Method for detecting MTBR tau isoforms and its use

Abstract

A method is provided for processing pre-obtained biological samples to measure the relative or absolute concentration of tau by mass spectrometry. [Solution] A method for measuring tau in a biological sample, comprising: (a) preparing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then desalting the cleavage product obtained optionally by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect at least one tau proteolytic peptide and measure its concentration.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Application 62 / 886,165 filed August 13, 2019, U.S. Provisional Application 62 / 970,950 filed February 6, 2020, and U.S. Provisional Application 63 / 044,836 filed June 26, 2020, each of which is incorporated herein by reference as a whole.

[0002] Government rights This invention was made with government support under NS095773, approved by the National Institutes of Health (NIH). The government has certain rights to this invention.

[0003] Reference to sequence listings This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy created on [date] is named "665135_ST25.txt" and is 14KB in size.

[0004] field The present invention encompasses a method for converting a blood or CSF (cerebrospinal fluid) sample into a sample suitable for quantifying MTBR (microtubule binding region) tau species by mass spectrometry, immunoassay, or other assays known in the art. The present invention also includes the use of MTBR tau species in blood or CSF for measuring the pathological features and / or clinical symptoms of 3R- and 4R- tauopathy for diagnosis, staging, and / or selection of appropriate treatment for a given disease stage. [Background technology]

[0005] background The accumulation of tau protein as insoluble aggregates in the brain is characteristic of Alzheimer's disease and other neurodegenerative diseases known as tauopathies. Tau pathology is thought to spread beyond brain regions and diffuse through cell-to-cell transmission of specific pathological tau species in a prion-like manner, although the properties and diffusion processes of these species (i.e., monomers, oligomers, and profibrillomas) remain unknown (Frost et al., 2009; Goedert et at., 2010, 2017; Sanders et at., 2014; Wu et at., 2016; Mirbaha et al., 2018; Lasagna-Reeves et at., 2012). Tau is a full-length protein with six different isoforms. Furthermore, tau has over 100 post-translational modification sites, including phosphorylation, and multiple shortening sites (Meredith et al., 2013; Sato et al., 2018; Barthelemy et al., 2019; Cicognola et al., 2019; Blennow et al., 2020). Therefore, identifying specific pathological tau species involved in tau diffusion is difficult. Several mass spectrometry (MS) studies have suggested that the microtubule-binding region (MTBR) of tau is enriched in aggregates in the brains of Alzheimer's disease patients (Taniguchi-Watanabe et al., 2016; Roberts et al., 2020). Furthermore, a series of cryo-electron microscopy (Cryo-EM) studies have shown that the core structure of tau aggregates consists of subsegments of the MTBR domain, and that specific morphologies are dependent on 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. The pathophysiology of corresponding extracellular MTBR-containing tau species in biological samples such as CSF and blood, which could serve as sarogative biomarkers for brain tau aggregates in living humans, remains largely unknown.

[0006] CSF is routinely obtained from participating patients via lumbar paracentesis during their hospital visits. Previous CSF tau biomarker studies have shown that MTBR tau is not present in CSF but is concentrated in the N-terminal and intermediate domain regions (Meredith et al., 2013; Sato et al., 2018). Species constituting the intermediate domain from the N-terminus appear to be actively secreted from neurons into the extracellular space after shortening between the intermediate domain and the MTBR domain (Sato et al., 2018). While the detection of MTBR tau species has been reported (Barthelemy et al., 2016 b, a), its correlation with disease has not yet been characterized. Recently, a tau species containing a cleavage of residue 368 (tau 368) within repeating region 4 (R4) was identified in CSF (Blennow et al., 2020). However, considering the regions not captured by antibodies, shortening, and conformational variations, it is unclear whether tau 368 reflects the overall pool of MTBR tau species.

[0007] Advances in high-resolution mass spectrometry technology have led to the development of new methods for measuring the abundance of numerous proteins in biological samples. Despite advances in instruments and data analysis software, sample preparation remains a significant challenge. The choice of sample preparation method affects the observed metabolite profile and data quality, ultimately impacting the results. This is especially true for proteins and peptides present in low abundance in biological samples. Peptides in this category include many proteolytic fragments of full-length proteins produced in various ways during diverse disease processes. [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, in this field, there remains a need for improvement in sample processing methods for quantifying low-abundance MTBR tau species in body fluids. [Means for solving the problem]

[0009] overview In various aspects of the present invention, a method is provided for processing a pre-obtained biological sample in order to measure the relative or absolute concentration of tau by mass spectrometry.

[0010] One aspect of the present invention is a method for measuring tau in a biological sample, comprising: (a) preparing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then optionally desalting the resulting cleavage product by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect at least one tau proteolytic peptide and measure its concentration.

[0011] Another aspect of the present invention is a method for evaluating tau in a biological sample, comprising: (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in a biological sample, such as a blood sample or a CSF sample, by affinity depletion; (b) removing further proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then desalting the cleavage product obtained optionally by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing the tau peptides to detect at least one tau proteolytic peptide and measure its concentration.

[0012] Another aspect of the present invention is a method for measuring tau in a biological sample, comprising: (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in a biological sample, such as a blood sample or a CSF sample, by affinity depletion; (b) affinity purifying MTBR tau; (c) cleaving the purified MTBR tau with a protease, and then desalting the cleavage product obtained optionally by solid-phase extraction to obtain a sample containing MTBR tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry on the sample containing MTBR tau proteolytic peptides to detect at least one of the MTBR tau proteolytic peptides and measure their concentration.

[0013] Another aspect of the present invention relates to a method for measuring tau in a biological sample, comprising: (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in a biological sample, which is a blood sample or a CSF sample, by affinity depletion, wherein affinity depletion involves contacting the biological sample with an epitope binder that specifically binds to an epitope (or a similarly defined region of another full-length isoform) within amino acids 1-221 (including both ends), preferably within amino acids 50-221 (including both ends), or more preferably within amino acids 104-221 (including both ends) of tau-441; and (b) MTBR The method comprises: affinity purification of tau, wherein affinity purification involves contacting the product of step (a) with an epitope binder that binds to the C-terminal epitope recognized by the epitope binder of step (a); (c) cleaving the purified MTBR tau with a protease, and then desalting the optionally obtained cleavage product by solid-phase extraction to obtain a sample containing MTBR tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry on the sample containing MTBR tau proteolytic peptides to detect at least one of the MTBR tau proteolytic peptides and to measure their concentration. In one embodiment, the epitope binder of step (b) specifically binds to an epitope within amino acids 221-441 of tau-441 (including both ends) (or a similarly defined region of another full-length isoform). In one embodiment, the epitope binder of step (b) specifically binds to an epitope within amino acids 235-441 of tau-441 (including both ends) (or within a similarly defined region of another full-length isoform). In another embodiment, the epitope binder of step (b) specifically binds to an epitope within amino acids 235-368 of tau-441 (including both ends) (or within a similarly defined region of another full-length isoform). In another embodiment, the epitope binder of step (b) specifically binds to an epitope within amino acids 244-368 of tau-441 (including both ends) (or within a similarly defined region of another full-length isoform).In one embodiment, the epitope binder of step (b) specifically binds to an epitope within amino acids 244-299 of tau-441 (including both ends) (or within a similarly defined region of other full-length isoforms).

[0014] Prior to use in the methods disclosed herein, biological samples may have been modified by removal of cell debris, addition of components (such as protease inhibitors, isotope-labeled internal standards, surfactants, chaotropic agents, etc.) and / or depletion of analytes (such as Aβ peptide, N-terminal tau, middle domain tau, etc.).

[0015] The methods disclosed herein are particularly suitable for the measurement of MTBR tau. In the specific embodiments described above, the methods of the invention can be used to measure the concentration of one or more tryptic digestion peptides of tau, including but not limited to IGST (SEQ ID NO: 2), VQII (SEQ ID NO: 4), LQTA (SEQ ID NO: 3), LDLS (SEQ ID NO: 5), HVPG (SEQ ID NO: 6), IGSL (SEQ ID NO: 7) and VQIV (SEQ ID NO: 9). In one example, it may be desirable to measure the concentration of two or more tryptic digestion peptides of tau and then calculate the ratio of the two values. As disclosed herein, the ratios of HVPG (SEQ ID NO: 6) to IGSL (SEQ ID NO: 7), LQTA (SEQ ID NO: 3) to IGSL (SEQ ID NO: 7), IGST (SEQ ID NO: 2) to IGSL (SEQ ID NO: 7), VQII (SEQ ID NO: 4) to IGSL (SEQ ID NO: 7), LDLS (SEQ ID NO: 5) to IGSL (SEQ ID NO: 7), IGST (SEQ ID NO: 2) to HVPG (SEQ ID NO: 6), VQII (SEQ ID NO: 4) to HVPG (SEQ ID NO: 6), LDLS (SEQ ID NO: 5) to HVPG (SEQ ID NO: 6) and VQIV (SEQ ID NO: 7) to LDLS (SEQ ID NO: 5) can provide clinically significant information for guidance in tauopathy diagnosis and treatment decisions. In yet a further example, it may be desirable to determine the presence / absence of one or more additional proteins in a biological sample and / or measure the concentration of one or more additional proteins.

[0016] Another aspect of the present invention is a method for measuring tauopathy-related pathology in a subject, comprising quantifying one or more intermediate domain-independent MTBR tau species in a biological sample obtained from the subject, such as a blood sample or a CSF sample, wherein the amount or ratio of the quantified intermediate domain-independent MTRB tau species represents tauopathy-related pathology in the brain of the subject. The tauopathy can be 3R-tauopathy, mixed 3R / 4R-tauopathy or 4R-tauopathy. The disease-related pathology can be tau deposition, post-translational modification of tau, amyloid plaques in the brain and / or arteries of the brain or other pathological characteristics known in the art. The subject may or may not have clinical symptoms of tauopathy.

[0017] Another aspect of the present invention is a method for diagnosing tauopathy in a subject, comprising quantifying one or more intermediate domain-independent MTBR tau species in a biological sample obtained from the subject, such as a blood sample or a CSF sample, and diagnosing tauopathy when the quantified intermediate domain-independent MTBR tau species differ by about 1.5σ or more, wherein σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. The tauopathy can be 3R-tauopathy, mixed 3R / 4R-tauopathy or 4R-tauopathy. The subject may or may not have clinical symptoms of the disease.

[0018] Another aspect of the present invention provides a method for measuring disease stability in a subject, comprising quantifying one or more intermediate domain-independent MTBR tau species in a first biological sample obtained from the subject, and then in a second biological sample obtained from the same subject, wherein the second biological sample is obtained after the first biological sample (e.g., several days, weeks, months, or years later), and calculating the difference in the quantified MTBR tau species between the samples, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates disease progression, a statistically significant decrease in the quantified MTBR tau species in the second sample indicates disease improvement, and no change indicates disease stability. The subject may or may not have clinical symptoms of the disease.

[0019] Another aspect of the present invention provides a method for treating a subject having tauopathy, comprising: quantifying one or more intermediate domain-independent MTBR tau species in a biological sample obtained from the subject, such as a blood sample or a CSF sample; and treating the subject to improve a measure of disease-associated pathology and / or clinical symptoms, wherein the subject differs by about 1.5σ or more in the quantified MTBR tau species, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as assessed by PET contrast and / or Aβ42 / 40 measurement in CSF. The tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. Measures of disease-related pathology may include the amount of MTBR tau species and / or tau deposition assessed by PET contrast, post-translational modification of tau assessed by mass spectrometry or other appropriate method, amyloid plaques in the brain or cerebral arteries assessed by PET contrast, amyloid plaques measured by Aβ42 / 40 in CSF, or other pathological features known in this field. Clinical symptoms may include dementia or other clinical symptoms known in this field for 3R-, 3R / 4R-, and 4R- tauopathy, assessed by clinically validated devices (e.g., MMSE, CDR-SB, etc.).

[0020] These and other aspects and iterations of the present invention are described in further detail below. [Brief explanation of the drawing]

[0021] This application includes at least one color photograph. A copy of this patent application publication accompanied by the color photograph will be provided by the Office upon request and payment of the necessary fees.

[0022] [Figure 1] Figure 1 shows a schematic diagram of the longest human tau isoform (2N4R). The N-terminus, intermediate domain, MTBR, and C-terminus of this isoform are identified and vary in a predictable manner for other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R, and 0N3R).

[0023] [Figure 2A] Figure 2A is a schematic diagram illustrating several methods of the present invention. The method detailed in the blue box (right) is one method. The combination of the red box (left) and the blue box (right) represents other methods.

[0024] [Figure 2B] Figure 2B is a schematic diagram illustrating several methods of the present invention. The method shown in the blue box (right) is one method. The combination of the red box (left) and the blue box (right) represents other methods.

[0025] [Figure 3A]Figure 3A is a graph comparing the effectiveness of three sample processing methods in quantifying tau peptides from a single CSF test sample. The CSF test samples were not from a single individual, and CSF-related disease states were unavailable. The x-axis represents the tau-441 peptide, and the y-axis represents the 14N / 15N ratio. The relative positions of epitopes recognized by antibodies HJ8.5 and Tau1 (indicated as "Y") are shown. In samples processed by the IP method (green triangles), the trypsin-digested tau peptide from the MTBR region was detectable, but the signal was much weaker than that of the trypsin-digested tau peptide from the N-terminal to intermediate domains, and it was unquantifiable in human CSF from chronic neurodegenerative diseases, including AD, and from healthy volunteers. In contrast, these peptides were readily detectable in samples processed by the CX method (blue circles) or the PostIP-CX method (red squares).

[0026] [Figure 3B] Figure 3B illustrates how sample processing may affect the population of tau proteins detected by downstream methods. In the IP method (circled in green dotted line), tau species with N-terminal and intermediate domain epitopes recognized by antibodies (exemplified by HJ8.5 and Tau1, respectively) are immunoprecipitated. In the PostIP-CX method (circled in red dotted line), tau species present after immunoprecipitation do not have epitopes recognized by the antibodies used in immunoprecipitation (exemplified by the "MTBR-C" figure), or the epitopes are not accessible (exemplified by the figure of linear morphology tau). The CX method (circled in blue dotted line), performed without prior immunoprecipitation, yields a sample containing tau species derived from both the IP and PostIP-CX methods.

[0027] [Figure 4]Figure 4 shows a graph of pT217% (x-axis) versus Aβ42 / 40 concentration (y-axis) evaluated for LOAD100 and LOAD60 CSF samples. The horizontal dotted line defines the amyloid state as defined by the CSF Aβ42 / 40 concentration (CSF Aβ42 / 40 > 0.1389 = amyloid positive and CSF Aβ42 / 40 < 0.1389 = amyloid negative). As shown, p217% correlates very well with the amyloid state defined by this cutoff.

[0028] [Figure 5] Figure 5 shows graphs of two tau trypsin-digested peptides, TPPS and HVPG, quantified by mass spectrometry in CSF samples treated by the IP method described in Example 1 (IP_TPPS, left graph) or the PostIP-CX method described in Examples 1 and 2 (PostIP_HVPG, right graph). CSF samples are identified by CDR score and amyloid status. The graph between the two graphs shows the relative position of the trypsin-digested peptides in tau-441. NS - Not significant.

[0029] [Figure 6]Figure 6A is a graph showing the amounts of tau trypsin-digested peptide, HVPG versus Aβ42 / 40, in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. CSF samples were identified by amyloid status - amyloid-positive (red) or amyloid-negative (blue). The data demonstrate that the measurement of HVPG in CSF samples treated by the PostIP-CX method described in Examples 1 and 2 reproduces the amyloid status in the brain, as evidenced by the strong correlation with the amyloid status in terms of Aβ42 / 40. Figure 6B is a graph showing the amounts of tau trypsin-digested peptide, HVPG versus pT217%, in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. CSF samples were identified by amyloid status - amyloid-positive (red) or amyloid-negative (blue). The data demonstrate that the measurement of HVPG in CSF samples treated by the PostIP-CX method described in Examples 1 and 2 reproduces the amyloid state in the brain, as evidenced by the strong correlation with the amyloid state in terms of pT217%.

[0030] [Figure 7A] Figures 7A, 7B, and 7C are graphs showing the levels of three tau trypsin-digested peptides, LQTA (Figure 7A), HVPG (Figure 7B), and IGSL (Figure 7C), in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. CSF samples were grouped by CDR score and amyloid status. The data show that LQTA increases in amyloid-positive subjects compared to amyloid-negative subjects, even in the symptomatic stage; HVPG increases in amyloid-positive subjects compared to amyloid-negative subjects, especially in the asymptomatic stage; and IGSL increases in amyloid-positive subjects compared to amyloid-negative subjects and decreases after the symptomatic stage.

[0031] [Figure 7B] Same as above [Figure 7C] Same as above

[0032] [Figure 8A] Figures 8A, 8B, and 8C are graphs showing the amounts of three tau trypsin-digested peptides, LQTA (Figure 8A), HVPG (Figure 8B), and IGSL (Figure 8C), respectively, in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. Long-term samples from individual patients are shown along with their amyloid status, indicated by symbols (CDR0 = black circle, CDR0.5 = blue triangle, CDR1 = red square, CDR2 = purple inverted triangle). Paired t-tests were used for the results of the first and second visits of each participant. The amyloid-positive group showed significant changes in direction in each patient. The changes observed in participant A (indicated by the thick red line), who had a high tau-PET signal (>2 SUVR) and a change in CDR score from CDR1 to CDR2, are also noteworthy. In this patient, as tau pathology progressed, LQTA increased (Figure 8A), HVPG decreased (Figure 8B), and IGSL decreased (Figure 8C). [Figure 8B] Same as above [Figure 8C] Same as above

[0033] [Figure 9A] Figures 9A, 9B, and 9C are graphs showing the CDR-SB scores for the amount of three tau trypsin-digested peptides in CSF samples treated by the PostIP-CX method described in Examples 1 and 2, for the subjects from which the samples were obtained. The three tau trypsin-digested peptides are LQTA, HVPG, and IGSL, respectively. Amyloid-positive subjects are represented by blue circles, and amyloid-negative subjects by red squares. As shown in the accompanying statistical analysis, only LQTA shows a significant correlation with CDR-SB. [Figure 9B] Same as above [Figure 9C] Same as above

[0034] [Figure 10A]Figures 10A, 10B, and 10C are graphs showing the Mini-Mental State Examination (MMSE) scores of subjects for which samples were obtained, based on the amount of three tau trypsin-digested peptides in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. The three tau trypsin-digested peptides are LQTA, HVPG, and IGSL, respectively. Amyloid-positive subjects are represented by blue circles, and amyloid-negative subjects by red squares. As shown in the accompanying statistical analysis, only LQTA is significantly correlated with MMSE. [Figure 10B] Same as above [Figure 10C] Same as above

[0035] [Figure 11A] Figures 11A, 11B, and 11C are graphs showing the tau-PET scores of the three tau trypsin-digested peptides in CSF samples treated by the PostIP-CX method described in Examples 1 and 2, for the subjects from which the samples were obtained. The three tau trypsin-digested peptides are LQTA, HVPG, and IGSL, respectively. Amyloid-positive subjects are shown as blue circles, and amyloid-negative subjects as red squares. As shown in the accompanying statistical analysis, only LQTA significantly correlated with tau-PET. The other MTBR tau trypsin-digested peptides did not significantly correlate. [Figure 11B] Same as above [Figure 11C] Same as above

[0036] [Figure 12] Figures 12A and 12B are graphs showing the amount of tau trypsin-digested peptide, LQTA, versus CDR-SB (Figure 12A) and MMSE (Figure 12B) in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. The data were evaluated using two different measures of cognitive impairment, and LQTA showed a significant correlation with cognitive function.

[0037] [Figure 13A]Figure 13A is a graph showing the amount of tau trypsin digested peptide, LQTA, in samples treated by the IP method (x axis) and the PostIP-CX method (y axis). Amyloid-positive subjects are identified with red symbols, and amyloid-negative subjects are identified with blue symbols. As shown by the accompanying statistical analysis, only "MTBR-related LQTA" (measured in samples treated by the PostIP-CX method) increased in the amyloid-positive group compared to the amyloid-negative group.

[0038] [Figure 13B] Figures 13B and 13C are graphs showing the amount of LQTA, the trypsin-digested peptide of tau, in CSF samples treated by the PostIP-CX method or IP method described in Examples 1 and 2, respectively. CSF samples were grouped by CDR score and amyloid status. The data show that LQTA-specific features (i.e., linear increase after the symptomatic stage) were observed only in "MTBR-related LTQA". Data are shown as individual results (plots) and mean (bars). Significance in statistical testing: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS = not significant. Statistical differences were evaluated using a one-way ANOVA with multiple comparison correction using the Benjamin Hoshberg false positive rate (FDR) method, with the FDR set at 5%. [Figure 13C] Same as above

[0039] [Figure 14] Figure 14 is a graph showing receiver operating characteristic curves (ROCs) comparing the sensitivity and specificity of tau trypsin-digested peptides, LQTAs, measured by mass spectrometry following the IP method (blue (bottom) line) or the PostIP-CX method (red (top) line) for determining amyloid status. The curves show that PostIP-LQTA (MTBR-associated LQTA) identifies amyloid status better than IP-LQTA.

