across all disease groups (N = 54) (r = -0.27, p = 0.049, and r = -0.45, p = 0.0006, respectively), but were strongly correlated in 4R tauopathies (PSP, CBD, and AGD, N = 29) (r = -0.61, p = 0.0004, and r = 0.75, p < 0.0001, Figures 6C and 6D). This suggests that 4R-specific MTBR-tau species are inversely correlated in CSF and brain in 4R tauopathies. In CBD, MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from CSF and brain were moderately correlated but not statistically significant (N = 12, r = -0.25, p = 0.43, and r = -0.31, p = 0.33, respectively, Figure 3E, F). One CBD participant who did not have cognitive impairment (Clinical Dementia Rating plus National Alzheimer's Coordinating Center FTLD box sum) had a significant correlation. 23 Patients with (CDR plus NACC FTLD-SB)=0) had lower changes in MTBR-tau275 / t-tau and MTBR-tau282 / t-tau in both the brain and CSF (brain MTBR-tau275 / t-tau=0.321, brain MTBR-tau282 / t-tau=0.499, CSF MTBR-tau275 / t-tau=0.0071, CSF MTBR-tau282 / t-tau=0.0134), which may suggest that the changes in these biomarkers depend on the severity of the disease.

[0184] To assess whether CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau decreased with disease stage, we examined correlations between these biomarkers and duration (i.e., age at onset (AAO) and interval between CSF sampling) (Figure 2, Figure S13B). The mean duration of disease overall was 5 ± 4 years (N = 81) and 4 ± 1 year for CBD only (N = 18). For CBD, there was a negative correlation between duration and CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau (r = -0.37 and -0.39, respectively), although this was not statistically significant (p = 0.13 and 0.11, respectively). The results suggest that participants with a later pathological stage and longer duration had a greater degree of decline in CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau biomarkers.

[0185] CSF MTBR-tau is reproducible on repeat lumbar punctures Ongoing studies examining protein turnover kinetics to assess the reproducibility and stability of CSF MTBR-tau measurements within the same individual 37 As part of this study, we examined CSF MTBR-tau275 / t-tau in an independent cohort of 25 participants who underwent repeated lumbar punctures (LPs, 3–5 times) within approximately 4 months (Figure 7, Figure 9). These participants included individuals clinically diagnosed with PSP-Richardson syndrome (PSP-RS, N=7) or corticobasal ganglionic syndrome (CBS; a clinical syndrome associated with heterogeneous neuropathological substrates including AD, CBD, PSP, and FTLD-TDP, N=9). Two participants had autopsy-confirmed CBD. Participants included seven carriers of MAPT mutations (P301L, R406W, and IVS10+16) who were either symptomatic or asymptomatic, and two non-carrier family members who were normal controls (NC). The mean coefficient of variation (CV) of CSF MTBR-tau275 / t-tau in repeated LPs was 12±7%, establishing high reproducibility and stability of CSF MTBR-tau measurements within 4 months.

[0186] Consistent with the FTLD-MAPT cases analyzed in the pathologically confirmed cohort, CSF MTBR-tau275 / t-tau was decreased in FTLD-MAPT mutation carriers in the repeated LP cohort. Interestingly, CSF MTBR-tau275 / t-tau was decreased in two symptomatic FTLD-MAPT P301L mutation carriers (participants #02 and #03, 0.00381 ± 0.00021) and a symptomatic FTLD-MAPT R406W mutation carrier (#05, 0.00508) compared with control subjects (#01 and #04, 0.00666 ± 0.00027). However, CSF MTBR-tau275 / t-tau was unchanged in asymptomatic FTLD-MAPT R406W mutation carriers with LP (#06 and #07). The FTLD-MAPT variant IVS10+16 promotes splicing of tau exon 10, resulting in the production of more 4R isoforms than 3R isoforms. Indeed, symptomatic FTLD-MAPT IVS10+16 mutation carriers (#08, #09, 0.00921±0.00053) had a 1.38-fold higher CSF MTBR-tau275 / t-tau compared to NC, indicating that the increase in 4R isoforms is reflected in the CSF.

