Anti-tau MTBR antibodies and methods for detecting closed fragments of

Abstract

Provided herein are antibodies or fragments thereof that specifically bind to the microtubule binding region (MTBR) of tau, and uses thereof. Further provided are methods of detecting MTBR species in blood or cerebrospinal fluid, as well as the use of such detection for diagnosing, prognosing or staging pathological characteristics and / or clinical symptoms of tauopathy, and selecting a treatment suitable for a given disease stage.

Description

Technical Field

[0001] The present invention relates to antibodies or fragments thereof that specifically bind to the microtubule binding region (MTBR) of tau, and uses thereof. The present invention further relates to methods for detecting mature neurofibrillary tangles in patients suffering from tauopathy. The present invention also relates to detecting MTBR species in blood or cerebrospinal fluid, and the use of such detection for diagnosing, prognosing or staging pathological features and / or clinical symptoms of tauopathy, and selecting treatments suitable for a given disease stage. Background Art

[0002] Tau protein accumulation in the brain as insoluble aggregates is one of the hallmarks of Alzheimer's disease and other neurodegenerative diseases known as tauopathy. Tau pathology appears to spread across brain regions and spreads between cells in a prion-like manner through specific pathological tau species, although the properties and diffusion processes of these species (i.e. monomers, oligomers, and fibrils) are still uncertain (Frost et al., 2009; Goedert et al., 2010, 2017; Sanders et al., 2014; Wu et al., 2016; Mirbaha et al., 2018; Lasagna-Reeves et al., 2012). Tau has six different full-length protein subtypes. In addition, Tau has more than one hundred potential post-translational modification sites, including phosphorylation, plus multiple truncation sites (Meredith et al., 2013; Sato et al., 2018; Barthélemy et al., 2019; Cicognola et al., 2019; Blennow et al., 2020). Therefore, it is challenging to identify specific pathological tau species involved in tau spread. Several mass spectrometry (MS) studies suggest that the microtubule binding region (MTBR) of tau is enriched in aggregates in Alzheimer's disease brains (Taniguchi-Watanabe et al., 2016; Roberts et al., 2020). In addition, a series of cryo-electron microscopy (Cryo-EM) studies confirmed that the core structure of tau aggregates is composed of subsegments of the MTBR domain, and the specific conformation depends on the tau protein disease (Fitzpatrick et al., 2017; Falcon et al., 2018, 2019; Zhang et al., 2020). These findings strongly suggest that MTBR tau is critical for tau aggregation. However, these studies used postmortem brain tissue. Little is known about the pathophysiology of corresponding extracellular MTBR-containing tau species in biological samples such as CSF and blood, which can be used as surrogate biomarkers for brain tau aggregation in living humans.

[0003] CSF is routinely obtained from study participants via lumbar puncture during clinical visits. Previous CSF tau biomarker studies suggest that MTBR tau is absent in CSF and that they are concentrated in the N-terminal and middle domain regions (Meredith et al., 2013; Sato et al., 2018). Species consisting of the N-terminal to middle domain appear to be actively secreted from neurons into the extracellular space after truncations between the middle domain and the MTBR domain (Sato et al., 2018). Detection of MTBR tau species has been reported (Barthélemy et al., 2016b, a), but has not been characterized in relation to disease. Recently, a tau species containing a cleavage at residue 368 (tau368) within repeat region 4 (R4) was identified in CSF (Blennow et al., 2020). However, given that changes in region, truncation, and conformational structure were not captured by antibodies, it is unclear whether tau368 reflects the overall pool of MTBR tau species.

[0004] Advances in high-resolution mass spectrometry have led to new methods for measuring protein abundance in biological samples. Despite advances in instrumentation and data analysis software, sample preparation remains a huge challenge. The choice of sample preparation method affects the observed metabolite profile and data quality, and can ultimately affect reported results. This is especially true for low-abundance proteins and peptides in biological samples. Peptides that fall into this category include many proteolytic fragments of full-length proteins, which are differentially produced in various disease processes.

[0005] Thus, there remains a need in the art for improved compositions and methods that can be used to quantify low-abundance MTBR tau species in biological fluids. Summary of the invention

[0006] Provided herein is an isolated anti-MTBR tau antibody. The isolated anti-MTBR tau antibody may comprise (a) L1, L2 and L3 from the light chain variable region of the sequence shown in SEQ ID NO:6, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:7; (b) L1, L2 and L3 from the light chain variable region sequence shown in SEQ ID NO:2, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:3; (c) L1, L2 and L3 from the light chain variable region of the sequence shown in SEQ ID NO:4, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:5; or (d) L1, L2 and L3 from the light chain variable region of the sequence shown in SEQ ID NO:8, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:9. The isolated anti-MTBR tau antibody may comprise (a) a light chain variable region from the sequence shown in SEQ ID NO:6 and a heavy chain variable region from the sequence shown in SEQ ID NO:7; (b) a light chain variable region from the sequence shown in SEQ ID NO:2 and a heavy chain variable region from the sequence shown in SEQ ID NO:3; (c) a light chain variable region from the sequence shown in SEQ ID NO:4 and a heavy chain variable region from the sequence shown in SEQ ID NO:5; or (d) a light chain variable region from the sequence shown in SEQ ID NO:8 and a heavy chain variable region from the sequence shown in SEQ ID NO:9. The isolated antibody may be a monoclonal antibody or an antibody fragment. The isolated antibody may be a monoclonal antibody.

[0007] Provided herein is a method for detecting insoluble tau aggregates in a plasma sample, which may include (a) purifying endogenous cleavage fragments of tau by contacting the plasma sample with an epitope binder that specifically binds to an epitope within amino acids 235-242 of SEQ ID NO: 1, without first contacting the endogenous cleavage fragments of tau with a protease in vitro; (b) contacting the purified endogenous cleavage fragments of tau with an Arg-C endopeptidase to obtain a proteolytic MTBR-tau243 peptide comprising amino acids 243-254 of SEQ ID NO: 1; and (c) detecting the proteolytic MTBR-tau243 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein detection of the proteolytic MTBR-tau243 peptide indicates insoluble tau aggregates in the sample. The purification step may include immunoprecipitation. The proteolytically cleaved MTBR-tau243 peptide may comprise one or more of MTBR-tau243-254, MTBR-tau243-256V, MTBR-tau243-256V (dN), and MTBR-tau212-221. The step of detecting the proteolytically cleaved MTBR-tau243 peptide may comprise performing an immunoassay or liquid chromatography-mass spectrometry. The epitope binding agent may be an anti-tau antibody comprising a light chain variable region from the sequence shown in SEQ ID NO: 6 and a heavy chain variable region from the sequence shown in SEQ ID NO: 7. The anti-tau antibody may be HJ32.11. The solution comprising the proteolytically cleaved MTBR-tau243 peptide may be desalted by solid phase extraction prior to step (c). The method may further comprise one or more of the following: detecting and quantifying one or more of amyloid beta, N-terminal tau, middle domain tau, post-translational modifications of tau, and ApoE subtypes in a biological or CSF sample. The step of quantifying amyloid β may include quantifying the Aβ42 / 40 value or quantifying the post-translational modification of tau, wherein the post-translational modification of tau includes one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111, and phosphorylated tau208.