[0040] [Figure 15A]Figures 15A and 15B show that the IGSL to HVPG ratio enhances discriminative power. Figure 15A is a graph showing the amounts of tau trypsin-digested peptides, IGSL and LQTA, as a ratio (IGSL / LQTA), in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. CSF samples were grouped according to CDR score and amyloid status. Figure 15B is a graph showing the correlation between the IGSL / LQTA ratio and pT205%. pT205% was measured as previously described (Barthelemy, NR, Li, Y, Joseph-Mathurin, N. et al. Nat Med 26, 398-407 (2020)). IGSL / LQTA showed a very close correlation with pT205, which is regulated near the onset of AD. [Figure 15B] Same as above

[0041] [Figure 15C] Figures 15C and 15D show that the IGSL to HVPG ratio enhances discriminative power. Figure 15C is a graph showing the amounts of tau trypsin-digested peptide, IGSL, and HVPG, expressed as the ratio (IGSL / HVPG), in CSF samples treated by the PostIP-CX method described in Examples 1 and 2. CSF samples are grouped by CDR score and amyloid status. Figure 15D is a graph showing the correlation between the IGSL / LQTA ratio and pT217%. IGSL / HVPG shows a very close correlation with pT217, which reproduces the amyloid status. [Figure 15D] Same as above

[0042] [Figure 16]Figure 16 illustrates how tau pathology progresses through different phases of Alzheimer's disease. Measurements of four different soluble and insoluble tau species in participants with definitive Alzheimer's disease mutations revealed tau-related changes over approximately 40 years (x-axis), (y-axis), and showed differences based on disease stage and other measurable biomarkers. Starting with the progression of fibrillary amyloid pathology, phosphorylation at positions 217 (purple) and 181 (blue) begins to increase. As neuronal dysfunction increases (based on metabolic changes), phosphorylation at position 205 (green) increases along with soluble tau (orange). Finally, with the onset of neurodegeneration (based on brain atrophy and cognitive decline), Tau-PET concentrates (red) begin to progress, and phosphorylation at positions 217 and 181 begins to decrease. In summary, this highlights the close correlation between the dynamic and branching patterns of soluble and aggregated tau during the disease course and amyloid pathology.

[0043] [Figure 17] Figure 17 is a diagram of a theoretical model illustrating how accessibility to various regions of MTBR tau can change during Alzheimer's disease (AD) progression and why MTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK) are good surrogates of tau pathology throughout all AD stages. In the pre-symptomatic AD stage, brain tau aggregates are immature, allowing for broader protease access. MTBR tau species, including MTBR tau 243, MTBR tau 299, and MTBR tau 354, are secreted into the CSF. However, as the disease progresses, tau aggregates mature and form increasingly rigid cores, and protease access to MTBR tau 354, and subsequently MTBR tau 299, decreases, and MTBR species, including MTBR tau 354, and subsequently MTBR tau 299, are stabilized in the CSF. However, MTBR tau 243 remains exposed throughout all disease stages and is capable of protease digestion and release into the CSF. These three imbalances in CSF are observed as signs of brain tau aggregate formation. Note: Size differences between MTBR tau species are not depicted in this figure.

[0044] [Figure 18A] Figure 18A is a schematic diagram of the trypsin-digested peptides from tau (gray bars) quantified in Example 3, which are further described in Figures 18B and 18C.

[0045] [Figure 18B] Figures 18B and 18C are graphs showing that, compared to control brain extracts, aggregated Alzheimer's disease brain insoluble extracts are enriched with brain MTBR tau species including MTBR tau 243, 299, and 354, indicating that MTBR tau is specifically deposited in Alzheimer's disease brains. The graphs show (Figure 18B) the enrichment profiles of tau peptides from control and Alzheimer's disease brains (n=2, 6-8 brain region samples / group in the discovery cohort) and (Figure 18C) the enrichment profiles of tau peptides from control (amyloid-negative, n=8), very mild to moderate Alzheimer's disease (AD) (amyloid-positive, CDR=0.5-2, n=5), and severe AD brains (amyloid-positive, CDR=3, n=7) (total n=20 in the validation cohort). The relative abundance of tau peptides was quantified against intermediate domain (residues 181-190) peptides for internal normalization. Species containing the upstream region of the microtubule-binding domain (MTBR) domain (residues 243–254, MTBR tau 243) and repeating regions 2 (R2) through R3 and R4 (residues 299–317, MTBR tau 299 and 354–369, MTBR tau 354, respectively) were highly enriched in the insoluble fraction of Alzheimer's disease brains compared to controls, as assessed by CDR, and were particularly enriched by the clinical stage of disease progression. MTBR tau 299 and MTBR tau 354 are located inside the fibrous core, while MTBR tau 243 is located outside the Alzheimer's disease aggregate core (Fitzpatrick et al., 2017). Notably, residues 195–209 are reduced in Alzheimer's disease brains, likely due to high phosphorylation. Data are presented as Tukey box plots showing individual points for median, interquartile interval, minimum, maximum, and outliers. Significance in statistical tests: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. [Figure 18C] Same as above

[0046] [Figure 19] Figure 19A is a schematic diagram of the general binding sites of trypsin-digested peptides from tau (gray bars) and antibodies HJ8.5 and Tau1, which were quantified in Example 3 and further described in Figures 19B and 19C. Figure 19B is a graph showing the tau profile in control human CSF. Tau peptides in control human CSF from a cross-sectional cohort (n=30) of amyloid-negative and CDR=0 patients were quantified by Tau1 / FIJ8.5 immunoprecipitation focused on the N-terminal to intermediate domain tau. For quantification of species including the microtubule-binding region (MTBR) and C-terminal region, CSF samples were chemically extracted after immunoprecipitation and analyzed sequentially. Using the Tau1 / FIJ8.5 immunoprecipitation method (blue circles), peptide recovery decreased dramatically after residue 222, and therefore only the N-terminal to intermediate domain tau (residues 6-23 to 243-254) peptide was quantified by this method (Sato et al., 2018). In contrast, the chemical extraction method of CSF after immunoprecipitation (red square) allows for the quantification of the entire region, including the C-terminal region, from the MTBR at concentrations of 0.4–7 ng / mL. The data are shown as averages.

[0047] [Figure 20A]Figures 20A, 20B, and 20C are graphs showing the amounts of intermediate domain-independent MTBR tau 243 (Figure 20A), intermediate domain-independent MTBR tau 299 (Figure 20B), and intermediate domain-independent MTBR tau 354 (Figure 20C) in PostlP-CX CSF from a cross-sectional cohort. Intermediate domain-independent MTBR tau 243, intermediate domain-independent MTBR tau 299, and intermediate domain-independent MTBR tau 354 show different profiles for amyloid plaques and clinical dementia stage. Amyloid-negative CDR=0 (control, n=30), amyloid-positive CDR=0 (pre-symptomatic AD, n=18), amyloid-positive CDR=0.5 (very mild AD, n=28), amyloid-positive CDR>1 (mild to moderate AD, n=12), and amyloid-negative CDR>0.5 (non-AD cognitive impairment, n=12). Intermediate domain-independent MTBR tau 243 continues to increase with Alzheimer's disease progression throughout all clinical stages. Intermediate domain-independent MTBR tau 299 and intermediate domain-independent MTBR tau 354 concentrations continue to increase up to the very mild Alzheimer's disease stage (amyloid-positive and CDR=0.5), but then saturate (MTBR tau 299) or decrease (MTBR tau 354) at CDR>1. p-values ​​in red or blue text indicate significant increases or decreases, respectively. Data are presented as individual results (plots) and means (bars). Statistical significance in the statistical test: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS = Not significant.

[0048] [Figure 20B] Same as above [Figure 20C] Same as above

[0049] [Figure 21]Figures 21A, 21B, and 21C show graphs illustrating the long-term rate of change (ng / mL / year) of intermediate domain-independent MTBR tau 243 (Figure 21A), intermediate domain-independent MTBR tau 299 (Figure 21B), and intermediate domain-independent MTBR tau 354 (Figure 21C) in the CSF of amyloid-negative (-) or positive (+) patients (total n=28 from the long-term cohort). The amyloid-positive group is further divided into various CDR changes, including CDR=0~0 (n=7), CDR=0~0.5 (n=2), CDR=0~1 (n=1), CDR=0.5~0.5 (n=2), CDR=0.5~1 (n=1), and CDR=1~2 (n=1, participant A). Participants in the amyloid-negative group did not show significant long-term changes (mean value close to 0), but most participants in the amyloid-positive group showed long-term increases in MTBR tau concentration. Notably, participant A, who experienced the greatest change in post-clinical-onset cognitive changes (CDR=1-2), showed only an increase of CSF MTBR tau 243 as he progressed from mild AD (CDR=1) to moderate AD (CDR=2), while MTBR tau levels 299 and 354 decreased. These data suggest that CSF intermediate domain-independent MTBR tau species increase long-term with clinical progression of Alzheimer's disease.

[0050] [Figure 22-1] Figures 22A, 22B, and 22C are graphs showing (x-axis) Tau-PET (AV-1451) SUVR and (y-axis) intermediate domain-independent concentrations of (Figure 22A) MTBR tau-243, (Figure 22B) MTBR tau-299, and (Figure 22C) MTBR tau-354 in PostIP-CX CSF (n=15 controls and n=20 from the Alzheimer's disease (AD) Tau-PET cohort). White circles: controls, black squares: AD. Intermediate domain-independent MTBR tau-243 showed the most significant correlation with Tau-PET SUVR (r=0.7588, p<0.0001). These data indicate that CSF intermediate domain-independent MTBR tau species, including sequence number 3 (LQTAPVPMPDLK), are highly correlated with Tau-PET SUVR measurements of tau-enriched organisms, while other intermediate domain-independent MTBR tau regions show low correlation with tau-enriched organisms. [Figure 22-2] Same as above

[0051] [Figure 23A] Figures 23A and 23B are graphs showing that cellular MTBR tau 243, MTBR tau 299, and MTBR tau 354 were not enriched in soluble extracts of Alzheimer's disease brain compared to control brain extracts. Figure 23A shows the enrichment profiles of tau peptides from control and Alzheimer's disease brains (n=2 and 8-10 brain region samples / group in the discovery cohort), and Figure 23B shows the enrichment profiles of tau peptides from control (amyloid-negative, n=8), very mild to moderate Alzheimer's disease (AD) (amyloid-positive, CDR=0.5-2, n=5), and severe AD brain (amyloid-positive, CDR=3, n=7) in the validation cohort (total n=20). The relative peptide abundance of tau peptides was quantified against the intermediate domain (residues 181-190) peptide for internal normalization. No changes were observed in soluble tau species containing the microtubule-binding region (MTBR) domain, in contrast to insoluble MTBR tau species, which were increased in Alzheimer's brains (Figure 18). Data are presented as Tukey box plots showing individual points for median, interquartile interval, minimum, maximum, and outliers. Statistical significance: **p<0.01, *p<0.05. [Figure 23B] Same as above

[0052] [Figure 24]Figure 24 is a graph showing the correlation between MTBR tau 354 and tau 368 in brain insoluble extracts, suggesting that the different species are not distinguishable as tau pathology progresses. Mass spectrometry analysis of MTBR tau 354 and tau 368 species in brain insoluble extracts from controls and Alzheimer's disease patients was performed using discovery cohort samples (23 brain samples in total: 6 brain regions from Alzheimer's disease #1 participants, 8 brain regions from Alzheimer's disease #2 participants, 5 brain regions from control #1 participants, and 4 brain regions from control #2 participants). MTBR tau 354 (residues 354-369) and its cleavage morphology, and tau 368 (residues 354-368) showed close correlations in brain insoluble extracts (Spearman r = 0.9783).

[0053] [Figure 25-1] Figures 25A, 25B, 25C, 25D, 25E, and 25F are graphs showing the quantification of trypsin-digested peptides of MTBR tau in human CSF samples after processing by the PostIP-CX method and subsequent mass spectrometry (MS) analysis. Extracted MS chromatograms of intermediate domain-independent MTBR tau 243 (Figures 25A and 25D), intermediate domain-independent MTBR tau 299 (Figures 25B and 25E), and intermediate domain-independent MTBR tau 354 (Figures 25C and 25F) are shown. Human CSF was obtained from amyloid-positive and CDR=0.5 very mild Alzheimer's disease participants from a cross-sectional cohort. Figures 25A, 25B, and 25C show peaks from endogenous trypsin-digested peptides, while Figures 25D, 25E, and 25F show peaks from the internal standard (15N-labeled tau). The X and Y axes represent the retention time and MS intensity of each peak, respectively. [Figure 25-2] Same as above [Figure 25-3] Same as above

[0054] [Figure 26-1]Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26I, 26J, and 26K are graphs showing the concentrations of trypsin-digested peptides of N-terminal tau and intermediate-domain tau in human CSF after sample processing by IP method and subsequent MS analysis. N-terminal and intermediate-domain CSF tau species distinguish between very early dementia and normal, but do not correlate with dementia stage. Tau species and residue concentrations in CSF from groups within a cross-sectional cohort: (Figure 26A) 6-23, (Figure 26B) 25-44, (Figure 26C) 45-67, (Figure 26D) 68-87, (Figure 26E) 88-126, (Figure 26F) 151-155, (Figure 26G) 181-190, (Figure 26H) 195-209, (Figure 26I) 212-221, (Figure 26J) 226-230 and (Figure 26K) 243-254 (residue numbering based on tau 441). Amyloid-negative CDR=0 (control, n=29), amyloid-positive CDR=0 (pre-symptomatic AD, n=18), amyloid-positive CDR=0.5 (very mild AD, n=27), amyloid-positive CDR>1 (mild to moderate AD, n=12), and amyloid-negative CDR>0.5 (non-AD clinical impairment, n=12). These tau species were isolated using the Tau1 / FIJ8.5 immunoprecipitation method (IP method). p values ​​in red indicate significance. Data are shown as individual results (plots) and means (bars). Significance in statistical tests: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS = not significant. [Figure 26-2] Same as above [Figure 26-3] Same as above [Figure 26-4] Same as above [Figure 26-5] Same as above [Figure 26-6] Same as above

[0055] [Figure 27-1]Figures 27A, 27B, 27C, 27D, 27E, 27F, 27G, and 27H are graphs showing the concentrations of trypsin-digested peptides of MTBR tau in human CSF after sample processing by the PostIP-CX method and subsequent MS analysis. The characteristic Alzheimer's disease amyloid and clinical stage patterns are specific to intermediate-domain-independent MTBR tau species in CSF. Only intermediate-domain-independent MTBR tau 243 identifies further advanced clinical stages. Tau species and residue concentrations in CSF obtained by chemical extraction from immunoprecipitation-precipitated samples from participants in a cross-sectional cohort: (Figure 27A) 243-254 (MTBR tau 243), (Figure 27B) 260-267, (Figure 27C) 275-280, (Figure 27D) 282-290, (Figure 27E) 299-317 (MTBR tau 299), (Figure 27F) 354-369 (MTBR tau 354), (Figure 27G) 386-395 and (Figure 27H) 396-406 (residue numbering based on tau 441). Amyloid-negative CDR=0 (control, n=30), amyloid-positive CDR=0 (pre-symptomatic AD, n=18), amyloid-positive CDR=0.5 (very mild AD, n=28), amyloid-positive CDR>1 (mild to moderate AD, n=12), and amyloid-negative CDR>0.5 (non-AD clinical impairment, n=12). p-values ​​in red or blue indicate significant increases or decreases, respectively. Data are presented as individual results (plots) and means (bars). Statistical significance in the test: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS = not significant. [Figure 27-2] Same as above [Figure 27-3] Same as above

[0056] [Figure 28-1]Figures 28A, 28B, 28C, 28D, 28E, 28F, 28G, 28H, 28I, and 28J are graphs showing the concentrations of trypsin-digested peptides of N-terminal tau and intermediate-domain tau in human CSF after sample processing by the PostIP-CX method and subsequent MS analysis. N-terminal and intermediate-domain CSF tau species do not correlate with dementia stage, regardless of the purification method (see also Figure 26). Tau species, residue concentrations in CSF from groups within a cross-sectional cohort (Figure 28A) 6-23, (Figure 28B) 25-44, (Figure 28C) 45-67, (Figure 28D) 68-87, (Figure 28E) 88-126, (Figure 28F) 151-155, (Figure 28G) 181-190, (Figure 28H) 195-209, (Figure 28I) 212-221, and (Figure 28J) 226-230. Amyloid-negative CDR=0 (control, n=29), amyloid-positive CDR=0 (pre-symptomatic AD, n=18), amyloid-positive CDR=0.5 (very mild AD, n=27), amyloid-positive CDR>1 (mild to moderate AD, n=12), and amyloid-negative CDR>0.5 (non-AD clinical impairment, n=12). These tau species were isolated using the Tau1 / FIJ8.5 immunoprecipitation method followed by chemical extraction of post-immunoprecipitation CSF (PostIP-CX). The sum of concentrations from the immunoprecipitation method and the chemical extraction of post-immunoprecipitation CSF is shown for N-terminal to intermediate domain tau species (as total concentration). p values ​​in red indicate statistical significance. Data are shown as individual results (plots) and means (bars). Significance in statistical tests: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS=not significant. [Figure 28-2] Same as above [Figure 28-3] Same as above

[0057] [Figure 28-4] Same as above. Figure 28K is a graph showing the total concentration of tau species containing residues 243-254 (total concentration from IP method and PostIP-CX method).

[0058] [Figure 29]Figure 29 is a graph showing that intermediate domain-independent MTBR tau 354 correlates with intermediate domain-independent tau 368 in CSF. CSF was processed by the PostIP-CX method as generally described in Example 3. Intermediate domain-independent MTBR tau 354 (residues 354-369) and its cleavage morphology, and tau 368 (residues 354-368) show a close correlation (Spearman r = 0.8382). Results were obtained from a cross-sectional cohort sample (n = 100, including all clinical stages).

[0059] [Figure 30-1] Figures 30A, 30B, and 30C are graphs showing that CSF intermediate domain-independent MTBR tau 243 is highly dependent on the clinical dementia grade-box summation (CDR-SB), while intermediate domain-independent MTBR tau 299 and intermediate domain-independent MTBR tau 354 are not. The graphs show the CDR-SB correlation between (Figure 30A) intermediate domain-independent MTBR tau 243, (Figure 30B) intermediate domain-independent MTBR tau 299, and (Figure 30C) intermediate domain-independent MTBR tau 354 concentrations in CSF. Intermediate domain-independent MTBR tau 243 in CSF from the amyloid-positive group showed a high correlation with CDR-SB (r=0.5562, p<0.0001). Results were obtained from a cross-sectional cohort sample (amyloid-negative n=42 and amyloid-positive n=58). [Figure 30-2] Same as above

[0060] [Figure 31-1]Figures 31A, 31B, and 31C are graphs showing that CSF intermediate domain-independent MTBR tau 243 correlates more strongly with the Mini-Mental State Examination (MMSE) than intermediate domain-independent MTBR tau 299 and intermediate domain-independent MTBR tau 354. The graphs show the correlation of MMSE with concentrations of intermediate domain-independent MTBR tau 243 (Figure 31A), intermediate domain-independent MTBR tau 299 (Figure 31B), and intermediate domain-independent MTBR tau 354 (Figure 31C) in CSF. Intermediate domain-independent MTBR tau 243 in CSF from the amyloid-positive group showed a high correlation with MMSE (r=0.5433, p<0.0001). Results were obtained from a cross-sectional cohort sample (amyloid-negative n=42 and amyloid-positive n=58). [Figure 31-2] Same as above

[0061] [Figure 32-1] Figures 32A, 32B, and 32C are graphs showing the long-term increases in CSF intermediate domain-independent MTBR tau 243, intermediate domain-independent MTBR tau 299, and intermediate domain-independent MTBR tau 354 with clinical progression of Alzheimer's disease. Long-term changes in CSF intermediate domain-independent tau concentrations (Figure 32A) MTBR tau 243, (Figure 32B) MTBR tau 299, and (Figure 32C) MTBR tau 354 in amyloid-negative (-) or positive (+) patients are shown. Black circles: CDR=0, blue triangles: CDR=0.5, red squares: CDR=1, purple inverted triangles: CDR=2. Participants with a stable (or decreasing) CDR trajectory are shown by dotted lines. Participants with an increased CDR between the first and second visits are shown by solid lines. The thick red line in the amyloid-positive group shows the long-term trajectory of a specific participant (Participant A) who showed the greatest cognitive change after the onset of Alzheimer's disease (CDR=1-2), suggesting that CSF MTBR tau 243 increases from mild AD (CDR=1) to moderate AD (CDR=2). Statistical significance was evaluated by paired t-tests for the first and second visits (amyloid-negative n=14 and amyloid-positive n=14). **p<0.01. NS = not significant. [Figure 32-2] Same as above

[0062] [Figure 33] Figure 33 is a schematic diagram illustrating the method of the present invention.

[0063] [Figure 34A] Figures 34A, 34B, and 34C are graphs showing the amounts of trypsin-digested peptides LQTA (Figure 34A), HVPG (Figure 34B), and IGSL (Figure 34C) measured in CSF samples treated with the PostIP-CX method (top) versus the PostIP-CX method (bottom). [Figure 34B] Same as above [Figure 34C] Same as above

[0064] [Figure 35A] Figure 35A is a graph showing the amounts of trypsin digested peptides LQTA (left), IGST (center), and VQII (right) measured in samples treated by the PostIP-IP method (y-axis) versus the PostIP-CX method (x-axis). The top of the graph shows the relative position of each trypsin digested peptide in tau 441. Both axes show absolute concentrations (ng / mL).

[0065] [Figure 35B] Figure 35B is a graph showing the amounts of trypsin digested peptides LDLS (left), HVPG (center), and IGSL (right) measured in samples treated by the PostIP-IP method (y-axis) versus the PostIP-CX method (x-axis). The top of the graph shows the relative position of each trypsin digested peptide in tau 441. Both axes show absolute concentrations (ng / mL).

[0066] [Figure 36A]Figure 36A is a graph showing the amounts of trypsin-digested peptides LQTA (left), IGST (center), and VQII (right) measured in samples treated by the PostIP-IP method (y axis) versus the PostIP-CX method (x axis). The top of the graph shows the relative position of each trypsin-digested peptide in tau 441. Both axes show absolute concentrations (ng / mL). Samples obtained from control subjects are blue circles; samples obtained from amyloid-positive subjects without cognitive impairment (CDR<0.5) are red squares; and samples obtained from amyloid-positive subjects with cognitive impairment (CDR>0.5) are black triangles.