[0187] CSF MTBR-tau275 / t-tau decreased in two participants with a clinical diagnosis of PSP-RS who later had autopsy-confirmed CBD (#10 (0.00396) and #11 (0.00535)), consistent with the pathologically confirmed CSF cohort results. However, CSF MTBR-tau275 / t-tau did not change in participants with a clinical diagnosis of PSP-RS (0.00779 ± 0.00052) or CBS (0.00748 ± 0.00187) without autopsy-confirmed disease during the repeated measures study. The mean duration of disease overall was 5 ± 3 years (N = 21) and 4 ± 2 years for CBS only (N = 9), similar to the pathologically confirmed cohort.

[0188] CSF MBSR-tau in clinically diagnosed primary tauopathies To estimate CSF MTBR-tau biomarker performance in clinically diagnosed primary tauopathies, CSF MTBR-tau275 / t-tau was measured in an additional independent cohort of 238 primary tauopathies using single LP (Figure 8). This cohort had previously been analyzed for CSF t-tau and phosphorylated tau. 24 , including clinically diagnosed AD, sporadic behavioral variant frontotemporal dementia (bvFTD), FTLD-MAPT, PSP-RS, CBS, and bvFTD secondary to the CBS-PSP continuum 25 Individuals with the CBS-PSP continuum are defined as those who initially exhibited CBS but subsequently developed clinical features of PSP-RS as the disease progressed. CSF MTBR-tau275 / t-tau was decreased in the CBS-PSP continuum and FTLD-MAPT compared with cognitive NC (p<0.05). However, CSF MTBR-tau275 / t-tau was not statistically altered in either AD or clinically diagnosed CBS compared with controls or other tauopathies.

[0189] Diagnostic accuracy of CSF MTBR-tau in primary tauopathies Finally, the diagnostic accuracy of CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau was examined in a cohort of pathologically confirmed primary tauopathies. First, CSF t-tau (central domain peptide 212-221) and phosphorylated tau (pT217 / T217) were examined in primary tauopathies for comparison (Figure 12C and Figure 14A). CSF t-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 distinguish FTLD-tau. CSF pT217 / T217 was increased in AD compared with NC, FTLD-TDP, and FTLD-tau (p<0.0001) and could distinguish AD from FTLD-tau with an AUC of 0.987 (Figure 14C). Co-pathology of AD in other neurodegenerative diseases (i.e., FTLD-TDP, CBD, PSP) also increased CSF pT217 / T217. These results suggest that CSF pT217 / T217 can be used to accurately identify individuals with AD pathology, regardless of co-pathology.

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

[0191] The diagnostic accuracy of CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau was examined to determine whether they distinguished 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 distinguished CBD from NC, other FTLD-tau (PSP, PiD, and AGD), PiD, and PSP with AUCs of 0.800-0.889. CBD also distinguished FTLD-TDP with AUCs of 0.701-0.770. When excluding cases of co-morbid AD, CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau were able to distinguish CBD from PSP with AUCs of 0.859 and 0.886, respectively (Figure 3, Figure S16K, and Figure S16L).

[0192] Finally, we retrospectively evaluated CSF MTBR-tau275 / t-tau and CSF MTBR-tau282 / t-tau by final clinical syndrome in a neuropathologically confirmed cohort to determine whether these biomarkers could facilitate the premortem diagnosis of primary tauopathies. The number of individuals within each clinical syndrome application of these biomarkers whose CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau levels were below the cutoffs (0.00563 and 0.01220, respectively, defined in Figure 3 for distinguishing CBD from PSP), including distinguishing CBD from PSP in PSP-RS, was 0 / 11 (0%) and 2 / 11 (18%) for PSP, respectively.

[0193] (Consideration) Despite a long search for antemortem biomarkers of FTLD-tau pathology, to date, no body fluid biomarkers have been identified that can distinguish subgroups of tauopathies other than AD. Previous studies have shown that CSF phosphorylated tau at T181 is decreased in PSP and FTLD-TDP compared with controls. 26~30 CSF pT217 / T217 was recently 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 therefore these studies may not capture important 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 present in CSF as a truncated C-terminal tau peptide. 19Using biochemical purification and quantitative mass spectrometry, the 4R isoform-specific MTBR-tau275 and MTBR-tau282, normalized to t-tau, were shown to decrease CSF soluble tau and increase brain insoluble tau in primary tauopathies, particularly CBD and FTLD-MAPT P301L. MTBR-tau / t-tau measurements were inversely correlated in CSF and brain, suggesting that an equilibrium or unidirectional shift may exist between soluble CSF MTBR-tau and insoluble brain MTBR-tau in these primary tauopathies. This study demonstrates the potential of the first biological fluid biomarkers to reflect brain pathology in primary tauopathies.