[0008] Provided herein is a method for detecting insoluble tau aggregates in a cerebrospinal fluid (CSF) sample, which may include (a) affinity depleting the CSF sample by contacting the sample with an affinity depleting agent, the affinity depleting agent comprising one or more epitope binding agents, each of which binds to one of N-terminal tau, middle domain tau, and long MTBR tau, but does not bind to an antigen within amino acids 235-256 of SEQ ID NO: 1, wherein the CSF sample comprises a cleavage fragment of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, middle domain tau, and long MTBR tau, and wherein the enriched sample is enriched for epitope binding agents comprising SEQ ID NO: 1. NO:1 amino acid 235-256 of tau endogenous cleavage fragment (endogenous MTBR-tau243 peptide); (b) by making the enriched sample contact with an immunoprecipitant and immunoprecipitate the enriched sample, the immunoprecipitant comprising an epitope binding agent that binds to the endogenous MTBR-tau243 peptide to obtain a purified sample; (c) the endogenous MTBR-tau243 peptide in the purified sample is contacted with a protease to obtain a sample containing proteolytic MTBR-tau243 peptides; and (d) by performing liquid chromatography-mass spectrometry (LC / MS) or immunoassay to detect proteolytic MTBR-tau243 peptides, wherein the detection of proteolytic MTBR-tau243 peptides can indicate insoluble tau aggregates in the CSF sample. The sample containing proteolytic MTBR-tau243 peptides can be desalted before step (d). The affinity depletion agent may comprise: an epitope binder that specifically binds to an epitope within amino acids 1 to 243 of SEQ ID NO: 1; a first epitope binder that specifically binds to an epitope within amino acids 1 to 103 of tau-441, and a second epitope binder that specifically binds to an epitope within amino acids 104 to 243 of SEQ ID NO: 1; or, a first epitope binder, a second epitope binder, and a third epitope binder that specifically binds to an epitope within amino acids 257-264 of SEQ ID NO: 1. The first epitope binder may be HJ8.5, the second epitope binder may be one or more of Tau1 and HJ8.7, and the third epitope binder may be HJ34.8. The affinity depletion agent may comprise Tau1, HJ8.5, and HJ8.7. The affinity depletion agent may further comprise HJ34.8. The immunoprecipitant may comprise HJ32.11.The proteolytic MTBR-tau243 peptide may comprise a cleaved tau fragment comprising one or more of MTBR-tau243-254, MTBR-tau243-255 (dN), MTBR-tau243-257K (dN), MTBR-tau243-256V, MTBR-tau243-256V (dN), MTBR-tau243-258S, and MTBR-tau212-221. The proteolytic MTBR-tau243 peptide may comprise one or more of MTBR-tau243-256V and MTBR-tau243-256V (dN). The protease may comprise trypsin. The CSF sample may comprise an internal standard. The internal standard may be. 15 N-0N3R-tau and 15 One or more of N-2N4R-tau. The method may further include one or more of the following: detecting and quantifying one or more of amyloid beta, N-terminal tau, middle domain tau, and post-translational modifications of tau, and may include identifying ApoE subtypes in biological or CSF samples. The step of quantifying amyloid beta may include quantifying one or more of Aβ42 / 40 values ​​and post-translational modifications of tau, and the post-translational modifications of tau may include one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111, and phosphorylated tau208.

[0009] Provided herein is a method for detecting insoluble tau aggregates in a plasma sample, which may include (a) affinity depleting the plasma sample by contacting the sample with an affinity depleting agent, the affinity depleting agent comprising one or more epitope binding agents, each of which binds to one of N-terminal tau, middle domain tau, and long MTBR tau, but does not bind to an antigen within amino acids 235-256 of SEQ ID NO: 1, wherein the plasma sample comprises cleavage fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, middle domain tau, and long MTBR tau, and wherein the enriched sample is enriched for epitope binding agents comprising SEQ ID NO: 1. (a) performing immunoprecipitation on the enriched sample by contacting the enriched sample with an immunoprecipitant, wherein the immunoprecipitant comprises an epitope binder that binds to the endogenous MTBR-tau243 peptide to obtain a purified sample; (c) contacting the endogenous MTBR-tau243 peptide with a protease including an Arg-C endopeptidase to obtain a sample comprising proteolytic MTBR-tau243 peptides; and (d) detecting the proteolytic MTBR-tau243 peptides by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein detection of one or more of MTBR-tau243-256V, MTBR-tau243-256V(dN) and MTBR-tau212-221 indicates insoluble tau aggregates in the plasma sample. The MTBR-tau243 peptide in step (d) may comprise one or more of MTBR-tau243-256V, MTBR-tau243-256V(dN), and MTBR-tau212-221. The sample comprising the proteolytically hydrolyzed MTBR-tau243 peptide may be desalted prior to step (d). The affinity depletion agent may comprise an epitope binding agent that specifically binds to an epitope within amino acids 1 to 243 of SEQ ID NO: 1; a first epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 of tau-441, and a second epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 of SEQ ID NO: 1; or, a first epitope binding agent, a second epitope binding agent, and a third epitope binding agent that specifically binds to an epitope within amino acids 257-264 of SEQ ID NO: 1. The first epitope binding agent may be HJ8.5, the second epitope binding agent may be one or more of Tau1 and HJ8.7, and the third epitope binding agent may be HJ34.8. The affinity depletion agent may comprise Tau1, HJ8.5, and HJ8.7. The affinity depletion agent may further comprise HJ34.8. The immunoprecipitant may comprise HJ32.11. The plasma sample may comprise an internal standard.Internal standards may be included. 15 N-2N4R-tau. Purified samples may contain an internal standard. Internal standards may contain 13 N 15 N-2N4R-tau and 15 One or more of N-0N3R-tau. The method may further include one or more of the following: one or more of the post-translational modifications of detection and quantification of amyloid beta, N-terminal tau, middle domain tau and tau, and may include identifying ApoE subtypes in biological or CSF samples. Quantification of the amyloid beta step may include quantification of the post-translational modification of Aβ42 / 40 value and tau, and the post-translational modification of the tau may include one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111 and phosphorylated tau208.

[0010] Provided herein is a method for detecting Alzheimer's disease (AD)-related pathology in a subject, which may include detecting insoluble tau aggregates in a CSF or plasma sample according to the above method, wherein the CSF or plasma sample is from a subject, and wherein detecting insoluble aggregates of tau indicates AD-related pathology in the subject. AD-related pathology may be tau deposition in the subject's brain. AD-related pathology may be amyloid beta deposition in the subject's brain or cerebral arteries.

[0011] Provided herein is a method for detecting Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, which may include detecting insoluble tau aggregates in a CSF or plasma sample according to the above method, wherein the CSF or plasma sample is from the subject, and wherein detection of insoluble aggregates of tau indicates AD-associated tau deposition in the brain of the subject.

[0012] Provided herein is a method for diagnosing Alzheimer's disease in a subject, which may include (a) detecting insoluble tau aggregates in a CSF or plasma sample according to any of the above methods, wherein the CSF or plasma sample is from the subject; and (b) diagnosing Alzheimer's disease when the amount of proteolytic MTBR-tau243 peptide detected is about 1.5σ or more higher than the mean of a healthy control population, wherein σ is the standard deviation defined by a normal distribution measured in a control population that is negative for amyloid as measured by PET imaging and / or Aβ42 / 40 measurement in CSF.

[0013] Provided herein is a method for measuring progression of Alzheimer's disease (AD) in a subject, which may include (a) detecting insoluble tau aggregates in a first and a second CSF or plasma sample according to any of the above methods; and (b) calculating the difference between the amount of proteolytic MTBR-tau243 peptide in the second sample and the first sample, wherein a statistically significant increase in the amount of proteolytic MTBR-tau243 peptide in the second sample compared to the first sample indicates progression of Alzheimer's disease in the subject.

[0014] The subject of any of the above methods may have the following characteristics. The subject may be amyloid negative or amyloid positive. The subject may not have dementia. The subject may suffer from dementia. The subject may have a Clinical Dementia Rating (CDR) score of 0.5 to 1.0. The subject may have a CDR score of >1.0 to 2.0 (moderate AD). The subject may have a CDR score of >2.0.