[0067] [Figure 36B] Figure 36B is a graph showing the amounts of trypsin digested peptides LDLS (left), HVPG (center), and IGSL (right) measured in samples treated by the PostIP-IP method (y axis) versus the PostIP-CX method (x axis). The top of the graph shows the relative position of each trypsin digested peptide in tau 441. Both axes show absolute concentrations (ng / mL). Samples obtained from control subjects are blue circles; samples obtained from amyloid-positive subjects without cognitive impairment (CDR<0.5) are red squares; and samples obtained from amyloid-positive subjects with cognitive impairment (CDR>0.5) are black triangles.

[0068] [Figure 37-1] Figure 37A shows various full-length tau isoforms. It illustrates the relative positions of several tau trypsin-digested peptides (e.g., LQTA, IGST, VQII, LDLS, HVPG, IGSL, VQIV). Each "Y" represents an antibody that specifically binds to the N-terminal (left), intermediate domain (center), and MTBR (right) regions. The major cleavage site of tau, amino acid 224 of tau 441, is shown as a dotted line.

[0069] [Figure 37-2]Figure 37B is a graph showing the ratios of trypsin-digested peptide VQIV and LDLS in samples obtained from control subjects (left, blue circles) and subjects with non-AD tauopathy (right, green circles), as determined by LC-MS after sample processing by the IP method described in Example 1. ns: Not significant; Tukey's multiple comparison test. Figure 37C is a graph showing the ratios of trypsin-digested peptide VQIV and LDLS in samples obtained from control subjects (left, blue circles) and subjects with non-AD tauopathy (right, green circles), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4.

[0070] [Figure 38] Figure 38 is a graph showing the amounts of trypsin-digested peptide VQIV (x axis) and LDLS (y axis) in samples obtained from control subjects (blue circles) and subjects with non-AD tauopathy (green triangles), as determined by LC-MS after sample processing using the PostIP-IP method described in Example 4. **** p<0.0001; Tukey's multiple comparison test.

[0071] [Figure 39] Figure 39 is a graph showing the ratios of trypsin-digested peptide VQIV and LDLS in samples obtained from control subjects (left, blue circles), subjects with AD (center, red circles), and subjects with non-AD tauopathy (right, green circles), as determined by LC-MS after sample processing using the PostIP-IP method described in Example 4. ns: Not significant;**** p<0.0001; Tukey's multiple comparison test.

[0072] [Figure 40-1]Figures 40A, 40B, 40C, 40D, 40E, and 40F are graphs showing a comparison of tau trypsin-digested peptides in samples obtained from control subjects (blue circles), subjects with AD (red squares), and subjects with non-AD tauopathy (green triangles), determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. In Figure 40A, the tau trypsin-digested peptides are IGST (x axis) and VQII (y axis); in Figure 40B, LDLS (x axis) and VQII (y axis); in Figure 40C, IGSL (x axis) and HVPG (y axis); in Figure 40E, IGST (x axis) and HPVG (y axis); in Figure 40E, VQII (x axis) and HPVG (y axis); and in Figure 40F, IGST (x axis) and HPVG (y axis). Both axes show absolute concentration (ng / mL).

[0073] [Figure 40-2] Same as above [Figure 40-3] Same as above

[0074] [Figure 41] Figure 41 is a graph comparing the amounts of tau trypsin-digested peptides IGST versus HVPG (upper left), VQII versus HVPG (upper right), and LDLS versus HVPG (lower) in samples obtained from subjects with non-AD tauopathy, as determined by LC-MS after sample processing using the PostIP-IP method described in Example 4. The key legend on the right indicates the diagnosis of non-AD tauopathy for each subject. Both axes show absolute concentrations (ng / mL).

[0075] [Figure 42]Figures 42A, 42B, and 42C are graphs showing the comparison of the amounts of tau trypsin digested peptides IGST vs. IGSL (Figure 42A), VQII vs. IGSL (Figure 42B), and LDLS vs. IGSL (Figure 42C) in samples obtained from control subjects (blue circles), subjects with AD (red squares), and subjects with non-AD tauopathy (green triangles) as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. Both axes show absolute concentration (ng / mL).

[0076] [Figure 43-1] Figure 43A is a diagram of the MTBR region of tau. The relative positions of the trypsin digested peptides IGST, VQII, LDLS, HVPG, and IGSL are shown as the relative positions of the epitopes to which antibody 77G7 specifically binds.

[0077] [Figure 43-2] Figures 43B, 43C, 43D, and 43E are graphs showing the ratios of tau trypsin-digested peptides IGSL / IGST (Figure 43B), IGSL / VQII (Figure 43C), IGSL / LDLS (Figure 43D), and IGSL / HVPG (Figure 43E) in samples obtained from non-AD subjects (left bars) and subjects with AD (right bars), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. For non-AD subjects, blue indicates subjects with PSP, green indicates subjects with FTD, red indicates subjects with CBD, and purple indicates subjects with continuous PSP-CBD. Statistical significance was determined by unpaired t-tests and Welch's correction. [Figure 43-3] Same as above

[0078] [Figure 44-1]Figures 44A, 44B, 44C, 44D, and 44E are graphs showing a comparison of the amounts of tau trypsin digested peptides IGSL vs. IGST (Figure 44A), IGSL vs. VQII (Figure 44B), IGSL vs. LDLS (Figure 44C), VQII vs. IGST (Figure 44D), VQII vs. LDLS (Figure 44E), and IGSL vs. HVPG (Figure 43E) in samples obtained from non-AD subjects (blue) and subjects with AD (red), determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. In Figures 44A, 44B, and 44C, non-AD subjects show a scatter plot, while in Figures 44D, 44E, and 44F, AD and non-AD showed the same correlation. [Figure 44-2] Same as above

[0079] [Figure 45] Figure 45 shows the correlation of various tau trypsin-digested peptides measured in CSF from subjects with AD and non-AD tauopathy. The boxed-up data suggest a branching point that distinguishes AD from non-AD tauopathy.

[0080] [Figure 46] Figure 46 shows a hypothesis on how CSF tau distinguishes non-AD tauopathy. As described, in CSF, non-AD tauopathy is characterized by (1) less R1-R2 and (2) more R3-R4 than AD, as signs of cerebral tau deposition.

[0081] [Figure 47] Figure 47 is a graph showing the amounts of various trypsin-digested peptides in brain-insoluble tau.

[0082] [Figure 48-1]Figures 48A, 48B, 48C, and 48D are graphs showing the ratios of tau trypsin digested peptides IGSL / IGST (Figure 48A), IGSL / VQII (Figure 48B), IGSL / LDLS (Figure 48C), and IGSL / HVPG (Figure 48D) in samples obtained from control subjects (left bar), subjects with AD (center bar), and non-AD subjects (right bar), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. For non-AD subjects, black triangles indicate subjects with genetically confirmed P301L FTLD (4R-tauopathy), and black squares indicate subjects with genetically confirmed R406W FTLD (3R / 4R mix). [Figure 48-2] Same as above

[0083] [Figure 49-1] Figures 49A, 49B, 49C, 49D, 49E, and 49F are graphs showing the comparison of the amounts of tau trypsin digested peptides IGSL vs. IGST (Figure 49A), IGSL vs. VQII (Figure 49B), IGSL vs. LDLS (Figure 49C), VQII vs. IGST (Figure 49D), VQII vs. LDLS (Figure 49E), and IGSL vs. HVPG (Figure 43F) in CSF samples from control subjects (blue), subjects with AD (red), and non-AD subjects (green), as determined by LC-MS after sample processing by the PostIP-IP method described in Example 4. In Figures 49A, 49B, and 49C, the non-AD subjects show a scatter plot, while in Figures 49D, 49E, and 49F, the control, AD, and non-AD subjects showed the same correlation. [Figure 49-2] Same as above [Modes for carrying out the invention]

[0084] Detailed description MTBR tau exists as multiple peptides in blood and CSF. Detection and quantification of MTBR tau in these biological samples is hindered because these polypeptides are present in very small amounts. The methods disclosed herein utilize a specific combination of processing steps to transform a biological sample into a sample suitable for the quantification of MTBR tau and other tau species. For example, in one method of the present invention, the processing step depletes a certain protein while simultaneously enriching multiple tau proteins. In another method of the present invention, the processing step depletes a certain protein while simultaneously enriching multiple MTBR tau proteins. One method disclosed herein is particularly suitable for the quantification of intermediate domain-independent MTBR tau species. The use of intermediate domain-independent MTBR tau species for clinical sign and symptom assessment of tauopathy, tauopathy diagnosis, and tauopathy treatment instruction is also described. These and other aspects and iterations of the present invention are described in more detail below.

[0085] I. Definition To make the present invention more easily understandable, some terms are defined first. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which embodiments of the present invention belong. Many similar, modified, or equivalent methods and materials can be used in carrying out embodiments of the present invention without excessive experimentation, and preferred materials and methods are described herein. In describing and claiming embodiments of the present invention, the following terms are defined and used as follows:

[0086] The term "approximately" as used herein includes, but is not limited to, any quantitative variation in mass, volume, time, distance, and quantity, including variations in quantity that may occur, for example, in common measuring techniques and apparatus. Furthermore, when assuming a certain method of handling solids and liquids in actual use, there may be variations due to certain careless errors and variations due to differences in the manufacture, source, or purity of the components used in the manufacture of a composition or the implementation of a method. The term "approximately" also includes these variations, which may be up to ±5%, and could be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "approximately," the claims include equivalent values ​​of quantities.

[0087] The antibodies used herein refer to complete antibodies as understood in this art, i.e., antibodies consisting of two heavy chains and two light chains, and also include, but are not limited to, antibody fragments such as Fab', Fab, F(ab')2, single-domain antibodies, Fv, and single-chain Fv, as well as any antibody-like molecules having an antigen-binding domain. The term antibody also refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies. Techniques for producing and using various antibody-based constructs and fragments are well known in this art. Means for producing and characterizing antibodies are also well known in this art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; which is incorporated herein in whole by reference).

[0088] The term "aptamer" as used herein refers to a polynucleotide, generally RNA or DNA, that possesses useful biological activity in terms of biochemical activity, molecular recognition, or binding attributes. Typically, aptamers have molecular activity such as binding to a target molecule at a specific epitope (region). It is generally recognized that aptamers specific to binding to polypeptides can be synthesized and / or identified by in vitro evolutionary methods. Means for producing and characterizing aptamers, including by in vitro evolutionary methods, are well known in the art. See, for example, US7,939,313, which is incorporated herein in whole by reference.

[0089] The term "Aβ" refers to peptides derived from the carboxyl-terminal region of a large protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are numerous Aβ forms that can have toxic effects: Aβ peptides are generally 37-43 amino acid sequences long, but can undergo shortening and modification that alters their overall size. They can be found intracellular or extracellularly in monomeric, oligomeric, and aggregated forms in soluble and insoluble compartments and can complex with other proteins or molecules. The harmful or toxic effects of Aβ may be due to any or all of the above forms, as well as others not specifically described. For example, two such Aβ isoforms include Aβ40 and Aβ42, with the Ap42 isoform being particularly fibrinogenic or insoluble and associated with disease states. The term "Aβ" generally refers to multiple Aβ species without distinguishing individual Aβ species. Specific Aβ species are identified by peptide size, e.g., Aβ42, Aβ40, Aβ38, etc.

[0090] The term "Aβ42 / Aβ40 value" used here refers to the ratio obtained by comparing the amount of Aβ42 in a sample from a subject with the amount of Aβ40 in the same sample.

[0091] Aβ amyloidosis is defined as clinically abnormal Aβ deposition in the brain. Subjects determined to have Aβ amyloidosis are referred to here as "amyloid-positive," while subjects determined not to have Aβ amyloidosis are referred to here as "amyloid-negative." There are recognized indicators of Aβ amyloidosis in this art. As of the present invention, Aβ amyloidosis is measured directly by amyloid imaging (e.g., PiB PET, florbetapir, or other contrast imaging methods known in this art) or indirectly by a decrease in cerebrospinal fluid (CSF) Aβ42 or a decrease in the CSF Aβ42 / 40 ratio. Mean cortical connectivity (MCBP) score > 0.18 11[C]PIB-PET contrast is an indicator of Aβ amyloidosis, similar to cerebrospinal fluid (CSF) Aβ42 concentration of approximately 1 ng / ml measured by immunoprecipitation and mass spectrometry (IP / MS). Alternatively, a CSF Aβ42 / 40 cutoff ratio can be used to maximize the accuracy of amyloid-positive predictions determined by PIB-PET. These values, or those used in other and / or examples known in the art, may be used alone or in combination for clinical confirmation of Aβ amyloidosis. For example, see Klunk WE et al. Ann Neurol 55(3) 2004, Fagan AM et al. Ann Neurol, 2006, 59(3), Patterson et al., Annals of Neurology, 2015, 78(3): 439-453 or Johnson et al., J. Nuc. Med., 2013, 54(7): 1011-1013, respectively, which are incorporated herein by reference in their entirety. Subjects with Aβ amyloidosis may or may not be symptomatic, and symptomatic subjects may or may not meet the clinical criteria for the disease associated with Aβ amyloidosis. Non-limiting examples of symptoms associated with Aβ amyloidosis may include cognitive impairment, behavioral changes, abnormal language function, emotional dysregulation, seizures, dementia, and structural or functional impairment of the nervous system. Diseases associated with Aβ-amyloidosis include, but are not limited to, Alzheimer's disease (AD), cerebral amyloid vascular disease (CAA), Lewy body dementia, and inclusion body myositis. Individuals with Aβ-amyloidosis have an increased risk of developing diseases associated with Aβ-amyloidosis.

[0092] "Clinical signs of Aβ amyloidosis" refers to the measurement of Aβ deposition as known in this field. Clinical signs of Aβ amyloidosis may include, but are not limited to, Aβ deposition identified by amyloidography (e.g., PiB PET, florbetapir, or other contrast methods known in this field) or by a decrease in cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio. See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, and Fagan AM et al. Ann Neurol 59(3) 2006, which are incorporated herein by reference in their entirety. The clinical signs of Aβ amyloidosis may also include measurements of Aβ metabolism, particularly measurements of Aβ42 metabolism alone or in comparison to measurements of other Aβ variants (e.g., Aβ37, Aβ38, Aβ39, Aβ40 and / or total Aβ), as described in U.S. Patent Applications 14 / 366,831, 14 / 523,148 and 14 / 747,453, which are incorporated herein by reference as a whole. Further methods are described in Albert et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 170-179; McKhann et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 263-269; and Sperling et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 280-292, respectively, which are incorporated herein by reference in their entirety. Importantly, subjects with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition. Nevertheless, subjects with clinical signs of Aβ amyloidosis have an increased risk of developing diseases associated with Aβ amyloidosis.

[0093] A "candidate for amyloidography" refers to a subject identified by a physician as an individual for whom amyloidography may be clinically justifiable. In non-limiting examples, amyloidography candidates may include subjects with one or more clinical signs of Aβ-amyloidosis, one or more Aβ-plaque-related symptoms, one or more CAA-related symptoms, or a combination thereof. A physician may recommend amyloidography and direct clinical care for such subjects. In other non-limiting examples, amyloidography candidates may include potential trial participants (control subjects or study subjects) for diseases associated with Aβ-amyloidosis.

[0094] "Aβ plaque-related symptoms" or "CAA-related symptoms" refer to any symptoms caused by or associated with the formation of amyloid plaques or CAA, respectively, which consist of regularly arranged fibrillary aggregates called amyloid fibrils. Examples of Aβ plaque-related symptoms may include, but are not limited to, neuronal degeneration, cognitive impairment, memory impairment, behavioral changes, emotional dysregulation, seizures, structural or functional impairment of the nervous system, and an increased risk of developing or worsening Alzheimer's disease or CAA. Neuronal degeneration may include structural changes in neurons (including molecular changes such as intracellular accumulation of toxic proteins and protein aggregates, and macroscopic changes such as changes in the shape or length of axons or dendrites, changes in myelin composition, loss of myelin, etc.), changes in neuronal function, loss of neuronal function, neuronal death, or any combination thereof. Cognitive impairment may include, but is not limited to, difficulties with memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and order. Behavioral changes may include, but are not limited to, violent behavior and language, impulsivity, decreased inhibition, apathy, decreased initiative, personality changes, alcohol, tobacco or substance abuse, and other addiction-related behaviors. Affective dysregulation may include, but are not limited to, depression, anxiety, mania, irritability, and emotional lability. Seizures may include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic and psychogenic seizures. Nervous system structural or functional disorders may include, but are not limited to, hydrocephalus, Parkinson's disease, sleep disorders, psychosis, and balance and coordination disorders. This may include motor dysfunctions such as monoparesis, hemiparesis, quadriplegia, ataxia, ballism, and tremor. This may also include loss or dysfunction of sensation, including olfactory, tactile, gustatory, visual, and auditory sensations. Furthermore, this may include autonomic nervous system dysfunctions such as bowel and bladder dysfunction, sexual dysfunction, and blood pressure and thermoregulatory dysfunction. Finally, this may include hormonal dysfunction resulting from hypothalamic and pituitary dysfunction, such as deficiencies and dysregulation of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin, and numerous other hormones and modulators.

[0095] The term "subject" as used herein means a mammal, preferably a human. Mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be awaiting medical treatment, under medical treatment, or receiving medical treatment.

[0096] The terms "control group," "normal group," or "healthy subjects" used herein refer to subjects or groups of subjects that have been clinically determined, based on qualitative or quantitative test results, to be free from tauopathy or Aβ amyloidosis or diseases associated with clinical Aβ amyloidosis (including, but not limited to, Alzheimer's disease).

[0097] The term "blood sample" as used herein refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. A blood sample may be whole blood, plasma, or serum, but plasma is generally preferred.

[0098] The term "isoform" as used herein refers to any of several different forms of the same protein variant resulting from alternative splicing of protein-coding mRNA, post-translational modification of proteins, proteolytic treatment of proteins, genetic mutation, and somatic recombination. The terms "isoform" and "variant" are interchangeable.

[0099] The term "tau" refers to the multiple isoforms and species encoded by the MAPT gene (or its homolog) that have undergone in vivo C-terminal cleavage, in vivo N-terminal cleavage, in vivo post-translational modification, or any combination thereof. The terms "tau," "tau protein," and "tau species" used herein are interchangeable. Tau is encoded by the MAPT gene in many animals, including but not limited to humans, non-human primates, rodents, fish, cattle, frogs, goats, and chickens. For animals where the gene has not been identified as MAPT, homologs can be identified by methods well known in this art.

[0100] In humans, there are six isoforms of tau produced by alternative splicing of MAPT exons 2, 3, and 10. These isoforms range in length from 352 to 441 amino acids. Exons 2 and 3 each encode a 29-amino acid insert at their N-terminus (referred to as N), and full-length human tau isoforms may have both inserts (2N), one insert (1N), or no insert (0N). All full-length human tau isoforms also have three repeats of a microtubule-binding domain (referred to as R). Insertion of exon 10 into the C-terminus leads to the insertion of a fourth microtubule-binding domain encoded by exon 10. Therefore, full-length human tau isoforms have either four repeats of the microtubule-binding domain (exon 10 contains R1, R2, R3, and R4) or three repeats of the microtubule-binding domain (exon 10 contains R1, R3, and R4). Human tau may or may not be post-translationally modified. For example, it is known in this field that tau can be phosphorylated, ubiquitinated, glycosylated, and glycated. Human tau can also be proteolytically treated in vivo at the C-terminus, N-terminus, or both C-terminus and N-terminus. Thus, the term "human tau" includes the 2N3R, 2N4R, 1N3R, 1N4R, 0N3R, and 0N4R isoforms as well as species that have undergone C-terminal cleavage in vivo, N-terminal cleavage in vivo, post-translational modification in vivo, or any combination thereof. Alternative splicing of the gene encoding tau occurs similarly in other animals.

[0101] The term "tau-441" as used herein refers to the longest human tau isoform (2N4R) with a length of 441 amino acids. The amino acid sequence of tau-441 is provided as Sequence ID No. 1. The N-term, intermediate domain, MTBR, and C-term are shown in Figure 1 for this isoform. These regions vary in a predictable manner for other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R, and 0N3R). Therefore, when the amino acid positions are identified for tau-441, those skilled in the art can determine the corresponding amino acid positions for other isoforms.

[0102] The term "N-terminal tau" as used herein refers to one or more tau proteins that contain two or more amino acids at the N-terminus of tau (for example, amino acids 1-103 of tau-441).

[0103] The term "intermediate domain tau" as used herein refers to one or more tau proteins that contain two or more amino acids from the intermediate domain of tau (for example, amino acids 104-243 of tau 441).

[0104] The term "MTBR tau" as used herein refers to one or more tau proteins that contain two or more amino acids from the microtubule-binding region (MTBR) of tau (for example, amino acids 244-368 of tau 441).

[0105] The term "C-terminal tau" as used herein refers to one or more tau proteins that contain two or more amino acids at the C-terminus of tau (for example, amino acids 369-441 of tau-441).

[0106] "Tau proteolytic peptides" refer to peptide fragments of the tau protein produced by in vitro proteolytic cleavage. "Tau trypsin-digested peptides" refer to peptide fragments of the tau protein produced by in vitro cleavage with trypsin. Tau trypsin-digested peptides may be indicated here by their first four amino acids. For example, "LQTA" refers to the trypsin-digested peptide LQTAPVPMPDLK (SEQ ID NO: 3). Non-limiting examples of other trypsin-digested peptides identified by their first four amino acids include IGST (SEQ ID NO: 2), VQII (SEQ ID NO: 4), LDLS (SEQ ID NO: 5), HVPG (SEQ ID NO: 6), IGSL (SEQ ID NO: 7), VQIV (SEQ ID NO: 9), and TPPS (SEQ ID NO: 10).