[0194] One interesting finding of this study was that 4R isoform-specific changes in MTBR-tau275 and MTBR-tau282 were observed only in a subset of 4R and 3R / 4R mixed tauopathies. As expected, these 4R isoform-specific measurements were not altered in 3R tauopathies (PiD) or non-tauopathic FTLD (FTLD-TDP). However, CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau were specifically decreased in CBD and FTLD-MAPT, but not in PSP and AGD, despite all being classified as 4R tauopathies. This may be due to greater variability in the extent of neocortical pathology in PSP and AGD cases compared with CBD and many FTLD-MAPT cases.

[0195] Many cases of PSP have 4R tau aggregates primarily in subcortical regions such as the thalamus and brainstem. 31 AGD pathology is most severe within the medial temporal lobe, even in advanced stages. 32 In contrast, CBD has more abundant and widespread tau pathology in the cerebrum. 33 FTLD-MAPT (e.g., P301L) can result in extremely high deposition of 4R tau aggregates in neurons and glia in multiple brain regions, including the hippocampus, neocortex, and substantia nigra. 34An alternative explanation is that CBD neurons have thin, filamentous inclusions within neuronal cell bodies, whereas PSP neurons tend to have a greater proportion of more compact tau aggregates. 35 CBD is generally associated with abundant cortical astrocytic plaque pathology and neuritic tau pathology in both gray and white matter, whereas PSP neuronal and astrocytic pathology (i.e., tufted astrocytes) is often restricted to the motor and premotor cortex and subcortical nuclei. It is possible that tau aggregates in PSP may have different physicochemical properties than CBD, and the equilibrium between insoluble and soluble forms may be different. Taken together, we speculate that the amount or total burden of 4R tau pathology in the whole brain may reflect changes in CSF 4R-specific MTBR-tau.

[0196] CSF MTBR-tau275 / t-tau and MTBR-tau282 / t-tau can potentially positively identify a subset of primary tauopathies and may be useful for assisting antemortem differential diagnosis. Our repeat lumbar puncture study confirmed that CSF MTBR-tau / t-tau measurements were reproducible and stable over a 4-month period, providing reliable biomarker value in hospitals or clinical trial settings. In clinically diagnosed cohorts without autopsy confirmation, CSF MTBR-tau275 / t-tau did not change in CBS, and there was a higher overlap between CBS and PSP-RS, which may be due to the lack of a one-to-one relationship between clinical syndromes and neuropathological diagnoses in FTLD. However, retrospective clinical syndrome analysis in a pathologically confirmed cohort has shown that the CSF MTBR-tau275 / t-tau and MTBRT-tau282 / t-tau biomarkers can identify individuals with CBD with an accuracy of as high as 83%, regardless of clinical syndrome (i.e., CBS, bvFTD, and PSP-RS), which is higher than the approximately 25–50% diagnostic accuracy of CBD without these biomarkers.

[0197] Further limitations of this study include the following: Tauopathies of shorter duration or during the asymptomatic phase may not yet show a decrease in these biomarkers. Future studies targeting larger cohorts with different severity and longitudinal samples with clinical measures will help address whether CSF MTBR-tau can capture disease at an earlier stage in primary tauopathies. The lack of orthogonal measures to identify brain tau pathology in living patients (i.e., tau PET imaging with tracers specific for primary tauopathies) limits our ability to assess the correlation between CSF and brain MTBR-tau premortem. The modest size of the cohort, with small sample sizes for subgroups such as AGD and PiD, may limit interpretation. The 77G7 MTBR-tau antibody used in this study for sequential immunoprecipitation may also target specific pools of truncated tau, and future technological advances and analytical method developments may reveal additional or novel populations of tau species in biological fluids that reflect qualitative and quantitative aspects of tau pathology in primary tauopathies. Overall, these findings advance our understanding of the heterogeneous pathophysiology of primary tauopathies and pave the way for therapeutic development and clinical trials targeting primary tauopathies.