[0015] These methods may further include quantifying amyloid beta, quantifying N-terminal tau, quantifying mid-domain tau, quantifying post-translational modifications of tau, or identifying ApoE isoforms in a biological or CSF sample. The step of quantifying amyloid beta may include quantifying Aβ42 / 40 values, and quantifying post-translational modifications of tau, including quantifying one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111, and phosphorylated tau208.

[0016] Provided herein is a method for treating tauopathy in a subject in need thereof, which may include (a) detecting insoluble tau aggregates in a CSF or plasma sample according to any of the above methods, wherein the CSF or plasma sample is from the subject; and (b) administering to the subject a treatment that alters tau pathology, wherein the amount of proteolytically cleaved MTBR-tau243 peptide detected may be about 1.5σ or more above the mean for a healthy control population, wherein σ is the standard deviation defined by a normal distribution measured in a control population that has no clinical signs or symptoms of tauopathy and may be amyloid negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF, and wherein the amount of proteolytically cleaved MTBR-tau243 peptide may indicate tauopathy.

[0017] Provided herein is a method for treating tauopathy in a subject in need thereof, comprising administering to a subject a treatment that changes tau pathology, wherein the subject has been identified as having an amount of proteolytically cleaved MTBR-tau243 peptide detected according to any of the above methods, wherein the amount is about 1.5σ or more higher than the mean value of a healthy control population, wherein σ is a standard deviation defined by a normal distribution measured in a control population that is negative for amyloid protein, without clinical signs or symptoms of tauopathy, and as measured by PET imaging and / or Aβ42 / 40 in CSF, and wherein the amount of proteolytically cleaved MTBR-tau243 peptides can indicate tauopathy. Treatment can change or stabilize the amount of proteolytically cleaved MTBR-tau243 peptides detected. The treatment can be selected from lecanemab, donanemab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, Bepranemab, Dasatinib + Quercetin, E2814, Epothilone D, Gosuranemab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, Semorinemab, TPI 287, Tideglusib, Tilavonemab, Zagotenemab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors and tau live vaccines (active vaccine). Treatment can be selected from anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, γ-secretase inhibitors, β-secretase inhibitors, kinase inhibitors, phosphatase activators, vaccines and tau protein aggregation inhibitors. Kinase inhibitors can be inhibitors of one thousand and one amino acid kinases (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. Phosphatase activators can increase the activity of protein phosphatase 2A. The vaccine can be CAD106 or AF20513. The anti-Aβ antibody can be aducanumab or another anti-amyloid antibody that can remove plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] This patent or patent application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0019] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. These drawings are not intended to limit the scope of the current teachings in any way.

[0020] Figure 1A -B shows the association between SF biomarkers and amyloid PET and tau PET. Figure 1A ) and tau-PET( Figure 1B The first two columns show scatter plots of MTBR-tau243 (first column) and pT217 / T217 (second column) and amyloid PET (n = 268) or tau-PET (n = 443) in BioFINDER-2 participants, color-coded by diagnosis and amyloid status. Linear regression models adjusted for age and sex were used to obtain the β, p-values ​​(asterisks), and R values ​​shown in the figures. 2 The scatter plots of all biomarkers in the two groups are shown in Figure 6 and 7A-B. The third and fourth columns show the standardized β of the association between each CSF biomarker and amyloid PET or tau-PET in BioFINDER-2 and Knight ADRC participants, respectively (n=219; except pT231 / T231, where n=184 for all cases). The solid and dashed lines show the standardized β (center point) and 95% CI when all participants or only amyloid-positive participants are included (BioFINDER-2: amyloid PET: n=172, tau-PET: n=287; Knight ADRC: n=136; except pT231 / T231, where n=117), respectively. The asterisk (cross) shows the highest or no significantly different standardized β for all (only amyloid-positive) participants in each group and based on the results of the bootstrap. Therefore, those biomarkers without asterisks or crosses have statistically lower correlations. Aβ-positive participants were selected based on previously validated cutoffs for CSF Aβ42 / 40 (CSF Aβ42 / 40 < 0.08 in BioFINDER-2 and CSF Aβ42 / 40 < 0.0673 in Knight ADRC). Association p values ​​are derived from two-sided tests, while bootstrapped p values ​​are from one-sided tests, all without adjustment for multiple comparisons. All p values ​​from associations between CSF biomarkers and amyloid PET and tau PET were < 0.001. Abbreviations: Aβ+, amyloid-positive; AD+, Alzheimer's disease dementia amyloid-positive; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid-negative; CU+, cognitively intact amyloid-positive; MCI+, mild cognitive impairment amyloid-positive; MTBR, microtubule-binding region; non-AD, non-Alzheimer's disease dementia; PET, positron emission tomography; SUVR, standardized uptake value ratio.

[0021] Figure 2A -B shows the proportion of the variation in CSF biomarker levels explained by amyloid-PET and tau-PET. 2 The values ​​are shown in the bars, while the partial R 2 The total R 2 The percentages are shown above each bar. These values ​​are calculated within each CSF biomarker and cohort in separate linear regression models adjusted for age and sex, using a single CSF biomarker as the outcome and amyloid and tau measures as predictors. Due to potential shared variance, the percentages may not add up to 100%. The biomarkers are arranged from left to right based on the contribution (%) of increasing tau to their levels.

[0022] Abbreviations: CSF, cerebrospinal fluid; MTBR, microtubule binding region; PET, positron emission tomography.

[0023] Figure 3 AB shows longitudinal CSF biomarker changes for baseline amyloid and tau status. The rate of change of CSF biomarkers for each baseline amyloid (A) and tau (T) status is depicted (pT231 / T231: n=218, the rest: n=220). Individual rates of change are represented by points. The trajectories for each group are shown as box plots generated using linear mixed models (the central band represents the median, the lower and upper edges correspond to the first and third quartiles, and the whiskers depict the maximum / minimum value or 1.5 times the interquartile range from the edge, whichever is lower). The differences between all groups were evaluated using the Kruskal-Wallis test, and post hoc comparisons were performed using paired Wilcoxon tests. Asterisks indicate p values ​​for two-sided tests, without correction for multiple comparisons. Longitudinal CSF data are available only in BioFINDER-2. Amyloid-positive participants were identified using a previously validated CSF Aβ42 / 40 cutoff (CSF Aβ42 / 40<0.08). Tau positivity was determined based on tau-PET SUVR in the meta-ROI (SUVR>1.32). Actual p-values ​​for AT- versus A+T- were: p=0.011 (pT205 / T205); actual p-values ​​for AT- versus A+T+ were: p=0.011 (pT181 / T181), p=0.014 (pT205 / T205), p=0.617 (pT217 / T217), and p=0.980 (pT231 / T231); and actual p-values ​​for A+T- versus A+T+ were: p=0.788 (pT205 / T205), p=0.007 (pT231 / T231). All other comparisons yielded p < 0.001. *, p < 0.050; **, p < 0.010; ***, p < 0.001. Abbreviations: AT-, amyloid and tau negative; A+T-, amyloid positive and tau negative; A+T+, amyloid positive, tau positive; CSF, cerebrospinal fluid; MTBR, microtubule binding region; PET, positron emission tomography; SUVR, standardized uptake value ratio.