[0107] Diseases associated with tau deposition in the brain are referred to here as "tauopathies." The term "tau deposition" includes all forms of pathological tau deposition, including but not limited to neurofibrillary condensations, villous filaments, and tau aggregates in degenerated neurites. Tauopathies known in this field include, but are not limited to, progressive supranuclear palsy (PSP), boxer's dementia, chronic traumatic encephalopathy, frontotemporal dementia and Parkinson's disease associated with chromosome 17, Ritico-Bodigg disease, Guam's Parkinson's dementia complex, neurofibrillary tangle dementia, ganglioglioma and gangliocytoma, meningeal hemangioma, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Haller-Holden-Spats disease, lipofuscinosis, Pick's disease, corticobasal degeneration (CBD), argyrophilic granule disease (AGD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and frontotemporal dementia (FTD).

[0108] Tauopathies are classified according to the dominance of tau isoforms found in pathological tau deposits. Tauopathies consisting of tau deposits primarily with three MTBRs are called "3R-tauopathies." Pick's disease is a non-limiting example of a 3R-tauopathy. For clarity, pathological tau deposits in some 3R-tauopathies may be a mixture of 3R and 4R tau isoforms with a 3R isoform dominance. Intracellular neurofibrillary enrichment (i.e., tau deposits) in the brains of individuals with Alzheimer's disease are generally thought to contain approximately equal amounts of both 3R and 4R isoforms. Tauopathies consisting of tau deposits primarily with four MTBRs are called "4R-tauopathies." PSP, CBD, and AGD are non-limiting examples of 4R-tauopathies, as are some forms of FTLD. Notably, pathological tau deposits in the brains of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, show a mixture of 3R and 4R isoforms.

[0109] The clinical signs of tauopathy may include, but are not limited to, tau aggregates in the brain, including neurofibrillary lumps. Methods for detecting and quantifying tau aggregates in the brain are known in this field (e.g., Tau-PET using tau-specific ligands such as [18F]THK5317, [18F]THK5351, [18F]AV1451, [11C]PBB3, [18F]MK-6240, [18F]RO-948, [18F]PI-2620, [18F]GTP1, [18F]PM-PBB3 and [18F]JNJ64349311, [18F]JNJ-067).

[0110] As used herein, the term “treatment” or “to treat” refers to the provision of medical care by a trained and licensed professional to a person in need of treatment. Medical care may include diagnostic tests, therapeutic treatments, and / or preventive or protective measures. The objective of therapeutic and preventive treatments is the prevention or slowing (reduction) of undesirable physiological changes or disease / disorder. Beneficial or desired clinical outcomes of therapeutic or preventive treatments include, but are not limited to, symptom relief, reduction of disease severity, stabilization of the disease state (i.e., no worsening), slowing or deceleration of disease progression, improvement or mitigation and remission of the disease state (whether partial or total), whether detectable or undetectable. “Treatment” may also mean an extension of survival compared to life expectancy without treatment. Those in need of treatment include those who already have a disease, condition or disorder, as well as those who are predisposed to or should be prevented from developing a disease, condition or disorder. Thus, those in need of treatment may or may not have any symptoms or clinical signs of the disease.

[0111] The term “tau therapy” collectively refers to any contrast agents, therapeutic procedures, and / or prophylactic or preventive measures intended for or used in subjects at risk of developing tauopathy or clinically diagnosed with tauopathy. Non-exclusive examples of contrast agents include functional contrast agents (e.g., fluorodeoxyglucose) and molecular contrast agents (e.g., Pittsburgh compound B, florbetaben, florbetapyr, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies).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-Aβ antibodies (including their antigen-binding fragments, variants, or derivatives), anti-tau antibodies (including their antigen-binding fragments, variants, or derivatives), stem cells, and nutritional therapy. 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, p38 alpha-MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthioninium chloride), and treatments to improve blood glucose control (e.g., insulin, exena). (e.g., liraglutide, pioglitazone), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, β-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A reverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, This includes, but is not limited to, glutaminyl-peptide cyclotransferase inhibitors, APP-producing selective inhibitors, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, neuroproliferative factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.

[0112] II. Tau Measurement Method The present invention provides a method for measuring tau in a biological sample by mass spectrometry. Generally speaking, the present invention's method for measuring tau in a biological sample involves processing the biological sample by preparing it, depleting one or more proteins, then purifying the tau, cleaving the purified tau with a protease, then optionally desalting the resulting cleavage product by solid-phase extraction to obtain a sample containing tau proteolytic peptides, and performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect at least one tau proteolytic peptide and measure its concentration (relative or absolute). Thus, in practice, the disclosed method uses at least one tau proteolytic peptide to detect and measure the amount of tau present in a biological sample.

[0113] In one example, the method of the present invention comprises (a) preparing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by protein precipitation and separating the precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then desalting the cleavage products obtained optionally by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect at least one tau proteolytic peptide and measure its concentration.

[0114] In other examples, the present invention includes (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in a biological sample, such as a blood sample or a CSF sample, by affinity depletion; (b) (i) removing further proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, and then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purifying MTBR tau, thereby producing (i) or (ii) enriched tau. The method further comprises: (c) enriching the tau remaining after affinity depletion, which may be called N-terminal independent tau and / or intermediate domain independent tau; (d) cleaving the enriched tau with a protease, and then optionally desalting the cleavage product obtained by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry (LC / MS) on the sample containing tau proteolytic peptides to detect at least one of the tau proteolytic peptides and to measure its concentration.

[0115] In other examples, the method of the present disclosure includes (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in a biological sample, such as a blood sample or a CSF sample, by affinity depletion; (b) removing further proteins from the biological sample by protein precipitation and separation of precipitated proteins to obtain a supernatant; (c) purifying tau from the supernatant by solid-phase extraction; (d) cleaving the purified tau with a protease, and then desalting the cleavage product obtained optionally by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry on the sample containing tau proteolytic peptides to detect at least one of the tau proteolytic peptides and to measure their concentration.

[0116] In other examples, the method of the present invention comprises (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in a biological sample, such as a blood sample or a CSF sample, by affinity depletion; (b) affinity purification of MTBR tau; (c) cleavage of the purified MTBR tau with a protease, and then desalting the cleavage product obtained optionally by solid-phase extraction to obtain a sample containing MTBR tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry on the sample containing MTBR tau proteolytic peptides to detect at least one of the MTBR tau proteolytic peptides and measure their concentration.

[0117] In other examples, the method of the present invention comprises (a) affinity purification of MTBR tau from a biological sample, such as a blood sample or a CSF sample; (b) cleavage of the purified MTBR tau with a protease, and then desalting the optionally obtained cleavage product by solid-phase extraction to obtain a sample containing MTBR tau proteolytic peptides; and (c) performing liquid chromatography-mass spectrometry on the sample containing MTBR tau proteolytic peptides to detect at least one of the MTBR tau proteolytic peptides and to measure their concentration.

[0118] The present invention further intends, in any of the above methods, to determine the presence or absence of one or more proteins in a biological sample and / or to measure the concentration of one or more further proteins in a biological sample. In some embodiments, the one or more proteins may be proteins depleted from the biological sample before tau purification. For example, in some embodiments, N-terminal tau and / or intermediate domain tau species may be identified and / or quantified separately from tau species (e.g., MTBR tau, C-terminal tau) by the quantification disclosed herein. Separately or in addition thereto, Aβ, ApoE, or any other protein of interest may be identified and / or quantified by depleting the protein of interest from the biological sample before use by the methods disclosed herein, or by depleting the protein of interest from the biological sample during the sample processing steps disclosed herein, by processing a portion of the biological sample in parallel.

[0119] The steps of biological sample preparation, appropriate internal standards, depletion of one or more proteins, tau purification, cleavage of purified tau with protease, and mass spectrometry are described in further detail below.

[0120] Biological samples Suitable biological samples include blood or cerebrospinal fluid (CSF) samples obtained from a subject. In one embodiment, the subject is a human. A human subject may be awaiting medical treatment or procedure, may be under medical treatment or procedure, or may be receiving medical treatment or procedure. In various embodiments, a human subject may be a healthy subject, a subject at risk of developing neurodegenerative disease, a subject with signs and / or symptoms of neurodegenerative disease, or a subject diagnosed with neurodegenerative disease. In further embodiments, the neurodegenerative disease may be a tauopathy. Specifically, a tauopathy may be Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), or frontotemporal lobar degeneration (FTLD). In other embodiments, the subject is an experimental animal. In further embodiments, the subject is an experimental animal genetically engineered to express human tau and optionally one or more additional human proteins (e.g., human Aβ, human ApoE, etc.).

[0121] CSF can be obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple blood or CSF samples collected simultaneously from a subject may be stored. Blood may be collected by venous puncture with or without an intravenous catheter or by fingertip collection (or equivalent procedures). Once collected, blood or CSF samples may be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cell debris; use of additives prepared to stabilize and preserve the specimens before analytical testing). Blood or CSF samples may be used immediately or frozen and stored indefinitely. Before use in the methods disclosed herein, biological samples may be processed, if necessary or desired, to add protease inhibitors, isotope-labeled internal standards, surfactants and chaotropic agents and / or to deplete other components (e.g., protein peptides, metabolites).

[0122] The sample size used may vary depending on the sample type, the health status of the subject from which the sample was obtained, and the specimen to be analyzed (in addition to tau). CSF sample volume may range from approximately 0.01 mL to 5 mL or approximately 0.05 mL to 5 mL. In specific examples, the sample size may be approximately 0.05 mL to 1 mL CSF. Plasma sample volume may range from approximately 0.01 mL to 20 mL.

[0123] (b) Isotope labeling, internal tau standard Isotope-labeled tau can be used as an internal standard to explain variations during sample processing and, if desired, to calculate absolute concentrations. Generally, the isotope-labeled internal tau standard may be added before significant sample processing, and may be added one or more times if necessary. See, for example, the methods described in Figures 2 and 33.

[0124] Several isotopically labeled internal tau standards are described here. All incorporate a heavy isotope label at least one amino acid residue. One or more full-length isoforms may be used. Separately or in addition to these, post-translationally modified tau isoforms and / or tau peptide fragments may also be used, as known in this art. Generally, the incorporated labeled amino acid residue increases the mass of the peptide without affecting its chemical properties, and the mass shift resulting from the presence of the isotopic label must be sufficient to distinguish the internal standard (IS) from the endogenous tau sample signal by mass spectrometry. As shown here, suitable heavy isotope labels are: 2 H, 13 C and 15 This includes, but is not limited to, N. Generally, an internal standard of about 1-10 ng is sufficient.

[0125] (c) Depletion of 1 or more proteins The method of the present invention comprises the step of depleting one or more proteins from a sample. The term "depletion" means a quantitative or numerical reduction. Thus, a protein-depleted sample may have an amount of protein that is measurably less than the amount in the original sample.

[0126] Proteins can be depleted from a sample 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 when downstream analysis of the protein is desired (e.g., identification, quantification, and analysis of post-translational modifications). For example, Aβ peptides can be identified and quantified by methods known in the art after affinity depletion of Aβ with a suitable epitope binder. As another non-limiting example, the apolipoprotein E (ApoE) state can be determined by methods known in the art after affinity depletion of ApoE and identification of ApoE isoforms. Targeted depletion includes, but is not limited to, apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, interleukin, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, NFL, prion protein, pNFH, and DJ-1, and can also be used for isolation for subsequent analysis of other proteins. Targeted depletion of certain tau proteins is also used here for enrichment of other tau proteins and / or removal of proteins that interfere with mass spectrometry analysis. For example, in one embodiment of the present invention, the N-terminal tau protein and / or intermediate domain tau protein are depleted from the sample before further sample processing for analysis by mass spectrometry. Downstream analysis of the depleted tau proteins is optional, but both options are considered in the method of the present invention.

[0127] In one embodiment, targeted depletion can be carried out by affinity depletion. Affinity depletion is a method of depleting a protein of interest from a sample by utilizing its specific binding ability to a molecule. Generally, the molecule is a ligand (referred to as an immobilized ligand) bound to a solid support such as beads, resins, or tissue culture plates. Immobilization of the ligand to the solid support can occur even after the ligand-protein interaction has occurred. Suitable ligands include antibodies, aptamers, and other epitope conjugates. The molecule may also be a polymer or other material that selectively absorbs the protein of interest. As a non-limiting example, fatty oxyethylated alcohol-substituted polyhydroxymethylene (e.g., PHM-L LIPOSORB, Sigma Aldrich) can selectively absorb lipoproteins (including ApoE) from serum. Two or more affinity depletors can be combined to deplete multiple proteins sequentially or simultaneously.

[0128] In one embodiment, the method of the present invention involves affinity depleting one or more proteins from a sample using at least one epitope-binding agent that specifically binds to an epitope (or a similarly defined region of the 0N or 1N isoform) within amino acids 1-243 (including both ends) of tau-441. 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.

[0129] In one embodiment, the method of the present invention involves affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to an epitope in the N-terminus of tau (e.g., amino acids 1-103 of tau 441, including both ends) and an epitope conjugate that specifically binds to an epitope in the intermediate domain of tau (e.g., amino acids 104-243 of tau 441, including both ends). The epitope conjugates may be used sequentially or simultaneously.

[0130] In one embodiment, the method of the present invention involves affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to epitope amino acids 1-35 of tau 441 (including both ends) and an epitope conjugate that specifically binds to epitope amino acids 104-243 of tau 441 (including both ends) (or within similarly defined regions of the 0N or 1N isoform). The epitope conjugates may be used sequentially or simultaneously.

[0131] In one embodiment, the method of the present invention comprises affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to epitope amino acids 1-103 of tau 441 (including both ends) (or within similarly defined regions of the 0N or 1N isoform); an epitope conjugate that specifically binds to epitope amino acids 104-243 of tau 441 (including both ends) (or within similarly defined regions of the 0N or 1N isoform); and an epitope conjugate that specifically binds to an amyloid-beta epitope. The epitope conjugates may be used sequentially or simultaneously.

[0132] In one embodiment, the method of the present invention comprises affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to epitope amino acids 1-35 of tau 441 (including both ends) (or similarly defined regions of the 0N or 1N isoform); an epitope conjugate that specifically binds to epitope amino acids 104-243 of tau 441 (including both ends) (or similarly defined regions of the 0N or 1N isoform); and an epitope conjugate that specifically binds to an amyloid-beta epitope. The epitope conjugates may be used sequentially or simultaneously.

[0133] In one embodiment, the method of the present invention comprises affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to epitope amino acids 1-103 of tau 441 (including both ends) (or within similarly defined regions of the 0N or 1N isoform); and an epitope conjugate that specifically binds to an amyloid-beta epitope. The epitope conjugates may be used sequentially or simultaneously.

[0134] In one embodiment, the method of the present invention comprises affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to epitope amino acids 1-35 of tau 441 (including both ends) (or similarly defined regions of the 0N or 1N isoform); and an epitope conjugate that specifically binds to an amyloid-beta epitope. The epitope conjugates may be used sequentially or simultaneously.

[0135] In one embodiment, the method of the present invention comprises affinity depleting one or more proteins from a sample using an epitope conjugate that specifically binds to epitope amino acids 104-243 of tau 441 (including both ends) (or within similarly defined regions of the 0N or 1N isoform); and an epitope conjugate that specifically binds to an amyloid-beta epitope. The epitope conjugates may be used sequentially or simultaneously.

[0136] In any of the above embodiments, the epitope conjugate may include an antibody or an aptamer. In one embodiment, an epitope conjugate that specifically binds to amyloid beta is HJ5.1 or an epitope that binds to the same epitope as HJ5.1 and / or competitively inhibits HJ5.1. In one embodiment, an epitope conjugate that specifically binds to epitope amino acids 1-103 (including both ends) of tau 441 is HJ8.5 or an epitope that binds to the same epitope as HJ8.5 and / or competitively inhibits HJ8.5. In one embodiment, an epitope conjugate that specifically binds to epitope amino acids 104-221 (including both ends) of tau 441 is an epitope conjugate that binds to Tau1 or the same epitope as Tau1 and / or competitively inhibits Tau1. Methods for identifying epitopes to which antibodies specifically bind and assays for evaluating competitive inhibition between two antibodies are known in the art.

[0137] Alternatively, proteins can be depleted from a sample by more common methods, such as ultrafiltration or protein precipitation with acid, organic solvent, or salt. Generally speaking, the use of these methods reliably reduces high-abundance and high-molecular-weight proteins, which then enriches low-molecular-weight and / or low-volume proteins and peptides (e.g., tau, Aβ, etc.).

[0138] In one embodiment, proteins may be depleted from a sample by precipitation. That is, precipitation involves adding a precipitant to the sample, thoroughly mixing it, and using the sample and precipitant as an incubation to precipitate the proteins, and then separating the precipitated proteins by centrifugation or filtration. The resulting supernatant can then be used for downstream application. The required amount of agent 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.

[0139] As a non-limiting example, proteins can be depleted from a sample by acid precipitation using perchloric acid. Here, "perchloric acid" refers to 70% perchloric acid unless otherwise specified. In one embodiment, perchloric acid is added to a final concentration of approximately 1% v / v to approximately 15% v / v. In another embodiment, perchloric acid is added to a final concentration of approximately 1% v / v to approximately 10% v / v. In yet another embodiment, perchloric acid is added to a final concentration of approximately 1% v / v to approximately 5% v / v. In yet another embodiment, perchloric acid is added to a final concentration of approximately 3% v / v to approximately 15% v / v. In yet another embodiment, perchloric acid is added to a final concentration of approximately 3% v / v to approximately 10% v / v. In yet another embodiment, perchloric acid is added to a final concentration of approximately 3% v / v to approximately 5% v / v. In other embodiments, perchloric acid is added to a final concentration of 3.5% v / v to about 15% v / v, 3.5% v / v to about 10% v / v, or 3.5% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3.5% v / v. After adding perchloric acid, the sample is thoroughly mixed (e.g., by a vortex mixer) and kept at a low temperature for at least about 10 minutes 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. Next, the sample is centrifuged at a low temperature to pelletize the precipitated protein, and the supernatant containing soluble tau (i.e., the acid-soluble fraction) is transferred to a new container. As used above, "low temperature" refers to a temperature of 10°C or lower. For example, low temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C. In some embodiments, a narrow temperature range, for example, about 3°C ​​to about 5°C or about 4°C, is preferred. In some embodiments, low temperature can be achieved by placing the sample on ice.

[0140] Multiple proteins can be depleted sequentially or simultaneously by combining two or more methods from one or both of the above approaches. For example, one or more proteins can be selectively depleted (targeted depletion), followed by the depletion of high-abundance / molecular-weight proteins. Alternatively, high-abundance / molecular-weight proteins can be depleted first, followed by targeted depletion of one or more proteins. In yet another option, high-abundance / molecular-weight proteins can be depleted first, followed by a first round of targeted depletion of one or more proteins, and then a second round of targeted depletion of one or more proteins different from the target of the first round. Other iterations are readily apparent to those skilled in the art.

[0141] (d) Tau purification Other steps in the method disclosed herein include the purification of tau, particularly MTBR tau. In some examples, the MTBR tau is N-terminal independent and / or intermediate domain independent MTBR tau. The purification of the tau may be partial or complete.

[0142] In one embodiment, the method of the present invention includes purification of tau by solid-phase extraction. Purification of tau by solid-phase extraction includes contact of a tau-containing sample with a solid phase containing an adsorbent that adsorbs tau, one or more washing steps, and elution of 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-linked silica, aryl-linked 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 contact with the tau-containing supernatant, the adsorbent is preconditioned (e.g., with a water-miscible organic solvent, then with a mobile phase-containing buffer). Furthermore, the supernatant may be acidified as desired so that one reversed-phase material retains the ionized sample more strongly than others. Using volatile components in the mobile phase and for elution is preferred because it promotes sample drying. In exemplary embodiments, the washing step may involve using a liquid phase containing about 0.05% v / v trifluoroacetic acid (TFA) to about 1% v / v TFA or an equivalent. In one example, washing may use 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 another example, washing may use 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. The bound tau is then eluted with a liquid phase containing about 20% v / v to about 50% v / v acetonitrile (ACN) or an equivalent. In some cases, tau can be eluted in a liquid phase containing approximately 20% v / v to 40% v / v ACN or approximately 20% v / v to 30% v / v ACN. In some cases, tau can be eluted in a liquid phase containing approximately 30% v / v to 50% v / v ACN or approximately 30% v / v to 40% v / v ACN. The eluent can be dried by methods known in the art (e.g., vacuum drying (e.g., Speed-vac), freeze-drying, evaporation under a nitrogen stream, etc.).

[0143] In one embodiment, the method of the present invention includes the purification of MTBR tau by affinity purification. Affinity purification is a method of enriching a protein of interest by its specific binding ability to a particular molecule. Generally, the molecule is a ligand (referred to as an immobilized ligand) that binds to a solid support such as beads, resins, or tissue culture plates. Immobilization of the ligand to the solid support may occur even after the ligand-protein interaction has taken place. Suitable ligands include antibodies, aptamers, and other epitope binders. Purification of MTBR tau by affinity purification includes contact between a tau-containing sample and a suitable immobilized ligand, one or more washing steps, and elution of MTBR tau from the immobilized ligand.

[0144] In one embodiment, the method of the present invention includes purification of MTBR tau by affinity purification using at least one epitope binder that specifically binds to the epitope amino acids 235-368 of tau 441 (including both ends) or to the amino acids 244-368 of tau 441 (including both ends) (or to a similarly defined region of another full-length isoform). In various embodiments, one, two, three or more epitope binders may be used. When two or more epitope binders are used, they may be used sequentially or simultaneously. Non-limiting examples of suitable epitope conjugates include antibodies 77G7, RD3, RD4, UCB1017, and PT76 described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, and antibodies E2814 and 7G6 described in Roberts et al., Acta Neuropathol Commun, 2020, 8:13, as well as other epitope conjugates that specifically bind to the same epitopes as these antibodies. In a further embodiment, the method of the present invention includes purification of MTBR tau by affinity purification using an epitope binder that specifically binds to an epitope in R1 of MTBR tau, an epitope binder that specifically binds to an epitope in R2 of MTBR tau, an epitope binder that specifically binds to an epitope in R3 of MTBR tau, an epitope binder that specifically binds to an epitope in R4 of MTBR tau, an epitope binder that specifically binds to an epitope specific to 3R tau, an epitope binder that specifically binds to an epitope specific to 4R tau, an epitope binder that specifically binds to an epitope extending to R1 and R2 of MTBR tau, an epitope binder that specifically binds to an epitope extending to R2 and R3 of MTBR tau, an epitope binder that specifically binds to an epitope extending to R3 and R4 of MTBR tau, or any combination thereof. In specific examples, the present invention includes the purification of MTBR tau by affinity purification using an epitope binder that specifically binds to an epitope containing amino acids 316-355 of tau 441 (or the same region of another full-length isoform). In various embodiments, one, two, three or more epitope binders may be used.When using two or more epitope binders, they may be used sequentially or simultaneously.