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Claims

1. 1. A method for detecting tau in a cerebrospinal fluid (CSF) sample, 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; A method comprising:

2. 1. A method for detecting 4R tau aggregates in a subject, comprising: (a) purifying endogenous cleaved tau fragments from a CSF sample from a subject without contacting the endogenous cleaved tau fragments with a protease in vitro; (b) contacting the purified endogenously cleaved tau fragment 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 proteolytic MTBR-tau275 peptides and / or proteolytic MTBR-tau282 peptides by performing liquid chromatography mass spectrometry (LC / MS) or an immunoassay, wherein measuring the proteolytic MTBR-tau275 peptides and / or proteolytic MTBR-tau282 peptides indicates 4R tau aggregates in the subject. A method comprising:

3. 3. The method of claim 2, wherein the solution containing the proteolytic MTBR-tau275 or proteolytic MTBR-tau282 peptide is desalted prior to step (c), and the desalting may be achieved by solid phase extraction.

4. 3. 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 according to any one of claims 2 to 5, wherein the purification in step (a) comprises immunoprecipitation.

7. 7. The method of claim 2, wherein the purification in step (a) comprises immunoprecipitating the N-terminal to mid-domain of tau to deplete the N-terminal to mid-domain of tau from the biological sample.

8. 8. The method of claim 7, wherein immunoprecipitation of N-terminal to central 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 to 243 of the sequence set forth in SEQ ID NO:

1.

9. the epitope binding agent is (a) an anti-tau antibody tau1 or an antigen-binding fragment thereof; (b) the anti-tau antibody HJ8.5 or an antigen-binding fragment thereof; and (c) anti-tau antibody HJ8.7 or an antigen-binding fragment thereof The method according to any one of claims 4 to 9, wherein the method is one or more of the following:

10. The method of any one of claims 2 to 9, wherein the purification in step (a) comprises immunoprecipitating one or more MTBR-tau species from the biological sample to capture one or more MTBR-tau species from the biological sample.

11. 11. The method of claim 10, wherein the one or more captured MTBR-tau species comprises MTBR-tau275 peptide and / or MTBR-tau282 peptide.

12. The method of claim 10 or 11, wherein immunoprecipitation of one or more MTBR-tau species comprises contacting the biological sample with one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244 to 368 relative to SEQ ID NO:

1.

13. 13. The method of any one of claims 10 to 12, wherein immunoprecipitation of one or more MTBR-tau species comprises contacting the CSF sample with one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 316 to 335 of the sequence set forth in SEQ ID NO:

1.

14. The method of any one of claims 10 to 13, wherein the epitope binding agent is 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. 17. The method of any one of claims 1 to 16, further comprising one or more of detecting and quantifying one or more of amyloid beta, N-terminal tau, central domain tau, post-translational modifications of tau, and ApoE isoforms in the CSF sample.

18. 18. The method of claim 17, wherein mid-domain tau is quantified, and mid-domain tau comprises tau 212-221 relative to SEQ ID NO: 1 (t-tau).

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 indicates 4R tau aggregates in the subject.

20. 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 compared to a threshold value indicates 4R tau aggregates in the subject.

21. 1. A method for detecting 4R tau aggregates in a subject, comprising: (a) performing affinity depletion in cerebrospinal fluid (a CSF sample from a subject) by contacting the biological sample with one or more affinity depletion agents, including one or more epitope binding agents that each bind to one of N-terminal tau, mid-domain tau, or C-terminal tau but do not bind to an antigen within microtubule-binding region (MTBR) tau, to obtain a depleted sample and an enriched sample, wherein the CSF sample comprises endogenous cleaved tau fragments, the depleted sample comprises N-terminal tau, mid-domain tau, and / or C-terminal tau, and the enriched sample is enriched for endogenous cleaved tau fragments comprising one or more of endogenous MTBR-tau275 peptide comprising amino acids 275-280 relative to SEQ ID NO:1 and endogenous MTBR-tau282 peptide comprising amino acids 282-290 relative to SEQ ID NO:1; (b) performing immunoprecipitation on the enriched sample by contacting the enriched sample with one or more immunoprecipitation agents, including one or more epitope-binding agents, that bind to MTBR-tau and capture one or more of the endogenous MTBR-tau 275 peptide and the endogenous MTBR-tau 282 peptide to obtain a purified sample; (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 containing one or more of the proteolytic MTBR-tau275 peptide and the 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. A method comprising:

22. 22. The method of claim 21, wherein the sample containing one or more of the proteolytic MTBR-tau275 and proteolytic MTBR-tau282 peptides is desalted prior to step (d), and the desalting may be achieved by solid phase extraction.