[0024] Figure 4A -B shows the association between CSF biomarkers and MMSE. Depicts the BioFINDER-2 ( Figure 4A , n = 342) and Knight ADRC ( Figure 4BAssociations between CSF biomarkers and MMSE in participants with 2-month follow-up (n=184, pT231 / T231: rest: n=219). The first two columns show scatter plots of MTBR-tau243 (first column) and pT217 / T217 (second column) versus MMSE, color-coded by diagnosis and amyloid status. In the BioFINDER-2 cohort, orange dots represent MCI+ participants, while in the Knight ADRC cohort, they represent individuals with very mild AD. Linear regression models adjusted for age and sex were used to obtain the beta coefficients, p-values, and R values ​​shown in the figures. 2 The scatter plots of all biomarkers in the two groups can be seen in Fig.9A -B. The third column shows the standardized beta coefficients for all biomarkers, as well as the associations of amyloid PET and CSF Aβ42 / 40 (reverse) and tau-PET for comparison. The solid and dotted lines represent the standardized beta coefficients (center points) and 95% confidence intervals (95% CI) when all participants or only amyloid-positive participants are included (BioFINDER-2: n=261, Knight ADRC: n=136, except pT231 / T231, where n=117), respectively. Asterisks (crosses) indicate the highest or no significantly different standardized beta coefficients for all (amyloid-positive only) participants within each group and the results based on bootstrapping. Non-AD participants from BioFINDER-2 were excluded from these analyses. Amyloid-positive participants were selected using previously validated CSF Aβ42 / 40 cutoffs (CSF Aβ42 / 40 < 0.08 in BioFINDER-2 and CSF Aβ42 / 40 < 0.0673 in Knight ADRC). Association p values ​​are derived from two-sided tests, while bootstrapped p values ​​are from one-sided tests, all without adjustment for multiple comparisons. All p values ​​for associations between CSF biomarkers and MMSE were < 0.001. Abbreviations: Aβ, amyloid β; Aβ+, amyloid positive; AD+, Alzheimer's disease dementia amyloid positive; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid negative; CU+, cognitively intact amyloid positive; MCI+, mild cognitive impairment amyloid positive; MMSE, Mini-Mental State Examination; MTBR, microtubule binding region; PET, positron emission tomography; SUVR, standardized uptake ratio.

[0025] Figure 5A -B shows the prediction of quantified amyloid PET, tau PET and MMSE continuous measures by CSF biomarkers. Linear regression model was used to predict BioFINDER-2 ( Figure 5A ) and Knight ADRC( Figure 5B , n=184) in amyloid PET (first column, BioFINDER-2: n=256), tau-PET (second column, BioFINDER-2: n=422), and cognition (MMSE, third column, BioFINDER-2: n=342). The base model included age and sex (and education status for MMSE) as predictors. The parsimonious model was derived by using LASSO regression to identify the best combination of CSF biomarkers and demographic factors (age, sex, and / or education status). The biomarkers included in the parsimonious model are indicated by a black border and their names are shown in bold. The other models only used a single CSF biomarker as a predictor. For comparison, CSF Aβ42 / 40 and tau-PET were used as predictors in independent models, respectively, only for the prediction of all outcomes and cognition. Model comparisons were performed using the F test for nested models or the Vuong test for non-nested models. Non-AD cases were excluded from the BioFINDER-2 cohort for cognitive analysis. Abbreviations: Aβ, amyloid β; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid-negative; MMSE, Mini-Mental State Examination; LASSO, minimum absolute shrinkage and selection operator; MTBR, microtubule-binding region; PET, positron emission tomography.

[0026] Figure 6CSF biomarkers at diagnosis are shown. Levels of each CSF biomarker by clinical diagnosis and amyloid status in the BioFINDER-2 cohort (n=448). Amyloid-positive participants were selected based on CSF Aβ42 / 40 (CSF Aβ42 / 40<0.0 8). The larger pink dots represent two MAPT R40 6W mutation carriers (one CU- and the other in the FTD group), both of whom were amyloid-negative but had substantial tau-PET binding. In the boxplots, the central band represents the median of the group, the lower and upper edges correspond to the first and third quartiles, and the whiskers represent the maximum / minimum value or 1.5 IQR from the edge, whichever is lower). Differences in CSF biomarker levels between diagnostic groups were tested using ANCOVA adjusted for age and sex. Two-sided post hoc analyses were performed using the Tuckey test. Due to space reasons, the actual p values ​​are reported in Table 3. p<0.050; **, p<0.010; ***, p<0.001. Abbreviations: AD+, Alzheimer's disease dementia amyloid-positive; CBS, corticobasal syndrome; CU-, cognitively intact amyloid-negative; CU+, cognitively intact amyloid-positive; FTD, frontotemporal dementia; MCI+, mild cognitive impairment amyloid-positive; MTBR, microtubule binding region; PD, Parkinson disease; PDD, Parkinson disease dementia; PPA, primary progressive aphasia; PSP, progressive supranuclear palsy.

[0027] Fig. 7A -B shows the association between all CSF biomarkers and Aβ-PET. BioFINDER-2 ( Fig. 7A ) and Knight ADRC( Figure 7B ) participants. Linear regression models adjusted for age and sex were used to obtain the standardized beta and p values ​​shown in the figures. In the BioFINDER-2 cohort, orange points represent MCI+ participants and in the Kinght-ADRC cohort, very mild AD. *, p<0.050; **, p<0.010, ***, p<0.001. Abbreviations: AD+, Alzheimer's disease dementia amyloid positive; CL, Centiloid, CSF, cerebrospinal fluid; CU-, cognitively intact amyloid negative; CU+, cognitively intact amyloid positive; MCI+, mild cognitive impairment amyloid positive; MTBR, microtubule binding region; non-AD, non-Alzheimer's disease dementia; PET, positron emission tomography.

[0028] Fig. 8A-B shows the association between all CSF biomarkers and tau-PET of Braak I-IV ROIs. The association between all CSF biomarkers and tau-PET of Braak I-IV ROIs. BioFINDER-2 ( Fig. 8A ) and KnightADRC( Figure 8B Figure 2 Association between CSF biomarkers and tau-PET (SUVR) of Braak I-IV ROIs in participants with 24-month follow-up. Linear regression models adjusted for age and sex were used to obtain the standardized beta and p values ​​shown in the figures. Orange dots represent MCI+ participants in the BioFINDER-2 cohort and very mild AD in the Kinght-ADRC cohort. *, p<0.050; **, p<0.010; ***, p<0.001. Abbreviations: AD+, Alzheimer's disease dementia amyloid-positive; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid-negative; CU+, cognitively intact amyloid-positive; MCI+, mild cognitive impairment amyloid-positive; MTBR, microtubule binding region; non-AD, non-Alzheimer's disease dementia; PET, positron emission tomography; SUVR, standardized uptake value ratio.

[0029] Fig.9A -C shows the association between CSF biomarkers and tau-PET in different Braak regions. Fig.9A )、Braak III-IV( Fig. 9B ) and Braak V-VI( Fig. 9C The first two columns show scatter plots of MTBR-tau243 (first column) and pT217 / T217 (second column) and tau-PET in BioFINDER-2 participants (n = 443), colored by diagnosis and amyloid status. Linear regression models adjusted for age and sex were used to obtain the standardized β, p-values ​​(asterisks), and R values ​​shown in the figures. 2. The third and fourth columns show the standardized β for the association between each CSF biomarker and tau-PET in BioFINDER-2 and Knight ADRC participants, respectively (n=219; except for pT231 / T231, where n=184). The solid and dashed lines show the standardized β (center point) and 95% CI when all participants or only amyloid-positive participants were included (BioFINDER-2: n=287; Knight ADRC, n=136), respectively. Asterisks (crosses) show the highest or no significantly different standardized β for all (amyloid-positive only) participants in each cohort and outcome. Amyloid-positive participants were selected based on previously validated cutoffs for CSF Aβ42 / 40 (CSF Aβ42 / 40<0.08 in BioFINDER-2 and CSF Aβ42 / 40<0.0673 in Knight ADRC). Association p values ​​are based on two-sided tests and bootstrapped p values ​​are from one-sided tests, all without adjustment for multiple comparisons. All p values ​​from associations between CSF biomarkers and tau-PET shown in the scatter plots are <0.001. *, p<0.050; **, p<0.010; ***, p<0.001. Abbreviations: Aβ+, amyloid positive; AD+, Alzheimer's dementia amyloid positive; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid negative; CU+, cognitively intact amyloid positive; MCI+, mild cognitive impairment amyloid positive; MTBR, microtubule binding region; non-AD, non-Alzheimer's dementia; PET, positron emission tomography; SUVR, standardized uptake value ratio.