[0145] In any of the above embodiments, the epitope conjugate may include an antibody or an aptamer. In one embodiment, the epitope conjugate that specifically binds to epitopes in R3 and R4 of MTBR tau is 77G7 or an epitope conjugate that binds to the same epitope as 77G7 and / or competitively inhibits 77G7 (BioLegend). In one embodiment, the epitope conjugate that specifically binds to epitopes specific to 3R tau is RD3 (de Silva et al., Neuropathology and Applied Neurobiology, 2003, 29: 288-302) or an epitope conjugate that binds to the same epitope as RD3 and / or competitively inhibits RD3. In one embodiment, the epitope conjugate that specifically binds to an epitope specific to 4R tau is RD4 (de Silva et al., Neuropathology and Applied Neurobiology, 2003, 29: 288-302) or an epitope conjugate that binds to the same epitope as RD4 and / or competitively inhibits RD4.

[0146] (e) Cleavage of purified tau with protease Other steps in the method disclosed herein include cleavage of purified tau with a protease. Cleavage of purified tau with a protease involves contacting a sample containing purified tau with the protease under conditions suitable for tau digestion. When affinity purification is used, digestion may be performed after elution of tau from the immobilized ligand or while tau is bound. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C, and Arg-N. In a preferred embodiment, the protease is trypsin. The resulting cleavage product is a composition containing a proteolytic peptide of tau. When the protease is trypsin, the resulting cleavage product contains a trypsin-digested peptide of tau. After proteolytic cleavage, the resulting cleavage product is generally desalted by solid-phase extraction.

[0147] (f) LC-MS Other steps of the method disclosed herein include performing liquid chromatography-mass spectrometry (LC-MS) on the proteolytic peptides of a tau-containing sample to detect and measure the concentration of at least one of the proteolytic peptides of tau. Thus, in practice, the disclosed method includes the use of one or more proteolytic peptides of tau for the detection and measurement of the amount of tau protein present in a biological sample.

[0148] In embodiments where trypsin is a protease, the tau proteolytic peptides indicating the presence of MTBR tau include, but are not limited to, the peptides listed in Table A. When a different enzyme is used for digestion, the resulting proteolytic peptides may differ slightly, but this can be easily determined by those skilled in the art. While we do not wish to be bound by theory, variations in the amount of trypsin-digested peptides between two biological samples of the same type are thought to reflect differences in the MTBR tau species constituting these biological samples. As disclosed herein, the amounts of specific proteolytic peptides of MTBR tau and the ratios of specific proteolytic peptides of MTBR tau provide clinically significant information to guide treatment decisions. Therefore, methods enabling the detection and quantification of trypsin-digested peptides of MTBR tau are useful in the diagnosis and treatment of many neurodegenerative diseases. [Table 1]

[0149] Tau proteolytic peptides can be separated by a liquid chromatography system compatible with a high-resolution mass spectrometer. A suitable LC-MS system may include the use of a <1.0 mm ID column and a flow rate of less than about 100 μl / min. In a preferred embodiment, a nanoflow LC-MS system is used (e.g., a column with an ID of about 50 - 100 μm and a flow rate <1 μL / min, preferably about 100 - 800 nL / min, more preferably about 200 - 600 nL / min). In an exemplary embodiment, the LC-MS system may include the use of a 0.05 mM ID column and a flow rate of about 400 nL / min.

[0150] As is known in the art, tandem mass spectrometry can be used to improve resolution or technologies for achieving the resolution of tandem mass spectrometry using a single mass analyzer can be improved. Suitable types of mass spectrometers are known in the art. These include quadrupole, time-of-flight, ion trap, and Orbitrap, as well as hybrid mass spectrometers that combine various types of mass analyzers in one structure (e.g., the Orbitrap Fusion TM Tribrid TM mass spectrometer or Orbitrap Fusion TM Lumos TM mass spectrometer, Orbitrap Tribrid TM Eclipse TM mass spectrometer, Q Exactive mass spectrometer), but are not limited thereto. In an exemplary embodiment, the LC-MS system is an Orbitrap Fusion TM Tribrid TM mass spectrometer, Orbitrap Fusion TM Lumos TM mass spectrometer, Orbitrap Tribrid TM Eclipse TMThis may include a mass spectrometer selected from a mass spectrometer or a mass spectrometer having ion focusing and ion transmission similar to or improved to a quadrupole. A suitable mass spectrometry protocol may be developed by optimizing the number of ions collected before analysis (e.g., AGC settings using bite trap) and / or injection time. In exemplary embodiments, the mass spectrometry protocol outlined in the Examples is used.

[0151] III. Use of MTBR Tau Measurement The present invention also includes the use of measuring MTBR tau species, particularly intermediate-domain-independent MTBR tau species, in blood or CSF as biomarkers of pathological features and / or clinical symptoms of tauopathy for diagnosis, staging, selection of appropriate treatment for a given disease stage, and modification of treatment regimens (e.g., dose changes, switching to different drugs or treatment modalities). Pathological features may be aspects of tau pathology (e.g., amount of tau deposition, presence / absence of post-translational modifications, amount of post-translational modifications, etc.). Pathological features may be tau-independent, either separately from or in addition to tau deposition. For example, when tauopathy is Alzheimer's disease, it may be amyloid-beta (Aβ) deposition in the brain or cerebral arteries. Clinical symptoms may be dementia, assessed by clinically validated devices (e.g., MMSE, CDR-SB, etc.) or any other clinical symptoms associated with tauopathy. As biomarkers for other pathological features and clinical symptoms known in this field as 3R- and 4R-tauopathy, evaluation of MTBR tau species in blood or CSF, particularly intermediate-domain-independent MTBR tau species, is also intended. Advantageously, MTBR tau species, including but not limited to intermediate-domain-independent MTBR tau species, not only distinguish between disease states and healthy states, but also differentiate between various tauopathy states.

[0152] Accordingly, in one embodiment, the present invention provides a method for evaluating tauopathy-related pathology in a subject, comprising quantifying one or more MTBR tau species in a biological sample obtained from the subject, such as a blood sample or a CSF sample, wherein the amount of quantified MTBR tau species represents tauopathy-related pathology in the subject's brain. Tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. Disease-related pathology may be tau deposition, post-translational modification of tau, amyloid plaques in the brain and / or cerebral arteries, or other pathological features known in the art. The subject may or may not have clinical symptoms of tauopathy. In a preferred embodiment, the quantification of at least one MTBR tau species is an intermediate domain-independent MTBR tau species. In a further embodiment, the quantification of two or more MTBR tau species is an intermediate domain-independent MTBR tau species. In a further embodiment, the quantification of each MTBR tau species is an intermediate domain-independent MTBR tau species.

[0153] In another embodiment, the present invention provides a method for diagnosing tauopathy in a subject, comprising quantifying one or more MTBR tau species in a biological sample obtained from the subject, such as a blood sample or a CSF sample, and diagnosing tauopathy when the quantified MTBR tau species is approximately 1.5σ or greater, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as evaluated by PET contrast and / or Aβ42 / 40 measurement in CSF. Tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The subject may or may not have clinical symptoms of the disease. In a preferred embodiment, at least one MTBR tau species quantified is an intermediate domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species quantified are intermediate domain-independent MTBR tau species. In a further embodiment, the quantification of each MTBR tau species is an intermediate domain-independent MTBR tau species.

[0154] In another embodiment, the present invention provides a method for measuring the stability of a tauopathic disease in a subject, comprising quantifying one or more MTBR tau species 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 (e.g., days, weeks, months, or years later), and calculating the difference in the quantified MTBR tau species between the samples, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates disease progression, a statistically significant decrease in the quantified MTBR tau species in the second sample indicates disease improvement, and no change indicates disease stability. The tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. The subject may or may not have clinical symptoms of the disease, and may or may not have received tau treatment. In some cases, tau treatment is administered to the subject once or more times during the period between the acquisition of the first and second biological samples, and the disease stability assessment indicates the effectiveness or lack thereof of tau treatment. In a preferred embodiment, at least one MTBR tau species quantified is an intermediate domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species quantified are intermediate domain-independent MTBR tau species. In yet another embodiment, each MTBR tau species quantified is an intermediate domain-independent MTBR tau species.

[0155] In other embodiments, the present invention provides a method for treating a subject having tauopathy, comprising: quantifying one or more MTBR tau species in a biological sample obtained from the subject, such as a blood sample or a CSF sample; and providing the subject with tau therapy to improve the assessment of disease-related pathology or clinical symptoms, wherein the quantified MTBR tau species of the subject is at least one standard deviation above or below the mean, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or more preferably at least 2 standard deviations above or below the mean (i.e., different by 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, where σ is the standard deviation defined by a normal distribution assessed in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as assessed by PET contrast and / or Aβ42 / 40 measurement in CSF). In addition to the use of a threshold (e.g., at least one standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used as a criterion for treating the subject. Tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy. Measures of disease-related pathology may include tau deposition measured by PET contrast, post-translational modification of tau measured by mass spectrometry or other appropriate methods, amyloid plaques in the brain or cerebral arteries measured by PET contrast, amyloid plaques measured by Aβ42 / 40 in CSF, or other pathological features known in the art. Clinical symptoms are dementia or other clinical symptoms known in the art for 3R- and 4R-tauopathy, assessed by clinically validated devices (e.g., MMSE, CDR-SB). In a preferred embodiment, at least one MTBR tau species quantification is an intermediate-domain-independent MTBR tau species. In a further embodiment, two or more MTBR tau species quantifications are intermediate-domain-independent MTBR tau species. In a further embodiment, each MTBR tau species quantification is an intermediate-domain-independent MTBR tau species. Many tau therapies target specific pathophysiological changes.For example, Aβ-targeting therapies are generally designed to reduce Aβ production, antagonize Aβ aggregation, or increase cerebral Aβ clearance; tau-targeting therapies are generally designed to alter tau phosphorylation patterns, antagonize tau aggregation (general antagonism of tau or antagonism of specific tau isoforms), or increase NFT clearance; and various therapies are designed for CNS inflammation or reduced cerebral insulin resistance, among others. However, not all tauopathies share the same pathophysiological changes. Therefore, the effectiveness of these various tau therapies may be improved by administering them to subjects precisely identified as having 3R-tauopathy, mixed 3R / 4R-tauopathy, or 4R-tauopathy.

[0156] The term “intermediate domain-independent MTBR tau” refers to several MTBR tau species that lack all or substantially all of the intermediate domain region of tau and therefore also the N-terminal region. These intermediate domain-independent MTBR tau species remain even after tau species containing intermediate domain tau have been partially or completely depleted from a biological sample, preferably a blood or CSF sample. Suitable biological samples are described in Section II(a), and their disclosure is incorporated herein by reference. Depletion of intermediate domain tau can be carried out by targeted depletion of these tau species, for example, by affinity depletion using epitope conjugates that specifically bind to epitopes in the N-terminus or intermediate domain of tau. Multiple epitope conjugates may also be used—for example, a primary epitope conjugate that specifically binds to an epitope in the N-terminus of tau and a secondary epitope conjugate that specifically binds to an epitope in the intermediate domain of tau. Further details can be found in Section II(c), and their disclosure is incorporated herein by reference. Generally, at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, or more) of the target protein in the starting material is depleted. In one embodiment, about 70% or more, about 80% or more, or about 90% or more of the target protein in the starting material is depleted. After depletion of intermediate domain tau from the biological sample, steps may be taken to enrich the remaining tau species, including intermediate domain-independent MTBR tau, by (1) purification of tau proteins, e.g., by removal of other proteins by precipitation and / or by solid-phase extraction, or (2) selective enrichment of intermediate domain-independent MTBR tau species, e.g., by affinity purification using an epitope-binding agent that specifically binds to epitopes in the MTBR. The term “enrichment” means an increase in quantity or number. Further details can be found in Section II, the disclosure of which is incorporated herein by reference. Preferably, the intermediate domain-independent MTBR tau species are enriched at least 100 times the amount in the CSF.In some cases, intermediate domain-independent MTBR tau species can be enriched by approximately 100 to 1000 times – for example, approximately 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 times. In some cases, intermediate domain-independent MTBR tau species can be enriched by approximately 500 to 1000 times or more. MTBR tau can be quantified in a processed CSF or blood sample obtained from a subject, where the CSF or blood sample is depleted of intermediate domain tau and then enriched with MTBR tau by LC-MS, as described in Section II or the Examples or by other methods known in the art (e.g., multiplexing assays (e.g., xMAP technology by Luminex, single-molecule protein detection (e.g., Simoa® bead technology)), etc.). In embodiments where intermediate domain tau is not depleted from the biological sample, tau is generally enriched by the above method and to the above extent.

[0157] In each of the above embodiments, the appropriate MTBR tau species is the amino acid sequence numbers 2 (IGSTENLK), 3 (LQTAPVPMPDLK), 4 (VQIINK), 5 (LDLSNVQSK), 6 (HVPGGGSVQIVYKPVDLSK), 8 (IGSLDNITHVPGGGN), 9 (VQIVYKPVDLSK), or combinations thereof. This may include, but is not limited to, MTBR tau species containing sequences and / or intermediate domain-independent MTBR species. The selection of MTBR species to evaluate may depend on the intended purpose of the method. For example, when the tauopathy is 3R-tauopathy, MTBR tau species containing SEQ ID NO: 9 (VQIVYKPVDLSK) may be reduced compared to mixed 3R / 4R-tauopathy or 4R-tauopathy, while MTBR tau species containing SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and / or SEQ ID NO: 8 (IGSLDNITHVPGGGN) may remain unchanged or increase compared to other tauopathy. Conversely, when tauopathy is 4R-tauopathy, MTBR species containing SEQ ID NO: 9 (VQIVYKPVDLSK) may increase compared to mixed 3R / 4R-tauopathy or 3R-tauopathy, while MTBR tau species containing SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and / or SEQ ID NO: 8 (IGSLDNITHVPGGGN) may remain unchanged or decrease compared to other tauopathy. As a further example, 4R tauopathy can be distinguished from AD by quantification of MTBR tau species containing SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and / or SEQ ID NO: 8 (IGSLDNITHVPGGGN). The use of intermediate domain-independent MTBR tau species can boost discriminative power, which can be further enhanced by using ratios of two different intermediate domain-independent MTBR tau species. For example, when the tauopathy is 3R-tauopathy or mixed 3R / 4R-tauopathy, the ratios of SEQ ID NOs. 3 to SEQ ID NOs. 6, SEQ ID NOs. 3 to SEQ ID NOs. 8, or SEQ ID NOs. 8 to SEQ ID NOs. 8 can be used when the tauopathy is 4R-tauopathy. The ratios of SEQ ID NOs. 2, 4, 5, or 9 to SEQ ID NOs. 6, 7, or 8 can be used. Other mathematical methods besides ratios may also be used.

[0158] Examples of the use of intermediate domain-independent MTBR tau 243 may be helpful in describing the various embodiments described above, but such descriptions do not limit the scope of the invention. "Intermediate domain-independent MTBR tau 243" is described in detail in Example 3. It is a trypsin digest peptide of multiple intermediate domain-independent MTBR tau species having the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), all containing the amino acid sequence of SEQ ID NO: 3. Measuring the amount of intermediate domain-independent MTBR tau 243 is one means of measuring the amount of this particular group of intermediate domain-independent MTBR tau species in a given sample. As shown in Examples 2 and 3, an increase in the amount of CSF intermediate domain-independent MTBR tau 243 replicates a direct measurement of increased Aβ and tau deposition in the brain associated with Alzheimer's disease (AD). In other words, the amount of CSF intermediate domain-independent MTBR tau 243 (and therefore the amount of CSF intermediate domain-independent MTBR tau including SEQ ID NO: 3) represents AD-related pathology (e.g., tau deposition in the brain, Aβ deposition in the brain, etc.). These amounts can therefore be used to assess AD-related pathology, determine the amyloid status of a subject, and diagnose AD in subjects without clinical symptoms of the disease. The amount of CSF intermediate domain-independent MTBR tau 243 also reproduces changes measured over the clinical duration of AD as defined, for example, by the results of MMSE or CDR-SB tests. Thus, the amount of intermediate domain-independent MTBR tau 243 (and therefore the amount of intermediate domain-independent MTBR tau including SEQ ID NO: 3) can be used to diagnose and stage AD in subjects across the entire disease spectrum (e.g., preclinical to clinical). The usefulness for diagnosing and staging AD in subjects across the entire disease spectrum was observed with whole trypsin-digested peptides of intermediate domain-independent MTBR tau. For example, see the intermediate domain-independent MTBR tau 299 and intermediate domain-independent MTBR tau 354 data in Example 3.This supports the use of the abundance of SEQ ID NO: 3 (among others) as a disease-specific biomarker for AD (and possibly other tauopathy), regardless of the measurement method (e.g., mass spectrometry, ELISA, etc.), because the peptide group constituting "intermediate domain-independent MTBR tau including SEQ ID NO: 3" may differ (though they may overlap) from the peptide group constituting "intermediate domain-independent MTBR tau including SEQ ID NO: 6". After disease diagnosis and / or staging, treatment may be administered to prevent a decrease or further increase in the amount of intermediate domain-independent MTBR tau 243 in the CSF and / or to prevent a decrease or further increase in other clinical signs or symptoms of AD. The choice of treatment is further guided by knowledge of the specific disease stage indicated by the amount of intermediate domain-independent MTBR tau 243 – for example, treatments designed for Aβ deposition prevention, Aβ deposition reversal, prevention of tau deposition, tau deposition reversal, and improvement of clinical signs of the disease may overlap, but are used for subjects with different amounts of intermediate domain-independent MTBR tau 243. The examples further demonstrate that CSF intermediate domain-independent MTBR tau 243 is extremely useful as a biomarker for AD, but not for non-AD tauopathy. Non-AD tauopathy is distinguished by the quantification and proportion of intermediate domain-independent MTBR tau species, including SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), or SEQ ID NO: 8 (IGSLDNITHVPGGGN). The examples demonstrate the above principle using CSF samples, but blood samples are intended as a suitable alternative.

[0159] In certain embodiments, the present invention provides a method for evaluating Alzheimer's disease (AD)-related pathology in a subject, comprising: preparing a treated CSF or blood sample obtained from the subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying MTBR tau species in the treated sample that contain the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 7 (IGSLDNITHVPGGGNK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), or combinations thereof, wherein the amount or ratio of the quantified MTBR tau species represents AD-related pathology in the subject's brain.

[0160] In other specific embodiments, the present invention provides a method for measuring Alzheimer's disease (AD)-related tau precipitate in the brain of a subject, comprising: preparing a treated CSF or blood sample in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying an MTBR tau species in the treated sample that contains the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), wherein the amount of the quantified MTBR tau species represents the AD-related tau precipitate in the brain of the subject.

[0161] In other specific embodiments, the present invention provides a method for measuring Alzheimer's disease (AD)-related tau deposition in the brain of a subject, comprising: preparing a treated CSF or blood sample obtained from the subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying MTBR tau species in the treated sample that contain the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 7 (IGSLDNITHVPGGGNK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), or combinations thereof, wherein the amount or ratio thereof of the quantified MTBR tau species represents AD-related tau deposition in the brain of the subject.

[0162] In other specific embodiments, the present invention provides a method for determining the amyloid status of a subject, the method comprising: preparing a treated CSF or blood sample obtained from the subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying the MTBR tau species in the treated sample, which comprises the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), where the amount of quantified MTBR tau species represents AD-related amyloid-beta deposition in the subject's brain and predicts amyloid positivity as determined by PIB-PET, e.g., PiB-PET SUVR as described in Ann Neurol 2016; 80:379-387.

[0163] In other specific embodiments, the present invention relates to a method for diagnosing Alzheimer's disease, comprising: preparing a treated CSF or blood sample obtained from a subject, in which intermediate domain tau is depleted and MTBR tau is enriched; quantifying MTBR tau species in the treated sample that contain the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 7 (IGSLDNITHVPGGGNK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), or combinations thereof; and diagnosing Alzheimer's disease when the quantified MTBR tau species differ by approximately 1.5σ or more, where σ is the presence of clinical signs or symptoms of tauopathy and PET contrast (e.g., PiB-PET as described in Ann Neurol 2016; 80:379-387). The method provides a standard deviation defined by a normal distribution measured in a control population that is amyloid-negative by measurement (SUVR) and / or Aβ42 / 40 measurement in CSF (e.g., a cutoff value for CSF Aβ42 / 40 calculated from PiB-PET SUVR (Ann Neurol 2016; 80:379-387) that maximizes sensitivity % + specificity %).

[0164] In other specific embodiments, the present invention provides a method for evaluating the progression of Alzheimer's disease (AD) in a subject, comprising: preparing a first treated CSF or blood sample and a second treated CSF or blood sample, where each treated sample is obtained from the same subject, each treated sample is depleted of intermediate domain tau and enriched with MTBR tau; quantifying for each treated sample an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof; and calculating the difference between the quantified MTBR tau species in the second sample and the first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates the progression of Alzheimer's disease in the subject.

[0165] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising: preparing a treated CSF or blood sample obtained from a subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying in the treated sample (i) MTBR tau species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK) or MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN); wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0166] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising: preparing a treated CSF or blood sample obtained from a subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying in the treated sample (i) MTBR tau species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) MTBR tau species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), or any combination thereof; wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes 4R-tauopathy from Alzheimer's disease and a healthy state.