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

1.

24. 24. The method of any one of claims 21 to 23, wherein the epitope binding agent is an anti-tau antibody or an antigen-binding fragment thereof.

25. the epitope binding agent is (a) an anti-tau antibody tau1 or an antigen-binding fragment thereof; (b) the anti-tau antibody HJ8.5 or an antigen-binding fragment thereof; and (c) anti-tau antibody HJ8.7 or an antigen-binding fragment thereof The method according to any one of claims 21 to 24, wherein the method is one or more of the following:

26. 26. The method of any one of claims 21 to 25, wherein the one or more immunoprecipitation agents comprise one or more anti-tau epitope binding agents that specifically bind to an epitope within amino acids 244 to 368 of the sequence set forth in SEQ ID NO:

1.

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

1.

28. 28. The method of any one of claims 21 to 27, wherein the one or more immunoprecipitation agents comprise the anti-tau antibody 77G7 or an antigen-binding fragment thereof.

29. 29. The method of any one of claims 21 to 28, wherein the endopeptidase comprises trypsin.

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

31. 31. The method of claim 30, wherein mid-domain tau is quantified, and mid-domain tau comprises tau 212-221 relative to SEQ ID NO: 1 (t-tau).

32. The method of claim 31, 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 indicates 4R tau aggregates in the subject.

33. 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 compared to a threshold value indicates 4R tau aggregates in the subject.

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

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

36. 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 compared to a threshold value indicates one or more of frontotemporal lobar degeneration (FTLD)-MAPT and corticobasal degeneration (CBD).

37. 34. 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 the method of any one of claims 1 to 33.

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

39. 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 compared to a threshold value indicates frontotemporal lobar degeneration (FTLD)-MAPT or corticobasal degeneration (CBD).

40. 1. A method for diagnosing frontotemporal lobar degeneration (FTLD)-MAPT in a subject, comprising: (a) detecting tau or 4R tau aggregates in a CSF sample from a subject according to the method of any one of claims 1 to 33, or providing an amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from the CSF sample; and (b) If the amount of one or more of the detected MTBR-tau275 / t-tau and MTBR-tau282 / t-tau is decreased compared to the threshold, FTLD-MAPT is diagnosed. A method comprising:

41. 1. A method for measuring progression of FTLD-MAPT in a subject, comprising: (a) detecting tau or 4R tau aggregates in a first CSF sample and a second CSF sample from a subject according to the method of any one of claims 1 to 33, or providing an amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from the CSF samples; 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. A method comprising:

42. 1. A method for diagnosing corticobasal degeneration (CBD) in a subject, comprising: (a) detecting tau or 4R tau aggregates in a CSF sample from a subject according to the method of any one of claims 1 to 33, or providing an amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from the CSF sample; and (b) diagnosing CBD when the amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau detected is decreased compared to a threshold value; A method comprising:

43. 1. A method for 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 a subject according to the method of any one of claims 1 to 33, or providing an amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau from the CSF samples; 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. A method comprising:

44. 1. A method of treating a primary tauopathy in a subject in need thereof, comprising: (a) detecting tau or 4R tau aggregates in a CSF sample from a subject according to the method of any one of claims 1 to 33, or providing an 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 decreased compared to a threshold. A method comprising:

45. A method of treating a primary tauopathy in a subject in need thereof, the method comprising administering to the subject a treatment that alters tau pathology, wherein the subject has been identified as having a decreased amount of one or more of MTBR-tau275 / t-tau and MTBR-tau282 / t-tau compared to a threshold, optionally measured according to the method of any one of claims 1 to 33.

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

47. Treatment included lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, bepranemab, dasatinib + quercetin, E2814, epothilone D, goslanemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, semolinemab, TPI 287, tideglusib, tirabonemab, zagotenemab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNAs, tau production inhibitors and tau-activating vaccines.

48. 48. The method of any one of claims 44 to 47, wherein the treatment is selected from the group consisting of an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma-secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, and a tau protein aggregation inhibitor.

49. 49. The method of claim 48, wherein the kinase inhibitor is an inhibitor of thousand and one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn.

50. 49. The method of claim 48, wherein the phosphatase activator increases the activity of protein phosphatase 2A.

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

52. 49. The method of claim 48, wherein the anti-Aβ antibody is aducanumab or another anti-amyloid antibody that clears plaques.

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