[0030] Fig.10 Longitudinal CSF biomarker trajectories for baseline "A" and "T" status are shown. Trajectories of individual (hatched) and group (bold) CSF biomarker levels over time based on their A / T baseline status. Statistical differences from the reference group (AT-) are shown with asterisks in appropriate colors. Longitudinal CSF data are only available in BioFINDER-2. Amyloid-positive participants were selected based on a previously validated cutoff for CSF Aβ42 / 40 (CSF Aβ42 / 40<0.08). Tau positivity was assessed based on tau-PET SUVR in the meta-ROI (Braak I-IV SUVR>1.32). *, p<0.050; **, p<0.010; ***, p<0.001. Abbreviations: AT-, amyloid and tau-negative; A+T-, amyloid-positive and tau-negative; A+T+, amyloid-positive and tau-positive; CSF, cerebrospinal fluid; MTBR, microtubule-binding region; PET, positron emission tomography.

[0031] Fig.11A -B shows the association between all CSF biomarkers and MMSE. In the BioFINDER-2 cohort ( Fig.11A ), orange dots represent MCI+ participants, while in the Kinght-ADRC group ( Fig. 11B ) represents very mild AD. Linear regression models adjusted for age, sex, and years of education were used to obtain the standardized β and p values ​​shown in the figures. *, p < 0.50; **, p < 0.010; ***, p < 0.001. Abbreviations: AD+, Alzheimer's disease dementia amyloid positive; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid negative; CU+, cognitively intact amyloid positive; MCI+, mild cognitive impairment amyloid positive; MMSE, Mini-Mental State Examination; MTBR, microtubule binding region; PET, positron emission tomography, SD, standard deviation; SUVR, standardized uptake value ratio.

[0032] Fig. 12A -B shows the association between all CSF biomarkers and CSF Aβ42 / 40. BioFINDER-2 ( Fig. 12A , n = 427) and Knight ADRC ( Fig. 12B Associations between CSF biomarkers and CSF Aβ42 / 40 in participants with ≥ 2 years of follow-up (n = 219; except for pT231 / T231, where n = 184). Linear regression models adjusted for age and sex were used to obtain the standardized β values, p values ​​(asterisks), and R values ​​shown in the figures. 2 . P values ​​are based on two-sided tests without adjustment for multiple comparisons. *, p < 0.050; **, p < 0.010; ***, p < 0.001. Abbreviations: AD+, Alzheimer's disease dementia amyloid-positive; CL, Centiloid, CSF, cerebrospinal fluid; CU-, cognitively intact amyloid-negative; CU+, cognitively intact amyloid-positive; MCI+, mild cognitive impairment amyloid-positive; MTBR, microtubule-binding region; non-AD, non-Alzheimer's disease dementia; PET, positron emission tomography.

[0033] Fig.13A -B shows the proportion of the variation in CSF biomarker levels explained by CSF Aβ42 / 40 and tau-PET. Fig.13A , n = 422) and Knight ADRC ( Fig. 13B , n = 184), the proportion of the variation in CSF biomarker levels explained by CSF Aβ42 / 40 and tau-PET is shown as the partial R for each biomarker 2. Partial R 2 The values ​​are shown inside the bars. 2 The total R 2 The percentage is shown at the top of each bar. Partial R was calculated using each CSF biomarker as outcome and amyloid and tau measures as predictors in linear regression models adjusted for age and sex in separate models for each CSF biomarker and cohort. 2 and total R 2 Because of possible shared variance, percentages do not add up to 100%. Biomarkers are ranked (left to right) by increasing tau contribution (%) to their levels. Abbreviations: CSF, cerebrospinal fluid; CU-, cognitively intact amyloid-negative; MTBR, microtubule-binding region; PET, positron emission tomography.

[0034] Fig.14Longitudinal CSF biomarker changes of baseline amyloid and tau status are shown. Each baseline amyloid (A) state as measured by PET and the CSF biomarker change rate of tau (T) state as measured by PET (n=174). Points represent individual change rates. The box plot represents the trajectory of each group as evaluated by a linear mixed model (the central band of the box plot represents the median of the group, the lower and upper edges correspond to the first and third quartiles, and the whiskers represent the maximum / minimum value or 1.5 IQR from the edge, whichever is lower). The differences between all groups were tested using the Kruskal-Wallis test and the paired Wilcoxon test for post hoc comparisons. The P value is from a two-sided test that is not corrected for multiple comparisons and is indicated by an asterisk. Longitudinal CSF data are only available in BioFINDER-2. Amyloid-positive participants were selected based on the cutoff value (SUVR>1.03) previously validated by amyloid PET. Tau positivity was evaluated based on tau-PET SUVR in meta-ROI (SUVR>1.32). The actual p-values ​​of AT- relative to A+T- were: p=0.024 (pT205 / T205); the actual p-values ​​of AT- relative to A+T+ were: p=0.277 (pT181 / T181), p=0.041 (pT205 / T205), p=0.178 (pT217 / T217) and p=0.368 (pT231 / T231); and the actual p-values ​​of A+T- relative to A+T+ were: p=0.001 (pT181 / T181), p=0.738 (pT205 / T205), p=0.011 (pT217 / T217), p=0.023 (pT231 / T231). The remaining comparisons provide p < 0.001. *, p < 0.050; **, p < 0.010; ***, p < 0.001. Abbreviations: AT-, amyloid and tau negative; A+T-, amyloid positive and tau negative; A+T+, amyloid positive, tau positive; CSF, cerebrospinal fluid; MTBR, microtubule binding region; PET, positron emission tomography; SUVR, standardized uptake value ratio.

[0035] Fig.15A -B shows the prediction of AD-related continuous measures by CSF biomarkers in amyloid-positive participants. Linear regression models were used to predict the AD-related continuous measures in BioFINDER-2 ( Fig.15A ) and Knight ADRC( Fig. 15B, n=117) amyloid-positive participants predicted amyloid PET (first column, n=172), tau-PET (second column, n=287) and MMSE (third column, n=261). The base model included age and sex (and education status for MMSE) as predictors. The parsimonious model was obtained with the best combination of CSF biomarkers and demographics (age and / or sex and / or education status) evaluated using LASSO regression. The biomarkers included in the parsimonious model are depicted with a black border and their names are shown in bold. Other models used only a single CSF biomarker as a predictor. As a comparison, CSF Aβ42 / 40 and tau-PET were used as predictors in independent models for predicting cognition. Models were compared using the F test (nested model) or the Vuong test (non-nested model). Non-AD cases were excluded from the BioFINDER-2 cohort for cognitive analysis. Amyloid-positive participants were selected based on previously validated cutoffs for CSF Aβ42 / 40 (Aβ42 / 40 < 0.08 in BioFINDER-2 and CSF Aβ42 / 40 < 0.0673 in Knight ADRC). Abbreviations: Aβ, amyloid β; CSF, cerebrospinal fluid; CU-, cognitively intact amyloid-negative; MMSE, Mini-Mental State Examination; LASSO, minimum absolute shrinkage and selection operator; MTBR, microtubule-binding region; PET, positron emission tomography.