[0167] In other specific embodiments, the present invention relates to a method for identifying 4R-tauopathy, comprising: preparing a treated CSF or blood sample obtained from a subject, wherein (a) N-terminal tau and intermediate domain tau are depleted and (b) MTBR tau is enriched; and in the treated sample, (i) MTBR tau species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or these The present invention provides a method comprising (i) quantifying a combination of (i) and (ii) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0168] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising: preparing a treated CSF or blood sample obtained from a subject, wherein (a) N-terminal tau and intermediate domain tau are depleted and (b) MTBR tau are enriched; and quantifying in the treated sample (i) MTBR tau species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or combinations thereof; and (ii) MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or combinations thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes 4R-tauopathy from Alzheimer's disease and a healthy state.

[0169] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising: preparing a treated CSF or blood sample obtained from a subject, wherein (a) N-terminal tau and intermediate domain tau are depleted and (b) MTBR tau is enriched; and quantifying in the treated sample (a) MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and (b) MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (a) and (b) distinguishes 4R-tauopathy from other tauopathy and a healthy state.

[0170] In other specific embodiments, the present invention provides a method for identifying 3R-tauopathy, comprising: preparing a treated CSF or blood sample obtained from a subject, wherein (a) N-terminal tau and intermediate domain tau are depleted and (b) MTBR tau are enriched; and quantifying in the treated sample (a) MTBR tau species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (b) MTBR tau species containing the amino acid sequences of SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 7 (IGSLDNITHVPGGGNK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), or combinations thereof, wherein the ratio of the quantified MTBR species from (a) and (b) distinguishes 3R-tauopathy from other tauopathy and a healthy state.

[0171] In other specific embodiments, the present invention provides a method for evaluating tauopathy-related pathology in a subject, comprising: preparing a treated CSF or blood sample obtained from the subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying in the treated sample MTBR species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 3 (LQTAPVPMPDLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), SEQ ID NO: 9 (VQIVYKPVDLSK), or combinations thereof, wherein the amount or ratio of the quantified MTBR species represents tauopathy-related pathology in the subject's brain.

[0172] In other specific embodiments, the present invention provides a method for evaluating tau deposition in a subject, comprising: preparing a treated CSF or blood sample obtained from the subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying in the treated sample an MTBR species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), an MTBR species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), an MTBR species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount or ratio of the quantified MTBR tau species represents tau deposition in the subject's brain.

[0173] In other specific embodiments, the present invention provides a method for evaluating tau deposition in a subject, comprising: preparing a treated CSF or blood sample obtained from the subject, in which intermediate domain tau is depleted and MTBR tau is enriched; and quantifying in the treated sample an MTBR species containing the amiic acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), an MTBR species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), an MTBR species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount or ratio of the quantified MTBR tau species represents tau deposition in the brain of a subject having 3R-tauopathy or 4R-tauopathy (i.e., non-AD tauopathy).

[0174] The following specific embodiments relate to methods for evaluating tau in a biological sample. In each of these embodiments, the method for measuring tau in a biological sample includes (a) reducing N-terminal tau, intermediate domain tau, or N-terminal tau and intermediate domain tau in the biological sample by affinity depletion, optionally by affinity depletion of amyloid beta, where the biological sample is a blood sample or a CSF sample, and the biological sample optionally contains isotope labeling, an internal tau standard; (b)(i) removing further proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then The method comprises enriching tau by (i) purifying tau from the supernatant by solid-phase extraction or (ii) affinity-purifying MTBR tau to produce (i) or (ii) enriched tau; (c) cleaving the enriched tau with a protease, and then optionally desalting the cleavage product obtained by solid-phase extraction to obtain a sample containing tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides to detect and measure the amount of at least one tau proteolytic peptide. Further details of each of steps (a) to (d) can be found in Section II, which are incorporated herein by reference.

[0175] In other specific embodiments, the present invention provides a method for evaluating Alzheimer's disease (AD)-related pathology in a subject, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the amount of MTBR tau species represents AD-related pathology in the subject's brain.

[0176] In other specific embodiments, the present invention provides a method for measuring Alzheimer's disease (AD)-related tau deposition in the brain of a subject, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is an MTRB tau species containing the amino acid sequence number 3 (LQTAPVPMPDLK), wherein the amount of the MTRB tau species represents AD-related pathology in the brain of the subject.

[0177] In other specific embodiments, the present invention provides a method for evaluating Alzheimer's disease (AD)-related tau deposition in the brain of a subject, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the amount of MTBR tau species represents AD-related pathology in the brain of a subject.

[0178] In other specific embodiments, the present invention provides a method for determining the amyloid status of a subject, comprising measuring tau in a biological sample by the method described above, wherein the tau measured is an MTRB tau species comprising the amino acid sequence SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), wherein the amount of MTRB tau species represents AD-related amyloid-beta deposition in the brain of the subject and predicts amyloid positivity as determined by PIB-PET.

[0179] In other specific embodiments, the present invention provides a method for diagnosing Alzheimer's disease, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof; and a diagnosis of Alzheimer's disease is made when the quantified MTBR tau species differ by approximately 1.5σ or more, wherein σ is the standard deviation defined by a normal distribution measured in a control population that does not have the clinical signs or symptoms of tauopathy and is amyloid-negative as measured by PET contrast and / or Aβ42 / 40 measurement in CSF.

[0180] In other specific embodiments, the present invention provides a method for evaluating the progression of Alzheimer's disease (AD) in a subject, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is an MTBR tau species containing the amino acid sequence number 3 (LQTAPVPMPDLK), an MTBR tau species containing the amino acid sequence number 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence number 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence number 8 (IGSLDNITHVPGGGN), or a combination thereof; and comprising calculating the difference between the quantified MTBR tau species in a second sample and a first sample, wherein a statistically significant increase in the quantified MTBR tau species in the second sample indicates the progression of Alzheimer's disease in the subject.

[0181] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK) or an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN); wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0182] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK) and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), or any combination thereof; wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0183] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is (i) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or a combination thereof, and (ii) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0184] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is (i) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), or a combination thereof, and (ii) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0185] In other specific embodiments, the present invention provides a method for identifying 4R-tauopathy, comprising measuring tau in a biological sample by the method described above, wherein the tau to be measured is (i) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK) and (ii) an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), an MTBR tau species comprising the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), or a combination thereof, wherein the ratio of the quantified MTBR species from (i) and (ii) distinguishes between 4R-tauopathy, Alzheimer's disease, and a healthy state.

[0186] In other specific embodiments, the present invention provides a method for evaluating tauopathy-related pathology in a subject, comprising measuring tau in a biological sample in which intermediate domain tau is depleted and MTBR tau is enriched by the method described above; and comprising quantifying in the treated sample MTBR species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), SEQ ID NO: 3 (LQTAPVPMPDLK), SEQ ID NO: 4 (VQIINK), SEQ ID NO: 5 (LDLSNVQSK), SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), SEQ ID NO: 8 (IGSLDNITHVPGGGN), SEQ ID NO: 9 (VQIVYKPVDLSK), or combinations thereof, wherein the amount or ratio of the quantified MTBR tau species represents tauopathy-related pathology in the brain of the subject.

[0187] In a particular embodiment, the present invention provides a method for measuring tau deposition in a subject, comprising measuring tau in a biological sample in which intermediate domain tau is depleted and MTBR tau is enriched by the method described above; and further comprising quantifying in the treated sample an MTBR species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), an MTBR species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), an MTBR species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), an MTBR species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), an MTBR species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), an MTBR species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), an MTBR species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK), or a combination thereof, wherein the amount or ratio of the quantified MTBR tau species represents tau deposition in the brain of the subject.

[0188] In other specific embodiments, the present invention relates to a method for treating a subject requiring treatment, comprising: (a) preparing a treated CSF or blood sample obtained from the subject which is (i) depleted of intermediate domain tau and (ii) enriched with MTBR tau; and (b) in the treated sample, MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 2 (IGSTENLK), MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 4 (VQIINK), MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 5 (LDLSNVQSK), MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), and MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK). The present invention provides a method comprising: (c) quantifying MTBRMTBR tau species, MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN), MTBRMTBR tau species containing the amino acid sequence of SEQ ID NO: 9 (VQIVYKPVDLSK), or a combination thereof; and (c) administering a treatment to a subject to alter tau pathology, wherein the treated CSF or blood sample of the subject differs by 1.5σ or more in the amount or ratio of the quantified MTBRMTBR tau species, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as assessed by PET contrast and / or Aβ42 / 40 measurement in CSF, wherein the amount or ratio of the quantified MTBR tau species represents tau pathology in the subject's brain. In one embodiment, administering a treatment to a subject to alter tau pathology alters or stabilizes the amount of the quantified MTBR species.In one embodiment, the treatment involves cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-Aβ antibodies (including their antigen-binding fragments, variants, or derivatives), anti-tau antibodies (including their antigen-binding fragments, variants, or derivatives), anti-TREM2 antibodies (including their antigen-binding fragments, 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, p38 alpha-MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthioninium chloride), blood Therapies to improve glucose management (e.g., insulin, exenatide, liraglutide, pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, β-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A reverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT The pharmaceutical composition includes 1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, APP production selective inhibitors, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, neuroproliferative factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.In exemplary embodiments, the pharmaceutical composition may include a kinase inhibitor. A suitable kinase inhibitor may inhibit a wide range of amino acid kinases (TAOK), CDK, GSK-3p, MARK, CDK5, or Fyn. In other exemplary embodiments, the pharmaceutical composition may include a phosphatase activator. In non-limiting examples, a phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments, the treatment is a pharmaceutical composition comprising a tau-targeting therapy that includes, but is not limited to, an active pharmaceutical ingredient that modifies the tau phosphorylation pattern, antagonizes tau aggregation, or increases the clearance of pathological tau isoforms and / or aggregates. In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma-secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor.

[0189] In other specific embodiments, the present invention relates to a method for treating a subject requiring treatment, comprising: (a) preparing a treated CSF or blood sample obtained from the subject which is (i) depleted of intermediate domain tau and (ii) enriched with MTBR tau; and (b) in the treated sample, MTBR tau species comprising the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), MTBR tau species comprising the amino acid sequence of SEQ ID NO: 6 (HVPGGGSVQIVYKPVDLSK), MTBR tau species comprising the amino acid sequence of SEQ ID NO: 7 (IGSLDNITHVPGGGNK), and amino acid sequence of SEQ ID NO: 8 (IGSLDNITHVPGGGN) The method provides a method comprising (c) quantifying MTBR tau species or combinations thereof; and administering a treatment to a subject to alter tau pathology, wherein the treated CSF or blood sample of the subject differs by 1.5σ or more in the amount or ratio of quantified MTBR tau species, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as assessed by PET contrast and / or Aβ42 / 40 measurement in CSF, wherein the amount or ratio of quantified MTBR tau species represents tau pathology in the subject's brain. In one embodiment, administering a treatment to a subject to alter tau pathology alters or stabilizes the amount of quantified MTBR species.In one embodiment, the treatment involves cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-Aβ antibodies (including their antigen-binding fragments, variants, or derivatives), anti-tau antibodies (including their antigen-binding fragments, variants, or derivatives), anti-TREM2 antibodies (including their antigen-binding fragments, 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, p38 alpha-MAPK inhibitors, recombinant granulocyte-macrophage colony-stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513), tau protein aggregation inhibitors (e.g., TRx0237, methylthioninium chloride), blood Therapies to improve glucose management (e.g., insulin, exenatide, liraglutide, pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, β-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A reverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT The pharmaceutical composition includes 1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, APP production selective inhibitors, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, neuroproliferative factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.In exemplary embodiments, the pharmaceutical composition may include a kinase inhibitor. A suitable kinase inhibitor may inhibit a wide range of amino acid kinases (TAOK), CDK, GSK-3p, MARK, CDK5, or Fyn. In other exemplary embodiments, the pharmaceutical composition may include a phosphatase activator. In non-limiting examples, a phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments, the treatment is a pharmaceutical composition comprising a tau-targeting therapy that includes, but is not limited to, an active pharmaceutical ingredient that modifies the tau phosphorylation pattern, antagonizes tau aggregation, or increases the clearance of pathological tau isoforms and / or aggregates. In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma-secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor. [Examples]

[0190] The following embodiments illustrate various iterations of the present invention. Those skilled in the art will recognize that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in carrying out the present invention. However, those skilled in the art will recognize that, based on the description herein, the specific embodiments disclosed herein can be modified to obtain similar or analogous results without departing from the spirit and scope of the invention. Accordingly, all matters described or shown in the accompanying drawings should be construed as explanatory, not restrictive.

[0191] Example 1 Several sample processing methods have been developed to enrich MTBR tau: immunoprecipitation of N-terminal and intermediate domain tau (IP) as described in Sato et al., 2018; chemical extraction (CX); and a combination of IP and CX methods (PostIP-CX). The CX and PostIP-CX methods were specifically developed for the detection and quantification of MTBR tau. An overview of these methods is shown in Figure 2.

[0192] In short, CSF (approximately 475 μL) is used as the internal standard. 15 The samples were mixed with a solution containing homogeneous labeling with N-tau 441 (2N4R) (approximately 10 μL of 100 pg / μL solution or approximately 5 μL of 200 pg / μL solution). The N-terminal tau and intermediate domain tau species were immunoprecipitated with Tau1 and HJ8.5 antibodies, then treated and trypsin-digested as described above (Sato et al., 2018).

[0193] Regarding the CX method, CSF (approximately 475 μL) is used as the internal standard. 15 The mixture was mixed with a solution containing homogeneous labeling with N-tau 441 (2N4R) (approximately 10 μL of 100 pg / μL solution or approximately 5 μL of 200 pg / μL solution). Tau was then chemically extracted. High-abundance CSF protein was precipitated using 25 μL of perchloric acid. After mixing on ice and incubation for 15 minutes, the mixture was centrifuged at 20,000 g for 15 minutes at 4°C, and the supernatant was further purified using an Oasis HLB 96 plate μElution Plate (Waters) according to the following procedure: The plate was washed once with 300 μL of methanol and equilibrated once with 500 μL of 0.1% FA aqueous solution. The supernatant was added to the Oasis HLB 96 plate μElution Plate and adsorbed onto the solid phase. The solid phase was then washed once with 500 μL of 0.1% FA aqueous solution. Elution buffer (100 μL; 35% acetonitrile and 0.1% FA aqueous solution) was added, and the eluent was dried using Speed-vac. The dried sample was dissolved in 50 μL of trypsin solution (10 ng / μL) in 50 mM TEABC and incubated at 37°C for 20 hours.

[0194] Regarding the PostIP-CX method, the CSF after immunoprecipitation (i.e., the supernatant remaining after the IP method described above) was treated as described in the CX method.

[0195] After trypsin digestion, all samples were purified by solid-phase extraction using a C18 TopTip. During this purification process, 5 fmol each of AQUA internal standard peptides at residues 354-369 (MTBR tau 354) and 354-368 (tau 368) were added for discriminative quantification. Before elution, 3% hydrogen peroxide and 3% FA aqueous solution were added to the beads, followed by incubation overnight at 4°C to oxidize the methionine-containing peptides. The eluent was lyophilized and resuspended in 27.5 μL of 2% acetonitrile and 0.1% FA aqueous solution, and then MS analysis was performed using a nanoAcquity UPLC system coupled to an Orbitrap Fusion Lumos Tribrid or Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operating in PRM mode.

[0196] As shown in Figure 3A, the CX and PostIP-CX methods yielded MTBR tau-containing samples that were detectable and quantifiable by mass spectrometry. A quantifiable signal for MTBR tau was not obtained by the IP method. Although not proven, it is conceivable that other methods with similar sensitivity to detect and quantify MTBR tau could also be used.

[0197] Example 2 In this example, CSF samples were analyzed from two clinical cohorts (LOAD100 and LOAD60) of subjects with late-onset Alzheimer's disease. The clinical dementia grade (CDR) scores and amyloid status of the samples used in this analysis are shown in Tables 1 and 2. CSF samples (approximately 500 μl each) were treated with the PostIP-CX method as described in Example 1 and evaluated by mass spectrometry. CSF Aβ42 and Aβ40 immunoprecipitation from CSF was measured by mass spectrometry as previously described (Patterson BW, et al., Ann Neurol 2015, 78: 439-453). pT217% was measured by mass spectrometry as previously described (Barthelemy, NR, et al., Alz Res Therapy, 2020, 12: 26).

[0198] The cutoff value for CSF Aβ42 / 40 was calculated from the PiB-PET SUVR results used to determine the amyloid state. Based on the PiB-PET SUVR established cutoff > 1.42 (Ann Neurol 2016; 80:379-387), the (sensitivity % + specificity %) for CSF Aβ42 / 40 was maximized at 0.1389. In particular, pT217% showed excellent correlation with the amyloid state defined by the established cutoff, although it had one outlier (Figure 4).

[0199] As shown in Figures 5–7, tau trypsin digested peptides associated with MTBR, measured in PostIP-CX samples, were significantly increased in amyloid-positive subjects. HVPG increased most significantly, even in the clinically asymptomatic stage (Figures 5–6). The increases in HVPG and IGSL saturated after symptomatic onset, while LQTA continued to increase even after clinical onset (Figures 7–8). LQTA concentrations showed the best correlation with tau pathology assessed by positron emission tomography (PET) (Pearson r=0.84, n=35) and cognitive test assessments (Figures 9–12). In some of the above analyses, data from amyloid-positive CDR1 and CDR2 samples were combined as CDR>1, and data from amyloid-negative CDR0.5 and CDR1 were combined as CDR>0.5. Statistical analysis was performed by one-way ANOVA adjusted for multiple comparisons using the Benjamin Hoshberg FDR method with FDR set to 5%.

[0200] In particular, sample processing was shown to affect the diagnostic usefulness of tau. For example, the trypsin-digested peptide HVPG of MTBR tau distinguished between amyloid-positive and amyloid-negative subjects in the pre-symptomatic stage in PostIP-CX samples, but this discriminative ability was not observed with the trypsin-digested peptide TPPS of intermediate-domain tau in IP samples (Figure 5). The amino acid sequence of the TPPS trypsin-digested peptide is TPPSSGEPPK (SEQ ID NO: 10). Sample processing also showed a significant effect on the ability to distinguish changes in MTBR tau levels among various CSF samples. For example, the trypsin-digested peptide LQTA showed a linear increase in post-symptomatic CSF samples in PostIP-CX samples, but not in IP samples (Figure 13), indicating that PostIP-LQTA (intermediate-domain independent MTBR tau 243) distinguishes amyloid status better than IP-LQTA (Figure 14).

[0201] The above data suggest that MTBR tau, enriched in AD brain aggregates, also increases in AD CSFs. It is hypothesized that the trypsin-digested peptides LQTA and HVPG are part of the fuzzy coat, serving as starting points for tau filament aggregation, while IGSLs are located inside the core. The position of the LQTA peptide on the filament surface as part of the fuzzy coat is always exposed, increasing the likelihood of release into the CSF. Given the role of HVPG in aggregation, immature filaments still expose HPVG on their surface, while this peptide can be replenished in the core of mature filaments. The position of IGSLs in the filament core can be predicted at an early AD stage.

[0202] Regardless of the underlying mechanism, the data suggest that the trypsin-digested peptides HVPG and LQTA in CSF can be used as biomarkers to reproduce amyloid status and tau pathology in AD, respectively. In particular, only LQTA showed a continuous increase throughout disease progression in terms of Tau-PET, amyloid status, and cognitive decline, suggesting that this region is important for identifying tau pathology in AD. The use of these peptides in combination with the trypsin-digested peptide IGSL and / or other biomarkers enhances the discriminative power when staging the disease trajectory in question (Figures 15-17). Furthermore, LQTA and other MTBR tau peptides can be used as biomarkers to identify various tauopathies. [Table 2] [Table 3]

[0203] Example 3 In this example, the presence and potential utility of MTBR tau species as Alzheimer's disease biomarkers are detailed. The results indicate that a substantial amount of intermediate domain-independent MTBR tau is present in CSF - i.e., tau species that are cleaved around the center of the polypeptide sequence that gives rise to a C-terminal fragment lacking the N-terminal and intermediate domain regions (the "C-terminal truncation"), for example, around amino acid 224 of tau441. Furthermore, various regions of CSF MTBR tau stage disease progression and correlate with tau aggregation in the Alzheimer's disease brain. These findings provide new insights into the correlation between MTBR tau and CSF in the brain and support CSF tau as a fluid biomarker for Alzheimer's disease.

[0204] Materials: Two different cohorts of human brain samples were used in the experiments of this example – a discovery cohort and a validation cohort. The discovery cohort included postmortem frozen brain tissue samples from two participants with Alzheimer's disease pathology and two control participants without pathology, provided by the Knight ADRC Pathology Core at Washington University School of Medicine. Each sample was classified by the National Institute on Aging and Alzheimer's Association as amyloid stage A3 (Thai phase) for amyloid deposition and Tau Braak stages VI and B3 for tau aggregation. Samples from each participant were collected from 6–10 brain regions, including the cerebellum, superior frontal gyrus, frontal pole, temporal lobe, occipital lobe, thalamus, amygdala, pons, parietal lobe, and striatum. As a validation cohort, further postmortem frozen brain tissue samples from the parietal lobe of 20 participants (8 amyloid-negative and 12 amyloid-positive by CSF Aβ 42 / 40 ratio) were analyzed. Twelve amyloid-positive samples were further divided into clinical groups based on their Clinical Dementia Grade (CDR) scores, and classified as either very mild to moderate Alzheimer's disease (amyloid-positive, CDR=0.5–2, n=5) or severe Alzheimer's disease (amyloid-positive, CDR=3, n=7). These human studies were approved by the Washington University Institutional Review Board.