[0036] Fig.16A -B shows the CSF tau analysis method. Fig.16A ) Schematic diagram of the binding sites of the antibodies used in this study and ( Fig. 16B ) Sample preparation protocol. CSF tau species were immunoprecipitated by Tau1 and HJ series (clone names: HJ8.5, HJ8.7, HJ32.11 and HJ34.8) antibodies. For all antibodies, 3mg / gram agarose beads were generated. For Tau1 immunoprecipitation (IP), 1.1ug antibody / sample was used. For HJ8.5 and HJ8.7 IP, 2.3ug antibody / sample was used. For HJ32.11 and HJ34.8 IP, 11.25ug antibody / sample was used. CSF (450μL) was mixed with 20μL of a solution containing 100pg / μL each of 15N 0N3R-tau and 15N2N4R-tau as internal standards. Mainly from the N-terminal to the middle domain region and the late MTBR region (at R 1Tau species consisting of tau residues 260 and 260 (near residue 260 in the tau) were immunoprecipitated with Tau1, HJ8.5, HJ8.7, and HJ34.8 antibodies. Subsequently, 20 μL of 13C15N 2N4R-tau internal standard (100 pg / μL) was spiked into the immunoprecipitated CSF. Then, in the immunoprecipitated CSF, another novel antibody HJ32.11 was used to immunoprecipitate the MTBR-tau243 species containing residue 243. All immunoprecipitated tau species (p-tau and MTBR-tau243) were digested by trypsin and desalted by C18 toptip (Glygen). The resulting tau peptides were analyzed by a nanoAcquity ultra-high performance LC system (Waters) coupled to an OrbitrapTribrid Eclipse mass spectrometer (Thermo Scientific) operating in parallel reaction monitoring mode. MS transitions were extracted using Skyline v.22.2.2.278 (MacCoss Lab, University of Washington). Data were aggregated using Tableau v.2022.2.2 (Tableau Software) to calculate CSF biomarker levels.

[0037] Fig.17 Schematic diagram of the longest human tau isoform (2N4R). The N-terminus (N term), middle domain, MTBR and C-terminus (C term) are identified for this isoform and will vary in a predictable manner for other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R and 0N3R).

[0038] Fig.18A -B shows an exemplary sequential immunodepletion / immunoprecipitation scheme for isolating MTBR-tau243 fragments from CSF. ( Fig.18A ) Method 1 (design No. 1) uses Tau1 / HJ8.5 / HJ8.7 in the first step and HJ32.11 in the second step. ( Fig.18B ) Method 2 (Design No. 2) uses Tau1 / HJ8.5 / HJ8.7 and HJ34.8 in the first step and then uses HJ32.11 in the second step. Fig.18C The affinity of the antibodies to the MTBR-tau243 fragment is shown.

[0039] Fig.19It shows that the short MTBR-tau243 species are diverse and only the short fragments are associated with AD tau tangles in biological fluids. The figure also shows the tau regions to which the HJ8.5, HJ8.7, Tau1, E2814, 77G7 and HJ34.8 antibodies bind. The short MTBR-tau243 and MTBR-tau212-221 fragments are captured by the HJ32.11 antibody.

[0040] Fig. 20A -B shows the concentration of the fragments precipitated by the two methods in Figure 18. Fig. 20A The HJ34.8 antibody was shown to precipitate a subset of MTBR-tau species in CSF. Fig. 20B The depletion effect of HJ34.8 antibody on MTBR-tau243 in a second immunoprecipitation and / or immunodepletion step using HJ32.11 antibody is shown.

[0041] Fig.21A -B shows the correlation between MTBR-tau243 concentrations detected by chemical extraction (CX) and each of the two immunoprecipitation and / or immunodepletion methods in Figure 18. ( Fig.21A ) Correlation of MTBR-tau243 using immunoprecipitation and / or immunodepletion of Tau1 / HJ8.5 / HJ8.7. Fig. 21B ) Correlation using MTBR-tau243 immunoprecipitated and / or immunodepleted using Tau1 / HJ8.5 / HJ8.7 / HJ34.8. Tau species precipitated by HJ34.8 did not improve correlation with CSF MTBR-tau243 prepared by chemical extraction methods.

[0042] Fig.22A -B shows the correlation between MTBR-tau243 concentrations detected by Tau PET and each of the two previously described immunoprecipitation and / or immunodepletion methods. ( Fig.22A ) Correlation of MTBR-tau243 using immunoprecipitation and / or immunodepletion of Tau1 / HJ8.5 / HJ8.7. Fig. 22B ) Correlation with MTBR-tau243 using immunoprecipitation and / or immunodepletion with Tau1 / HJ8.5 / HJ8.7 / HJ34.8. Tau species precipitated by HJ34.8 did not correlate with tau-PET and were therefore depleted in the first step. Finally, a second immunoprecipitation using HJ32.11 showed better correlation with tau-PET.

[0043] Fig.23A-D shows that MTBR-tau243 correlates most with Tau-PET, even in the amyloid-only (+) population, but not with amyloid PET (a contrasting signature to pT217). pT205 is an intermediate signature (between MTBR243 and pT217) that recapitulates both amyloid and tau pathology.

[0044] Fig.24A -C shows that MTBR-tau243 retains the discriminatory ability to identify tau pathology in the amyloid-positive subset (B).

[0045] Fig.25 An exemplary sequential immunodepletion or immunoprecipitation protocol for detecting short fragments of MTBR-tau243 in plasma is shown.

[0046] Fig.26A -B shows that the development of plasma MTBR-tau243 is challenging compared to CSF.

[0047] Fig. 27 A protocol for identifying endogenous MTBR-tau243 species that replace AD ​​tau pathology.

[0048] Fig.28 The MTBR-tau243 sequence quantified in the CSF protocol using trypsin digestion is shown. MTBR-tau243-254 has multiple lysine (K) residues near MTBR-tau243. The tau sequence shown (SRLQTAPVPMPDLKNVKSKIGSTENLKHQP; SEQ ID NO: 22) is amino acids 241-270 of tau-441.

[0049] Fig.29 A modified one-step immunoprecipitation method for detection of MTBR-tau243 in CSF using HJ32.11 beads and ArgC digestion is shown.

[0050] Fig. 30A -B shows that the amounts of MTBR-tau243-256V (SEQ ID NO: 15) and 243-256V@255N-deamidated (SEQ ID NO: 16) fragments detected were dramatically increased in samples with high Tau-Pet levels.

[0051] Fig.31A -B shows MTBR-tau243-255 (SEQ ID NO: 17) in CSF ( Fig.31A ) and MTBR-tau 243-255 with @255N-deamidation (SEQ ID NO: 10) ( Fig.31B) Correlation with Tau-PET levels. MTBR-tau243-255 species with @255N-deamidation showed better specificity for low, medium and high Tau-PET levels compared to non-deamidated fragments.

[0052] Fig.32A -B shows MTBR-tau243-257 (SEQ ID NO: 18) in CSF ( Fig.32A ) and MTBR-tau 243-257 (SEQ ID NO: 11) with @255N-deamidation ( Fig.32B ) Correlation with Tau-PET. Compared with the non-deamidated fragment, the MTBR-tau243-257 species with @255N-deamidation showed better specificity for low, medium and high Tau-PET levels.

[0053] Fig.33A -B shows MTBR-tau243-252D (SEQ ID NO: 13) in CSF ( Fig.33A ) and MTBR-tau 243-258S with @255N-deamidation (SEQ ID NO: 14) ( Fig.33B ) Correlation with Tau-PET. Compared with the non-deamidated fragment, the MTBR-tau243-258S species with @255N-deamidation showed better specificity for low, medium and high Tau-PET levels.

[0054] Fig.34A -B shows MTBR-tau243-253L (SEQ ID NO: 12) in CSF ( Fig.34A ) and MTBR-tau 243-254K with @244-deamidation (SEQ ID NO: 14) ( Fig.34B Both categories stabilized from medium to high Tau-PET.

[0055] Fig.35A -B shows MTBR-tau243-254 (SEQ ID NO: 21) and MTBR-tau243-256V (SEQ ID NO: 15) detected in plasma samples. The amount of MTBR-tau243-256V detected in samples with high Tau-PET levels increased dramatically.

[0056] Fig.36A-B shows MTBR-tau243-254 (SEQ ID NO:21), MTBR-tau243-256V (SEQ ID NO:15), and MTBR-tau243-256V@255N-deamidation (SEQ ID NO:16) detected in plasma samples.

[0057] Fig.37A -F shows the LC chromatogram and mass spectrum of MTBR-tau243-256V@255N-deamidation (SEQ ID NO: 16).

[0058] Fig.38 Exemplary methods for detecting MTBR-tau243-256 (SEQ ID NO:21) and MTBR-tau243-256V-dN (SEQ ID NO:15) in plasma are shown.

[0059] Fig.39 Exemplary methods for detecting MTBR-tau243-256 (SEQ ID NO:21) and MTBR-tau243-256V-dN (SEQ ID NO:15) in plasma are shown.

[0060] Fig.40A -C shows how the results of MTBR-tau243 detection in CSF translate to results in plasma ( Fig.40A ), and MTBR-tau243 detection in plasma relative to plasma ( Fig.40B ) and CSF( Fig.40C ) in comparison of tau PET.

[0061] Fig.41 It is shown how the 243-256V@255N-deamidated (SEQ ID NO: 16) fragment can provide meaningful information about the presence of tangles based on the use of HJ32.11 and HJ34.8 in the plasma detection method compared to CSF.

[0062] Fig.42 A possible model explaining why plasma 243-256V@255N-deamidation (SEQ ID NO: 16) is extremely specific for mature tangles in plasma compared to detection of MTBR-tau243 in CSF is shown.

[0063] Fig.43A-C shows CSF pT217 / T217, CSF MTBR-tau243 and plasma MTBR-tau243-256dN for Alzheimer's disease staging (n=55). Plasma MTBR-tau243-256dN increased significantly after AD onset (CDR≥1).

[0064] Fig.44 It was shown that in samples from the Knight Alzheimer's Disease Research Center cohort, MTBR-tau212 was highly correlated with MTBR-tau243 (ρ>0.9), suggesting that MTBR-tau212-221 is equivalent to Tau-PET as a marker.

[0065] Fig.45 It was shown that in samples from the Biofinder2 cohort, MTBR-tau212 was highly correlated with MTBR-tau243 (ρ>0.9), suggesting that MTBR-tau212-221 is equivalent to Tau-PET as a marker. DETAILED DESCRIPTION

[0066] The inventors have found that tau fragments containing MTBR-tau243 (i.e., fragments containing the amino acids of tau-441 as disclosed herein) can be detected in cerebrospinal fluid (CSF) or plasma, and methods for detecting have been developed. They have also found that, unexpectedly, MTBR-tau243 is a specific marker for insoluble tau aggregates (e.g., neurofibrillary tangles) associated with tau diseases (including Alzheimer's disease (AD)). The MTBR-tau243 fragment disclosed herein can be used as an alternative to tau positron emission tomography (PET) assays. Therefore, the inventors have found a simpler, cheaper method for diagnosing tau diseases.

[0067] The inventors have also unexpectedly discovered that the MTBR-tau243 fragments disclosed herein can be used to determine whether a patient's symptoms are due to AD. About 20-30% of patients at least 65 years old have amyloid plaques but no symptoms. Detection of amyloid plaques using available markers such as Aβ42, pTau-217, pTau-181, and pTau-231 can lead to a false positive diagnosis of AD because the presence of amyloid plaques is not specific for AD symptoms. Detection of the presence of MTBR-tau243 fragments as disclosed herein indicates that the patient's symptoms are due to AD, not just associated with it.

[0068] 1. Definition

[0069] For the recitation of numerical ranges herein, each intervening number with the same degree of precision is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are expressly contemplated.

[0070] The following discusses the composition for preparing the disclosed composition and the composition itself to be used in the method disclosed herein. These and other materials are disclosed herein, and it should be understood that when the combination, subset, interaction, group, etc. of these materials are disclosed, although each different individual and common combination of these compounds and the specificity of the arrangement may not be explicitly disclosed, each is specifically considered and described herein. For example, if a specific compound is disclosed and discussed, and many modifications that can be made to many molecules of the compound are discussed, each combination and arrangement of the compound and possible modifications are specifically considered, unless specifically indicated to the contrary. Therefore, if a class of molecules A, B and C and a class of molecules D, E and F are disclosed and an example AD of the combined molecule is disclosed, then even if each is not narrated individually, each is also considered individually and collectively, which means that the combination AE, AF, BD, BE, BF, CD, CE and CF is considered to be disclosed. Similarly, any subset or combination of these is also disclosed. Therefore, for example, the subset of AE, BF and CE will be considered to be disclosed. This concept is applicable to all aspects of the application, including but not limited to the method steps for preparing and using the disclosed composition. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0071] Other aspects and iterations of the invention are described in more detail below.

[0072] In order to make the present invention easier to understand, some terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the embodiments of the present invention belong. Those similar, modified or equivalent to the many methods and materials described herein can be used in the practice of the embodiments of the present invention without excessive experimentation, and preferred materials and methods are described herein. When describing and requesting embodiments of the present invention, the following terms will be used according to the definition set forth below.

[0073] The term "a" or "an" entity refers to one or more of that entity; for example, a "polypeptide subunit" is understood to mean one or more polypeptide subunits. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0074] In addition, when used in this article, "and / or" should be regarded as a specific disclosure of each specific feature or component with or without other features or components. Therefore, the term "and / or" as used in this article with phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone).

[0075] As used herein, the term "about" refers to variations in numerical quantities that may occur, for example, by typical measurement techniques and equipment, with respect to any quantifiable variable (including, but not limited to, mass, volume, time, distance, and amount). Further, given the solid and liquid handling procedures used in the real world, there are certain unintentional errors and variations that are likely to exist through differences in the manufacture, source, or purity of the ingredients used to prepare the composition or perform the method, etc. The term "about" also includes these variations, which may be up to ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about", the claims include equivalents to the quantities.

[0076] As used herein, the term "non-naturally occurring" substances, compositions, entities and / or any combination of substances, compositions or entities, or any grammatical variations thereof, is a conditional term that expressly excludes, but only excludes, those forms of substances, compositions, entities and / or any combination of substances, compositions or entities that are fully understood by one of ordinary skill in the art to be "naturally occurring" or that are determined or interpreted at any time by a judge or administrative or judicial body to be or could be "naturally occurring".

[0077] "Specifically binds" means that a binding molecule (e.g., an antibody or antigen binding fragment thereof) binds to an epitope via its antigen binding domain, and such binding requires some recognition between the antigen binding domain and the epitope. According to this definition, when a binding molecule binds to an epitope via its antigen binding domain, the binding molecule is considered to "specifically bind" to the epitope more easily than it binds to a random, unrelated epitope. Thus, the term "specifically binds" as used herein with respect to an epitope binding agent means that the epitope binding agent generally does not cross-react to a significant extent with other epitopes on a target protein (e.g., Tau) or other proteins.

[0078] As used herein, the term "antibody" is used in the broadest sense and includes various antibodies and antibody-like structures, including but not limited to full-length monoclonal, polyclonal and multispecific (e.g., bispecific, trispecific, etc.) antibodies, as well as heavy chain antibodies and antibody fragments, as long as they exhibit the desired antigen-binding activity. The antibody domains involved in binding to antigens are called "variable regions" or "variable domains", and are described in further detail below. A single variable domain may be sufficient to confer antigen-binding specificity. Preferably, but not necessarily, the antibodies useful in discovery are recombinantly produced. Antibodies may or may not be glycosylated, but glycosylated antibodies may be preferred. "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some embodiments, the antibodies are purified to a purity greater than 95% or 99%, as determined by methods known in the art.