[0205] Three different cohorts of human CSF samples were also used in the experiments of this example - a cross-sectional cohort, a longitudinal cohort, and a Tau-PET cohort (Tables 3A and 3B). CSF samples from 100 participants were collected for analysis as the cross-sectional cohort from the amyloid-beta (Aβ) stable isotope labeling kinetics (SILK) test (Patterson et al., 2015). This cohort is also referred to as the LOAD100 cohort in Example 2. CSF collection was performed as described previously (Patterson et al., 2015). Briefly, CSF was collected at baseline. Next, the participants received a leucine bolus infusion over 10 minutes. 6 mL of CSF was obtained every 36 hours. The CSF aliquot collected at 30 hours was used for MS measurement of tau species in this test. The amyloid status was defined using the previously reported CSF Aβ 42 / 40 ratio (Patterson et al., 2015). The corresponding cut-off ratio (0.1389) maximized the predictive accuracy of amyloid positivity determined by Pittsburgh compound B (PiB) PET. The amyloid group was further divided into clinical groups by the CDR scores shown in Table 3A. From the cross-sectional cohort, 28 participants (14 amyloid positive and 14 amyloid negative) were followed for 2 to 9 years for evaluation of the long-term trajectory of tau species in CSF. CSF samples were collected and analyzed in the same manner as the cross-sectional cohort. The Tau-PET cohort consisted of 35 participants (20 amyloid positive and 15 amyloid negative, including 16 participants from the longitudinal cohort) in whom the Tau-PET AV-1451 standardized uptake value ratio (SUVR) was measured within 3 years from the CSF collection time point. PET scans were performed as described previously (Sato et al., 2018) and implemented for SUVR using the partial volume effect correction region diffusion function technique (Su et al., 2015). CSF samples were collected and analyzed in the same manner as the other cohorts.

Table 4

Table 5

[0206] Brain tau analysis by MS: Frozen brain tissue samples were sliced ​​using a cryostat at -20°C and collected in tubes. Tissue (300-400 mg) was sonicated at a brain tissue concentration of 0.3 mg / μL in ice-cold buffer containing 25 mM tris-hydrochloride (pH 7.4), 150 mM sodium chloride, 10 mM ethylenediaminetetraacetic acid, 10 mM ethylene glycoltetraacetic acid, a phosphatase inhibitor cocktail, and a protease inhibitor cocktail. The homogenate was purified by centrifugation at 11,000 g at 4°C for 20 minutes. The supernatant (whole brain extract) was divided equally into new tubes and maintained at -80°C until use. The whole brain extract was incubated with 1% sarcosyl on ice for 60 minutes, followed by ultracentrifugation at 100,000 g at 4°C for 60 minutes to obtain an insoluble pellet. The insoluble pellet was resuspended in 200 μL of PBS, followed by sonication, and maintained at -80°C until the insoluble suspension was used.

[0207] For soluble tau analysis, tau species in whole brain extracts were immunoprecipitated with Tau1 and HJ8.5 antibodies. The immunoprecipitated soluble tau species were treated and digested as described above (Sato et al., 2018).

[0208] For insoluble tau analysis, an insoluble suspension (10-20 μL containing 2.5 μg of total protein) is mixed with 200 μL of lysis buffer (7M urea, 2M thiourea, 3% 3-[(3-coramidopropyl)dimethylammonio]-1 propanesulfonate, 1.5% n-octyl glucoside, 100 mM triethylammonium bicarbonate (TEABC)), followed by an internal standard... 15A 5 μL solution containing homogeneous labeling with N-tau 441 (2N4R) (2 ng / μL, donated by Dr. Guy Lippens, Lille University, France) was added. 5 μL of 500 mM dithiothreitol was added to the suspension, followed by sonication. The resulting solution was mixed with 15 μL of 500 mM iodoacetamide and incubated at room temperature in the dark for 30 minutes. Protein digestion was performed using the previously reported filter-assisted sample preparation method (Roberts et al., 2020). Briefly, each prepared solution was packed into a Nanosep 10K filter unit (PALL) and centrifuged. After washing the sample on the filter unit with 8 M urea in 100 mM TEABC solution, the immobilized protein was digested on the filter at 37°C for 60 minutes using 0.25 μg of endoproteinase Lys-C. Next, the samples were further digested overnight at 37°C using 0.4 μg of trypsin.

[0209] Digested samples (soluble and insoluble tau species) were collected by centrifugation and then desalted with C18 TopTip (Glygen). During this purification process, 50 fmol each of AQUA internal standard peptides at residues 354-369 (MTBR tau 354) and 354-368 (tau 368) were added for discriminative quantification. Before elution of the samples, 3% hydrogen peroxide and 3% formic acid (FA) aqueous solutions were added to beads, followed by incubation overnight at 4°C to oxidize the methionine-containing peptides. The eluent was lyophilized and resuspended in 27.5 μL of 2% acetonitrile and 0.1% FA aqueous solutions, and then analyzed by MS using a nanoAcquity UPLC system (Waters) connected to an Orbitrap Fusion Tribrid or Orbitrap Tribrid Eclipse (Thermo Scientific) operating in parallel reaction monitoring (PRM) mode.

[0210] 16 brain tau peptides from both soluble and insoluble tau species, 15Quantitative analysis was performed by comparing the corresponding isotopomer signals from N or AQUA internal standards (Table 4). Peptide profile comparisons across brain samples were performed by normalizing the amount of each peptide using the intermediate domain tau peptide (residues 181-190). [Table 6]

[0211] MS analysis of CSF tau: CSF (455 μL) was used as an internal standard, and 10 μL of CSF tau was used. 15 The tau species were mixed with a solution containing homogeneous labeling with N-tau 441 (2N4R) (100 pg / μL). Tau species, mainly consisting of the N-terminal to intermediate domain regions, were immunoprecipitated with Tau1 and HJ8.5 antibodies. The immunoprecipitated tau species were treated and digested as previously described (Sato et al., 2018). Subsequently, 20 μL of 15 An N-tau internal standard (100 pg / μL) was added to immunoprecipitated CSF. Tau was then chemically extracted as described in a modified version of the previous report (Barthelemy et al., 2016b). High-abundance CSF proteins were precipitated using 25 μL of perchloric acid. After mixing on ice and incubation for 15 minutes, the mixture was centrifuged at 20,000 g for 15 minutes at 4°C, and the supernatant was further purified using an Oasis HLB 96 plate μElution Plate (Waters) according to the following procedure: The plate was washed once with 300 μL of methanol and equilibrated once with 500 μL of 0.1% FA aqueous solution. The supernatant was added to the Oasis HLB 96 plate μElution Plate and adsorbed onto the solid phase. The solid phase was then washed once with 500 μL of 0.1% FA aqueous solution. Elution buffer (100 μL; 35% acetonitrile and 0.1% FA aqueous solution) was added, and the eluent was dried using Speed-vac. The dried sample was dissolved in 50 μL of trypsin solution (10 ng / μL) in 50 mM TEABC and incubated at 37°C for 20 hours.

[0212] After incubation of both immunoprecipitation and chemical extraction samples, each trypsin digest was purified by solid-phase extraction using a C18 TopTip. During this purification process, 5 fmol each of AQUA internal standard peptides at residues 354-369 (MTBR tau 354) and 354-368 (tau 368) were added for discriminative quantification. Before elution of the samples, 3% hydrogen peroxide and 3% FA aqueous solution were added to the beads, followed by incubation overnight at 4°C to oxidize the methionine-containing peptides. The eluent was lyophilized and resuspended in 27.5 μL of 2% acetonitrile and 0.1% FA aqueous solution, and then MS analysis was performed using a nanoAcquity UPLC system coupled to an Orbitrap Fusion Lumos Tribrid or Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operating in PRM mode. The 19CSF tau peptide was quantified (Table 4). A schematic procedure for CSF tau analysis is shown in Figure 2.

[0213] Statistical analysis: Differences in biomarker values ​​were evaluated using one-way ANOVA unless otherwise specified. Two-sided p<0.05 was considered statistically significant, and adjustments were made for multiple comparisons using the Benjamin Hochberg false positive rate (FDR) method with a 5% FDR setting (Benjamini and Hochberg, 1995). Spearman correlation was used to evaluate the correlation between tau biomarkers and cognitive test measures and Tau-PET SUVR.

[0214] Results - Tau Species Enrichment Profiling in Alzheimer's Disease Brains: It was hypothesized that tau aggregation in Alzheimer's disease brains is reflected in the tau profile in CSF. Therefore, the tau profiles in insoluble extracts from Alzheimer's disease and control brains were first analyzed (Figure 18B: Discovery Cohort) and then compared with the CSF tau profile. Species containing residues 299-317 (MTBR tau 299) and 354-369 (MTBR tau 354), located between the R2 and R3 domains and within the R4 domain, respectively, were enriched 3-4 times more in insoluble extracts from Alzheimer's disease brains than in controls. The upstream region of the MTBR, including residues 243–254 (MTBR tau 243), was approximately three times more abundant in Alzheimer's disease brains compared to controls, while species containing residues 260–267 and 275–280, located within the R1 and R2 domains respectively, did not differ between Alzheimer's disease and control tissues. Other regions of tau were not enriched in Alzheimer's disease brains compared to controls. Notably, species containing residues 195–209 within the intermediate domain were particularly low in Alzheimer's disease brains compared to controls, possibly resulting from widespread hyperphosphorylation occurring at residues 199, 202, 205, and 208 in insoluble tau aggregates (Malia et al., 2016). Notably, no changes were observed in the identified MTBR tau species in soluble tau (whole brain extract) between controls and Alzheimer's disease (Figure 23A). These results were replicated in brain samples from control (amyloid-negative, n=8), very mild to moderate Alzheimer's disease (amyloid-positive, CDR=0.5-2, n=5), and severe Alzheimer's disease (amyloid-positive, CDR=3, n=7) participants (Figure 18C and Figure 23B: validation cohort), suggesting that MTBR tau species 243, 299, and 354 were particularly enriched in insoluble tau aggregates throughout the disease progression stages.

[0215] Next, we tested the recently reported cleaved tau 368 (residues 354-368) species produced by asparagine endopeptidase (Zhang et al., 2014; Blennow et al., 2020) against its uncleaved counterpart, MTBR tau 354, and quantified both species using brain-insoluble extracts (Figure 24). A high correlation was found between tau 368 and MTBR tau 354 (r=0.9783), suggesting that shortening at residue 368 occurs at the same rate at various stages of brain pathology.

[0216] Results - Quantification of MTBR tau in CSF: To determine whether MTBR tau enrichment in Alzheimer's disease brain aggregates correlates with the level of soluble tau species in CSF, we developed a method to analyze MTBR tau in CSF. The method involved chemical extraction of tau in CSF after immunoprecipitation (Tau1 / FIJ8.5), followed by MS analysis (Figure 2A). This method resulted in sufficient recovery for MTBR peptide quantification (Figure 25). The abundance of tau peptide recovered by the Tau1 / FIJ8.5 immunoprecipitation method before chemical extraction decreased dramatically after residue 222 (Sato et al., 2018). In contrast, the concentration of MTBR tau species quantified by the PostIP-CX method was relatively low compared to the N-terminal to intermediate domain region, but still comparable to other regions of tau obtained by immunoprecipitation (Figure 19). The CSF concentrations in normal control participants (calculated as the sum of values ​​from immunoprecipitation and chemical extraction methods) ranged from 8.2 to 32.0 ng / mL for intermediate domain species (residues 151-155, 181-190, 195-209, and 212-221), 0.4 to 3.7 ng / mL for MTBR tau species (residues 243-254, 260-267, 275-280, 282-290, 299-317, and 354-369), and 6.5 to 5.1 ng / mL for non-MTBR C-terminal tau species (residues 386-395 and 396-406). The CSF concentration of C-terminal cleaved tau species is in a similar range to that of species containing the intermediate domain (residues 195-209 and 212-221), suggesting that the C-terminal side of tau is cleaved in neuronal cells in the same way as N-terminal to intermediate domain tau and secreted extracellularly (Sato et al., 2018).

[0217] Results - CSF MTBR tau in a cross-sectional cohort of Alzheimer's disease: To determine whether MTBR-containing species present in the extracellular space reflect Alzheimer's disease-related changes, CSF was analyzed in a cross-sectional cohort of amyloid-negative and amyloid-positive participants at various clinical stages: amyloid-negative CDR=0 (controls, n=30), amyloid-positive CDR=0 (pre-symptomatic AD, n=18), amyloid-positive CDR=0.5 (very mild AD, n=28), amyloid-positive CDR>1 (mild to moderate AD, n=12), and amyloid-negative CDR>0.5 (non-AD cognitive impairment, n=12).

[0218] First, we examined the CSF levels of three MTBR tau species (MTBR tau 243, MTBR tau 299, and MTBR tau 354) that are particularly enriched in Alzheimer's disease brains (Figure 20). All three species were present in both Alzheimer's disease and control CSFs, and their levels were higher in the amyloid-positive group even at the asymptomatic stage (CDR=0) compared to the control group (MTBR tau 243 p=0.0170, MTBR tau 299 p=0.0002, and MTBR tau 354 p=0.0076). Notably, these species exhibited different characteristics in the CSF after clinical disease onset. MTBR tau 299 levels were 204% higher in pre-symptomatic AD compared to controls, but saturated between very mild AD (CDR=0.5) and mild to moderate AD (CDR>1) (p=0.2541), while MTBR tau 354 levels were significantly lower in samples collected after symptom onset (p=0.0345). In contrast, MTBR tau 243 levels gradually increased across all disease stages, including after symptom onset (p=0.0025). These results suggest that regional specificity within MTBR tau species can be identified across various stages of Alzheimer's disease, indicating that MTBR tau 243 is a good stage-specific marker for Alzheimer's disease.

[0219] Next, we investigated whether CSF MTBR tau species provide increased sensitivity and specificity to Alzheimer's disease staging compared to tau species containing other regions. Multiple species, including the N-terminal, intermediate, MTBR, and C-terminal domains, were quantified using region-specific methods (Figures 26, 27, and 28). N-terminal and intermediate domain species were quantified by immunoprecipitation (IP method) and chemical extraction of immunoprecipitation-treated CSF (PostIP-CX method). The MTBR of the C-terminal species was quantified only by chemical extraction of immunoprecipitation-treated CSF (PostIP-CX method) because no quantifiable signal was obtained by immunoprecipitation. The levels of species containing the N-terminal domain quantified by immunoprecipitation did not differ from the control and the asymptomatic stage (residues 6-23, p=0.0362) or other neighboring disease stages. Intermediate domain species levels, as determined by immunoprecipitation, were significantly higher than those of the control in the asymptomatic amyloid stage (except for residues 212-221, p=0.0762). However, the effective size was relatively moderate (123%-168% vs. control) compared to MTBR tau species (e.g., MTBR tau 299 level was >200% larger than the control in the pre-symptomatic AD stage), and did not differ from the later disease stages. Regardless of the extraction method, MTBR tau species 243, 299, and 354 showed significant differences between the control and disease stages compared to N-terminal to intermediate domain species (residues 6-23-226-230, Figure 28). Profiles of other species containing the C-terminal domain (residues 260-267, 275-280, 282-290, 386-395, and 396-406) relative to MTBR were similar to those of intermediate domain species and were not specific to the stage of clinical dementia in Alzheimer's disease.

[0220] In summary, three representative MTBRs (MTBR tau 243, MTBR tau 299, and MTBR tau 354) enriched in Alzheimer's disease brains (Figure 18) exhibited similar characteristics in the CSF, with MTBR tau 243 showing the highest specificity for the dementia stage of Alzheimer's disease. Notably, a high correlation was observed between the cleaved form of tau 368 and the uncleaved form of MTBR tau 354 in the CSF (r=0.8382) (Figure 29). MTBR tau 243 was the only species that reliably distinguished the clinical stage of Alzheimer's disease.

[0221] Results - Intermediate domain-independent MTBR tau-243 as a specific biomarker for Alzheimer's disease staging: The gradual increase in levels of MTBR tau-243 species across the clinical dementia stages of Alzheimer's disease suggests that it may be a reliable predictor of disease progression. Next, we tested whether those MTBR tau species (MTBR tau-243, MTBR tau-299, and MTBR tau-354) had the highest correlations with the results of cognitive tests such as the CDR-Sum of Boxes (CDR-SB) and Mini-Mental State Examination (MMSE). The intermediate domain-independent MTBR tau-243 species in the amyloid-positive group was found to be highly correlated with both CDR-SB and MMSE (r = 0.5562, p < 0.0001 and r = -0.5433, p < 0.0001, respectively) (Figures 30 and 31). Other species levels had much lower or no significant correlations with the cognitive tests (Table 5), suggesting that CSF MTBR tau-243 specifically discriminates clinical stages and overall disease progression through the progression of the clinical stages of Alzheimer's disease from the asymptomatic stage.

Table 7

[0222] Results - CSF MTBR tau in the long-term Alzheimer's disease cohort: From a cross-sectional cohort, a subset of participants (n = 28) was followed for 2 to 9 years to measure the long-term trajectory of MTBR tau in CSF (Table 6). MTBR tau species enriched in the Alzheimer's disease brain (MTBR tau-243, MTBR tau-299, and MTBR tau-354) increased significantly over time in the amyloid-positive group (p < 0.01 by two-sided paired t-test between the first and second hospital visits), but did not increase in the amyloid-negative group except for MTBR tau-243 (Figure 32). The amyloid-negative group also showed a slight long-term increase in MTBR tau-243, but it was lower than that observed in the amyloid-positive group (mean difference = 0.4926 and 2.208 in the amyloid-negative and positive groups, respectively).

[0223] Figure 21 shows the long-term rate of change in MTBR tau species concentrations in individual participants. Notably, one participant with the highest post-disease CDR (Participant A) (CDR changed from 1 to 2 over 7 years) showed specific trajectory profiles for each MTBR tau species. MTBR tau 243 continued to increase even in mild AD (CDR=1) to moderate AD (CDR=2), while MTBR tau 299 and MTBR tau 354 decreased in this participant's CSF after mild AD. Other participants in the amyloid-positive group were diagnosed with pre-disease AD or very mild AD (CDR=0 or 0.5, respectively) at their first visit, and the increasing trends at various levels were observed in the majority of participants, supporting the findings from the cross-sectional cohort. [Table 8]

[0224] Results - Correlation with Tau-PET contrast: Tau pathology measured by Tau-PET scanning was strongly correlated with cognitive decline and clinical stage of Alzheimer's disease (Arriagada et al., 1992; Johnson et al., 2016; Ossenkoppele et al., 2016; Bejanin et al., 2017; Jack et al., 2018; Gordon et al., 2019). Next, we investigated whether MTBR tau in CSF correlated with brain tau pathology assessed by Tau-PET (Figure 22). Intermediate domain-independent MTBR tau 243 was significantly correlated with Tau-PET SUVR (r=0.7588, p<0.0001), while MTBR tau 299 and MTBR tau 354 were much less correlated (r=0.4584, p=0.0056 and r=0.4375, p=0.0086, respectively). Tau species containing residues 226-230 also showed a high correlation with Tau-PET SUVR (r=0.6248, p<0.0001, Table 7), but lower than that observed with MTBR tau 243. This suggests that CSF MTBR tau 243 and surrounding regions may be surrogate biomarkers for tau aggregation in the brain. The ability to specifically and quantitatively track tau pathology in the brain is a highly desirable biomarker in Alzheimer's disease clinical trials. [Table 9]

[0225] Discussion: The MTBR region of tau has been primarily tested in brain aggregates, but not extensively in CSF. In this study, we demonstrated the presence and quantification of the MTBR region of tau in CSF samples from human participants using sensitivity and antibody-independent methods for analyzing CSF tau. Previous studies using antibody-dependent assays (Meredith et at., 2013; Sato et at., 2018) may not have been able to detect MTBR-containing tau species in CSF due to assay limitations, including antibody specificity or sensitivity or the ability to recover forms that may be selected by MTBR species in CSF. Alternatively, MTBR tau can be cleaved by various proteases, producing fragments that are undetectable by conventional immunoassays or immunoprecipitation, followed by MS assays (Gamblin et al., 2003; Cotman et al., 2005; Zhang et al., 2014; Zhao et al., 2016; Chen et al., 2018; Quinn et al., 2018). Surprisingly, in this study, robust concentrations of MTBR tau were detected at approximately 1%–10% compared to intermediate domain tau species using the PostIP-CX method followed by mass spectrometry (Figures 19 and 2A).

[0226] Until now, it was unclear whether MTBR tau is involved in extracellular tau proliferation, as extracellular levels were thought to be too low for pathological dissemination and diffusion. These new findings on the stoichiometry of MTBR in CSF support the hypothesis that MTBR-containing species diffuse extracellularly, similar to pathological species. These measurements provide information on potential targets for anti-tau drug development for Alzheimer's disease and offer quantitative measurements of targets, as indicated by the 2-3-fold increase in MTBR tau species in CSF from Alzheimer's patients. However, a limitation is that pathological species may be present in interstitial fluid (ISF) rather than CSF (Colin et al., 2020). While CSF tau is primarily derived from ISF (Reiber, 2001), and several reports have shown that human CSF from Alzheimer's disease patients can induce tau dissemination in transgenic mouse models (Skachokova et al., 2019), further research is needed to clarify whether the tau species detected in CSF reflect pathological tau that can proliferate in the human brain.

[0227] Previous studies have shown that inoculation of mouse brains with tau aggregates from Alzheimer's disease brains induced severe tau pathology (Guo et al., 2016; Narasimhan et al., 2017); however, there are no reports identifying extracellular pathological tau species linked to disease progression in humans. This led to the investigation of CSF MTBR tau species changes in Alzheimer's disease and the exploration of its suitability as a novel Alzheimer's disease biomarker. CSF MTBR tau levels were found to be elevated in Alzheimer's disease and to correspond to species enriched in the insoluble fraction of Alzheimer's disease brains. The finding that CSF MTBR tau correlates with the clinical stage of Alzheimer's disease and tau pathology suggests that MTBR tau is associated with tau proliferation in Alzheimer's disease, although the nature (i.e., monomer, oligomer, or fibrilloma) and origin of extracellular CSF MTBR tau remain unknown. CSF MTBR tau may originate from brain aggregates or neurons that actively secrete monomer species, and further studies should be designed to address this issue.

[0228] Interestingly, the trajectory of changes in CSF MTBR tau species was found to differ across various regions of the MTBR and across different clinical stages of Alzheimer's disease. This finding was hypothesized to be due to structural changes in tau determined by recent Cryo-EM findings. Cryo-EM analysis suggests an ordered β-sheet core of tau aggregates starting from residue 306 (Fitzpatrick et al., 2017). Therefore, MTBR tau 354 (containing residues 354-369), MTBR tau 299 (containing residues 299-317), and MTBR tau 243 (containing residues 243-254) represent the inner, borderal, and outer regions of the fibrous core, respectively. In contrast to both MTBR tau 354 and MTBR tau 299, MTBR tau 243 levels gradually increased across all disease stages. Levels of MTBR tau 243 and its neighboring region (i.e., residues 226-230) in CSF also correlated strongly with Tau-PET SUVR performance (Figure 22 and Table 7), supporting the hypothesis that MTBR tau 243 and possibly its neighboring region are deposited in brain tau aggregates and secreted extracellularly (Figure 17).

[0229] The finding that MTBR tau is highly correlated with Alzheimer's disease pathology and clinical progression stages provides important insights into promising targets for therapeutic anti-tau drugs for the treatment of tauopathy. For example, a novel tau antibody that recognizes an epitope in the upstream region of the MTBR (residues 235-250) shows significant and selective ability to mitigate tau dissemination from Alzheimer's disease and progressive supranuclear palsy brains in cell-based assays (Courade et al., 2018). These findings indicate that the upstream region of the MTBR is related to extracellular, pathological tau. This is supported by antibodies that mitigate tau pathological proliferation to distal brain regions in transgenic mice injected with human Alzheimer's disease brain extracts (Albert et al., 2019). Other novel tau antibodies that recognize the epitope in the upstream region of the MTBR (residues 249-258) show reduced induction of tau pathology in cells and in vivo transgenic mouse models seeded with human Alzheimer's disease brain extracts (Vandermeeren et al., 2018). Antibody-targeting MTBR tau 299 and MTBR tau 354 species also mitigate tau pathology induced by seeding with P301L tau or Alzheimer's disease brain extracts (Weisova et al., 2019; Roberts et al., 2020), supporting the hypothesis that species containing specific regions of the MTBR are responsible for the spread of tau pathology in tauopathy.

[0230] In summary, the findings indicate that MTBR tau species exist as C-terminal fragments in CSF, are specifically increased in Alzheimer's disease, and reflect enrichment observed in Alzheimer's brain aggregates. The findings suggest that specific MTBR-containing species (MTBR tau 299 and MTBR tau 243) are promising CSF biomarkers for evaluating amyloid and tau pathology in Alzheimer's disease. In particular, intermediate domain-independent MTBR tau 243 parallels disease progression and tau pathology in Alzheimer's disease and may be utilized as a biomarker for tau pathology and a target for novel anti-tau antibody therapies.

[0231] Example 4 A further sample processing method called "PostIP-IP" was developed and compared with the PostIP-CX method described in Examples 1 and 2. An exemplary workflow of the PostIP-IP method is shown in Figure 33.

[0232] CSF samples obtained from the LOAD100 cohort described in Example 2 were processed using the PostIP-CX method (Example 1) or the PostIP-IP method (This Example), and then analyzed by LC-MS as generally described in Example 2.

[0233] As shown in Figure 34A, the trypsin-digested peptide LQTA showed different profiles between the two samples. For example, the amount of LQTA that continued to increase even in clinically onset, as measured in samples treated with the PostIP-CX method, was not observed in samples treated with the PostIP-IP method. In contrast, the trypsin-digested peptides HVPG and IGSL showed similar profiles between samples treated with the PostIP-CX and PostIP-IP methods (Figures 34B and 34C). Further analysis of the trypsin-digested peptides suggests that there may be a significant cleavage event occurring at R1 in the amino acid sequence between LQTA and IGST peptides (Figure 35A).

[0234] The abundance of total trypsin digested peptides downstream of LQTA (i.e., C-terminus) showed a good correlation between sample processing methods, but (R 2 Based on the values ​​(see Figure 35), the R between trypsin-digested peptide HVPG and IGSL 2A significant increase in values ​​was observed. To explore this further, samples were grouped by CDR score – more specifically, cognitively impaired subjects (Cl, CDR > 0.5) and non-cognitively impaired subjects (CDR < 0.5). As shown in Figure 36B, only cognitively impaired subjects showed a low correlation between HVPG and IGSL trypsin-digested peptides between samples treated with the PostIP-CX versus PostIP-IP method. This may reflect the development of tau aggregates in the brain, which are supplemented in the tau region containing HVPG and IGSL trypsin-digested peptides in aggregates, thereby leading to changes in the amount of tau species containing these peptides in the CSF and other body fluids.

[0235] Overall, these data suggest that the choice of sample processing method affects the ability to detect MTBR tau species that reproduce tau pathology in the CNS in CSF and other bodily fluids.

[0236] Example 5 In this example, CSF samples from subjects in three clinical cohorts were treated by the IP method (Example 1) and the PostIP-IP method (Example 4) and evaluated by mass spectrometry as generally described in Example 3. Samples were obtained from control subjects (n=93), subjects with amyloid-positive AD (n=41), and subjects with non-AD tauopathy (n=87). Subjects with non-AD tauopathy were clinically diagnosed with CBD or CBD / PSP (n=20), FTD (n=29), FTLD (R406W n=7, P301L n=3), PSP (n=18), and unconfirmed non-AD dementia (n=3). Trypsin-digested peptides specific to the 3R and 4R isoforms were of particular interest. CSF Aβ42 / 40 was measured by mass spectrometry as commonly described in Ovod et al., Alzheimers Dement J. Alzheimers Assoc, 2017, 13:841-849. Amyloid status was defined using a cutoff value of 0.085 (i.e., amyloid-positive >0.085, amyloid-negative <0.085). pT217% was measured by mass spectrometry as previously described.

[0237] As shown in Figure 37, the trypsin-digested peptide VQIV / LDLS ratio in the samples distinguishes non-AD tauopathy from controls in samples treated by the PostIP-IP method (Figure 37C), but not in samples treated by the IP method (Figure 37B). Further analysis of samples treated by the PostIP-IP method showed that the abundance of LDLS in CSF from subjects with non-AD tauopathy was lower compared to controls (Figure 38).

[0238] An increase in the VQIV / LDLS ratio was measured in PostIP-IP treated samples obtained from subjects with non-AD tauopathy, but not in samples from subjects with AD or control subjects (Figure 39). Further analysis of trypsin digested peptides after PostIP-IP sample treatment identified a lower correlation between R1-R2 trypsin digested peptides (e.g., IGST, VQII, LDLS, etc.) and late R2-R3 trypsin digested peptides (e.g., HVPG, etc.) in subjects with non-AD tauopathy compared to subjects with AD or control subjects (Figure 40, Table 8). When comparing among non-AD tauopathy subjects, certain subjects with PSP, CBD, and FTD were outliers (Figure 41). Similar results were obtained when comparing with the trypsin digested peptide IGSL, rather than HPVG (Figure 42).

[0239] Overall, these data suggest that CSF tau profiles measured after PostIP-IP sample processing reflect the state of brain tau aggregation. For example, 4R-tauopathy contains brain-insoluble tau enriched with the R2 region, including the VQII trypsin-digested peptide, and some CSF samples from subjects with 4R-tauopathy showed reduced levels of trypsin-digested peptides IGST, VQII, and LDLS compared to HVPG or IGSL. This was not observed in subjects with AD. Based on these data, a method for identifying 4R-tauopathy should focus on enrichment of the R2 region of MTBR tau. [Table 10]

[0240] Example 6 In this example, further analysis of CSF and brain samples obtained from a single clinical cohort of subjects including non-AD tauopathy provided further evidence of the usefulness of intermediate-domain-independent MTBR tau for distinguishing CSF samples obtained from subjects with non-AD tauopathy from CSF samples obtained from subjects with AD. The subjects in this cohort included subjects with AD (n=28), subjects clinically diagnosed with CBD or CBD / PSP (n=20), subjects clinically diagnosed with FTD (n=22), and subjects clinically diagnosed with PSP (n=11). CSF samples obtained from these subjects were processed using the PostIP-IP method generally described in Example 4 and evaluated by mass spectrometry generally described in Example 3. Brain-insoluble tau was evaluated as described in Example 3.

[0241] As described in Example 5, analysis of trypsin-digested peptides after PostIP-IP sample treatment of CSF identified a low correlation between R1 and early R2 trypsin-digested peptides (e.g., IGST, VQII, LDLS, etc.) and late R2, R3, and / or R4 trypsin-digested peptides (e.g., IGSL, etc.) in subjects with non-AD tauopathy (Figures 43, 44, and 45). It was hypothesized that in CSF, tau species in non-AD tauopathy contain (1) fewer R1 and R2 and (2) more R3 and R4 than tau species in AD, and that this is an indication of cerebral tau deposition (Figure 46). To test this hypothesis, cerebral insoluble tau was analyzed. As shown in Figure 47, the trypsin-digested peptide VQII is enriched in cerebral tau aggregates in 4R-tauopathy. Trypsin-digested peptides FIVPG and IGSL are also enriched in brain tau aggregates, but less so compared to AD. These data further support the R1 or R2 to R3 or R4 ratio as a way to distinguish non-AD tauopathy from AD.

[0242] Next, the analysis was further expanded to include CSF samples obtained from genetically confirmed FTLD cases (R406W, n=7; P301L, n=3), CSF samples obtained from control subjects (n=44), and additional CSF samples obtained from subjects with AD (n=41). These CSF samples were processed using the PostIP-IP method generally described in Example 4 and evaluated by mass spectrometry generally described in Example 3. Analysis of trypsin digested peptides after PostIP-IP sample processing of CSF similarly identified low correlations between R1 and early R2 trypsin digested peptides (e.g., IGST, VQII, LDLS, etc.) and late R2, R3, and / or R4 trypsin digested peptides (e.g., IGSL, etc.) in subjects with non-AD tauopathy (Figures 48 and 49). These data confirm the applicability of the ratio of R1 or R2 to R3 or R4 levels as a method for distinguishing non-AD tauopathy from AD, and also demonstrate its ability to distinguish non-AD tauopathy from controls. In particular, CSF samples obtained from genetically confirmed FTLD cases further rigorize the analysis.

Claims

1. For use in methods for measuring tau in biological samples, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, Methods for measuring tau in biological samples are (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Performing liquid chromatography-mass spectrometry (LC / MS) on a sample containing tau proteolytic peptides to detect at least one tau proteolytic peptide and to measure its quantity, composition.

2. For use in methods for measuring Alzheimer's disease (AD)-related pathology in subjects, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method for measuring Alzheimer's disease (AD)-related pathology in the subject is (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in CSF or a blood sample by the following: Here, the tau being measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8, or a combination thereof. Here, the amount or ratio of the measured MTBR tau species represents the AD-related pathology in the brain of the subject. composition.

3. The composition according to claim 2, wherein the AD-related pathology is tau deposition in the target brain.

4. The composition according to claim 2, wherein the AD-related pathology is amyloid-beta deposition in the brain or cerebral arteries of the target.

5. For use in a method for measuring Alzheimer's disease (AD)-related tau deposition in the brain of a subject, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method for measuring Alzheimer's disease (AD)-related tau deposition in the brain of a subject is (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in CSF or a blood sample by the following: Here, the tau being measured is the MTBR tau species containing the amino acid sequence of SEQ ID NO: 3 (LQTAPVPMPDLK), and the amount of this MTBR tau species measured represents AD-related tau deposition in the brain of the subject. composition.

6. For use in a method for measuring Alzheimer's disease (AD)-related tau deposition in the brain of a subject, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method for measuring Alzheimer's disease (AD)-related tau deposition in the brain of a subject is (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in CSF or a blood sample by the following: Here, the tau being measured is an MTB-TAU tau species containing the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or a combination thereof. Here, the amount or ratio of the measured MTB-TAU species represents AD-related tau deposition in the brain of the subject. composition.

7. For use in assisting the diagnosis of Alzheimer's disease, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, Methods to assist in the diagnosis of Alzheimer's disease are (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in a CSF or blood sample, wherein the tau to be measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8, or a combination thereof; wherein, A difference of 1.5σ or more in the quantified MTBR tau species indicates Alzheimer's disease, where σ is the standard deviation defined by a normal distribution measured in an amyloid-negative control population using PET contrast and / or Aβ42 / 40 measurement in CSF. composition.

8. For use in a method to assist in measuring the progression of Alzheimer's disease (AD) in subjects, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method to assist in measuring the progression of Alzheimer's disease (AD) in subjects is: (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The tau in the first and second CSF or blood samples is measured by a method comprising the following: the tau being measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8, or a combination thereof; and The difference in quantified MTB-BR tau species between the second and first samples was calculated, where a statistically significant increase in quantified MTB-BR tau species in the second sample indicates the progression of Alzheimer's disease in the subject. composition.

9. A composition according to any one of claims 5 to 8, wherein the target is amyloid-negative.

10. The composition according to claim 9, wherein the subject does not have dementia.

11. The composition according to claim 9, wherein the subject has dementia.

12. A composition according to any one of claims 5 to 8, wherein the target is amyloid-positive.

13. The composition according to claim 12, wherein the subject does not have dementia.

14. The composition according to claim 12, wherein the subject has dementia.

15. The composition according to claim 9 or claim 12, wherein the target has a CDR score of 0.5 to 1.

0.

16. The composition according to claim 9 or claim 12, wherein the subject has a CDR score of >1.0 to 2.0 (moderate AD).

17. The composition according to claim 9 or claim 12, wherein the subject has a CDR score of >2.

0.

18. A composition according to any one of claims 5 to 17, wherein the method further comprises the quantification of amyloid beta, N-terminal tau, intermediate domain tau, post-translational modification of tau, or ApoE isoform identification in a biological or CSF sample.

19. For use in a method for measuring tau pathology in the brain of a subject, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method for measuring tau pathology in the target brain is (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in CSF or a blood sample, wherein the tau to be measured is an MTBR tau species containing the amino acid sequence of SEQ ID NO: 2, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 3, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 4, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 5, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 9, or a combination thereof, wherein the amount of quantified MTBR tau species or their ratios represents the tau pathology in the brain of the subject. composition.

20. For use in a method to aid in the identification of 4R-tauopathy, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method to aid in the identification of 4R-tauopathy is, (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in a biological sample, wherein the tau to be measured is (a) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 9 and (b) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 4, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 5, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, or an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8; Here, the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) assists in the identification of 4R-tauopathy. composition.

21. For use in a method to aid in the differentiation between 3R-tauopathy or 4R-tauopathy and Alzheimer's disease, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method to help distinguish between 3R-tauopathy or 4R-tauopathy and Alzheimer's disease is (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in a biological sample, wherein the tau being measured is (a) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 2, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 4, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 5, or a combination thereof, and (b) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8, or a combination thereof. Here, the ratio of the quantified MTBR species in (a) to the quantified MTBR species in (b) helps in distinguishing between 3R-tauopathy or 4R-tauopathy and Alzheimer's disease. composition.

22. For use in a method to assist in the diagnosis of 4R-tauopathy, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method to aid in the diagnosis of 4R-tauopathy is, (a) N-terminal tau, intermediate domain tau, N-terminal tau and intermediate domain tau, N-terminal tau and amyloid beta, intermediate domain tau and amyloid beta, or N-terminal tau, intermediate domain tau and amyloid beta are reduced by affinity depletion in a biological sample, where the biological sample is a blood sample or a CSF sample; (b) Enriching MTBR tau by a method comprising (i) removing further proteins from a biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, then purifying tau from the supernatant by solid-phase extraction, or (ii) affinity purification of MTBR tau to produce enriched MTBR tau of (i) or (ii); (c) Enriched MTBR tau was cleaved with protease to obtain a sample containing tau proteolytic peptides; and (d) Liquid chromatography-mass spectrometry (LC / MS) of the sample containing tau proteolytic peptides is performed to detect at least one tau proteolytic peptide and to measure its quantity. The method includes measuring tau in a CSF or blood sample, wherein the tau to be measured is (a) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 2, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 4, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 5, or a combination thereof, and (b) an MTBR tau species containing the amino acid sequence of SEQ ID NO: 6, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 7, an MTBR tau species containing the amino acid sequence of SEQ ID NO: 8, or a combination thereof; wherein, A difference of 1.5σ or more in the quantified MTBR tau species indicates 4R-tauopathy, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as measured by PET contrast and / or Aβ42 / 40 measurement in CSF. composition.

23. A composition according to any one of claims 19 to 22, wherein the subject does not have dementia.

24. The composition of claim 23, wherein 4R-tauopathy is corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia, or progressive supranuclear palsy.

25. A composition according to any one of claims 19 to 22, wherein the subject has dementia.

26. The composition of claim 25, wherein 4R-tauopathy is corticobasal degeneration, frontotemporal lobar degeneration, frontotemporal dementia, or progressive supranuclear palsy.

27. For use in assisting the determination of whether or not a subject requires treatment, (1) An epitope binder that specifically binds to the N-terminal tau epitope, (2) An epitope binder that specifically binds to the epitope of the intermediate domain tau, or (3) Epitope conjugates that specifically bind to amyloid-beta epitopes, A composition comprising, A method to help determine whether or not the subject needs treatment is, Prepare a treated CSF or blood sample obtained from a subject in which (a) intermediate domain tau is depleted and (b) MTBR tau is enriched; In the processed sample, the number of MTBR tau species containing the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or combinations thereof is quantified; here, A difference of 1.5σ or more in the quantified MTBR tau species or the ratio of quantified MTBR tau species indicates that the subject requires treatment to modify tau pathology, where σ is the standard deviation defined by a normal distribution measured in a control population that does not have clinical signs or symptoms of tauopathy and is amyloid-negative as measured by PET contrast and / or Aβ42 / 40 measurement in CSF, where the amount or ratio of quantified MTBR tau species represents tau pathology in the subject's brain. composition.

28. A composition according to any one of claims 1 to 27, comprising an epitope binder that specifically binds to the N-terminal tau epitope.

29. The composition of claim 28, wherein the epitope binder that specifically binds to the N-terminal tau epitope is an epitope binder that specifically binds to the epitope within amino acids 1 to 103 (including both ends) of tau 441.

30. The composition according to claim 29, wherein the epitope binder is HJ8.5, which specifically binds to the epitopes within amino acids 1 to 103 (including both ends) of tau 441.

31. A composition according to any one of claims 1 to 27, comprising an epitope binder that specifically binds to an epitope of the intermediate domain tau.

32. The composition of claim 31, wherein the epitope binding agent that specifically binds to the epitope of the intermediate domain tau is an epitope binding agent that specifically binds to the epitope within amino acids 104 to 243 (including both ends) of tau 441.

33. The composition according to claim 32, wherein Tau1 is an epitope binder that specifically binds to the epitopes within amino acids 104-243 (including both ends) of tau 441.

34. A composition according to any one of claims 1 to 27, comprising an epitope binder that specifically binds to an amyloid-beta epitope.

35. The composition of claim 34, wherein the epitope binder that specifically binds to amyloid beta is HJ5.

1.

36. A composition according to any one of claims 1 to 35, to be used in combination with a precipitating agent.

37. The composition of claim 36, wherein the precipitant is an acid.

38. A composition according to any one of claims 1 to 37, to be used in combination with a reverse-phase adsorbent that adsorbs tau.

39. The composition of claim 38, wherein the reverse-phase adsorbent is alkyl-bonded silica, aryl-bonded silica, styrene / divinylbenzene material, or N-vinylpyrrolidone / divinylbenzene material.

40. A composition according to any one of claims 1 to 39, to be used in combination with an epitope binder that specifically binds to the epitope of MTBR tau.

41. An epitope binder that specifically binds to the epitope of MTBR tau is Within amino acids 221-441 (including both ends) of tau 441 or Within amino acids 235-441 (including both ends) of tau 441 or Within amino acids 235-368 (including both ends) of tau 441 or Within amino acids 244-368 (including both ends) of tau 441 or Within amino acids 244-299 (including both ends) of tau 441 The composition of claim 40, which specifically binds to the epitope of .

42. The composition of claim 40 or 41, wherein the epitope conjugate that specifically binds to the epitope of MTBR tau is an antibody selected from the group consisting of 77G7, RD3, RD4, UCSB1017, PT76, E2814, and 7G6.

43. A composition according to any one of claims 1 to 42, to be used in combination with a protease.

44. The composition of claim 43, wherein the protease is trypsin.

45. For use in methods for measuring tau in biological samples, (1) Precipitant, (2) A reversed-phase adsorbent that adsorbs tau, or (3) Protease A composition comprising, Methods for measuring tau in biological samples (a) Prepare a biological sample selected from a blood sample or a CSF sample; (b) Remove proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain the supernatant; (c) Purify tau from the supernatant by solid-phase extraction; (d) Cleavage purified tau with protease to obtain a sample containing tau proteolytic peptides; and (e) Performing liquid chromatography-mass spectrometry on a sample containing tau proteolytic peptides to detect and measure the amount of at least one tau proteolytic peptide, composition.

46. The composition of claim 45, comprising a precipitating agent.

47. The composition of claim 46, wherein the precipitant is an acid.

48. The composition according to claim 45, comprising a reversed-phase adsorbent that adsorbs tau.

49. The composition of claim 48, wherein the reversed-phase adsorbent is alkyl-bonded silica, aryl-bonded silica, styrene / divinylbenzene material, or N-vinylpyrrolidone / divinylbenzene material.

50. The composition of claim 45, comprising a protease.

51. The composition according to claim 50, wherein the protease is trypsin.