[0079] In addition to the antibodies described herein, methods known in the art can also be used to design antibody mimetics or aptamers that have substantially the same functions as the antibodies of the present invention. "Antibody mimetics" refers to a polypeptide or protein that can be specifically bound to an antigen but is structurally unrelated to an antibody. The mass of an antibody mimetics is about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, high-affinity polymers (avimers), DARPins, and monobodies. An aptamer is a class of small nucleic acid ligands consisting of RNA or single-stranded DNA oligonucleotides, and has high specificity and affinity for its target. An aptamer interacts and binds to its target through structural recognition (which is a process similar to the antigen-antibody reaction process). The molecular weight of an aptamer is lower than that of an antibody, generally about 8-25 kDa. As used herein, the term "aptamer" refers to a polynucleotide with useful biological activity in terms of biochemical activity, molecular recognition, or binding properties, generally RNA or DNA. Typically, an aptamer has a molecular activity, such as binding to a target molecule at a specific epitope (region). It is generally believed that aptamers that are specific in binding to a polypeptide can be synthesized and / or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art. See, for example, US 7,939,313, which is incorporated herein by reference in its entirety.

[0080] The terms "full-length antibody" and "intact antibody" are used interchangeably and refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein. The basic structural unit of a natural antibody comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). Light chains are divided into γ, μ, α and λ. Heavy chains are divided into γ, μ, α, δ or ε, and define the isotype of the antibody as IgG, IgM, IgA, IgD and IgE, respectively. The amino terminal portion of each light and heavy chain includes a variable region (VL and VH, respectively) of about 100 to 110 or more amino acid sequences that are primarily responsible for antigen recognition. The carboxyl terminal portion of each chain defines a constant region that is primarily responsible for effector function. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. As is known in the art, intact antibodies are correctly cross-linked via disulfide bonds.

[0081] The variable domains of antibody heavy and light chains generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a library of complementary VL or VH domains, respectively, screened using a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0082] "Framework region" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is generally composed of the following four FR domains: FR1, FR2, FR3 and FR4. Accordingly, the HVR and FR sequences generally appear in the following order: FR1-HVR1-FR2-HVR2-FR3-HVR3-FR4. As is known in the art, the FR domains of the heavy chain and light chain can be different.

[0083] As used herein, the term "hypervariable region" or "HVR" refers to each variable domain region that is hypervariable in sequence (also often referred to as a "complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen-contact residues ("antigen contact points"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3), and three in VL (L1, L2, L3). As used herein, an "HVR derived from a variable region" refers to an HVR that has no more than two amino acid substitutions compared to the corresponding HVR from the original variable region. Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, 1994). Health, Bethesda, MD (1991)); (c) antigenic contact points at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol.262:732-745 (1996)); (d) CDR1-IMGT (positions 27-38), CDR2-IMGT (positions 56-65) and CDR3-IMGT regions (positions 105-116 or 105-117), which are based on the IMGT unique numbering (Lefranc, "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains," The Immunologist, 1999, 7:132-136; Lefranc et al., Nucleic Acids Research, 2009, 37 (Database issue): D1006-D1012; Ehrenmann et al., "Chapter 2: Standardized Sequence and Structure Analysis of Antibody Using IMGT," in Antibody Engineering, Vol. 2, Roland E. Kontermann and Stefan Dubel, eds., 2010, Springer-Verlag Berlin Heidelberg, doi:10.1007 / 978-3-642-01147-4; imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition), and (e) combinations of (a), (b), (c) and / or (d), as defined below for various antibodies of the present disclosure. Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain to which sequence identification numbers are assigned are numbered based on the IMGT unique numbering, supra. .

[0084] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0085] A "variant Fc region" comprises an amino acid sequence that, compared to a native Fc region or to the Fc region of a parent polypeptide, may differ from a native Fc region due to one or more amino acid substitutions and / or due to a modified glycosylation pattern. In one example, a variant Fc region may have about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology, at least about 90% homology, or at least about 95% homology with a native sequence Fc region and / or with the Fc region of a parent polypeptide.

[0086] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain forms of antibodies and higher-order variants thereof; single-domain antibodies and multispecific antibodies formed from antibody fragments.

[0087] The single-chain form of the antibody and its higher-order form may include, but are not limited to, single-domain antibodies, single-chain variant fragments (scFv), divalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFvs (tetra-scFv), double-chain antibodies (diabody) and three-chain antibodies (triabody) and four-chain antibodies (tetrabody). ScFv comprises a heavy chain and a light chain variable region connected by a joint. In most cases (but not all), the joint can be a peptide. The joint peptide is preferably about 5 to 30 amino acids in length, or about 10 to 25 amino acids in length. Generally, the joint allows the stable variable domain without interfering with the correct folding and generation of the active binding site. In a preferred embodiment, the joint peptide is rich in glycine and serine or threonine. ScFv can be used to promote phage display, or can be used for flow cytometry, immunohistochemistry or as a targeting domain. Methods for preparing and using scFv are known in the art. ScFv can also be conjugated to human constant domains (e.g., heavy constant domains derived from IgG domains (such as IgG1, IgG2, IgG3, or IgG4), or heavy chain constant domains derived from IgA, IgM, or IgE). Diabodies, triabodies, and tetrabodies, as well as higher-order variants, are generally produced by changing the length of the linker peptide from 0 to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be produced using self-assembling units connected to variable domains.

[0088] "Single domain antibody" refers to an antibody fragment consisting of a single monomeric variable antibody domain.

[0089] Multispecific antibodies include bispecific antibodies, trispecific antibodies, or antibodies with four or more specificities. Multispecific antibodies can be produced by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains can be covalently linked.

[0090] The antibody disclosed herein may be a dual-affinity re-targeting antibody (DART). The DART format is based on a double-chain antibody format, which separates the homologous variable domains of the heavy chain and light chain of two antigen-binding specificities on two separate polypeptide chains. Although the two polypeptide chains are non-covalently associated in the double-chain antibody format, the DART format provides additional stability through a C-terminal disulfide bridge. DART can be produced in high quantity and quality and shows excellent stability in both formulation buffer and human serum.

[0091] "Monoclonal antibody" refers to an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic or phage clone). "Monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma technology well known in the art, as well as recombinant technology, phage display technology, synthetic technology, or a combination of such technology with other technologies readily known in the art. In addition, monoclonal antibodies can be labeled with detectable markers immobilized on a solid phase and / or conjugated to a heterologous compound (e.g., an enzyme or toxin), according to methods known in the art.

[0092] A "heavy chain antibody" refers to an antibody composed of two heavy chains. A heavy chain antibody may be an IgG-like antibody from camels, llamas, alpacas, sharks, etc., or an IgNAR from cartilaginous fish.

[0093] "Humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of non-human antibodies inducing an immune response in humans after administration, but retains a binding specificity and affinity similar to the starting non-human antibody. A humanized antibody binds to an epitope that is identical or similar to a non-human antibody. The term "humanized antibody" includes antibodies that are partially or completely composed of an amino acid sequence derived from a human antibody germline by changing the sequence of an antibody with a non-human hypervariable region ("HVR"). The simplest such change may include simply replacing the mouse constant region with the constant region of a human antibody, thereby producing a human / mouse chimera that may have sufficiently low immunogenicity to make it useful for pharmaceutical purposes. Preferably, the variable region of the antibody is also humanized by techniques currently well known in the art. For example, the framework region of the variable region may be replaced with a corresponding human framework region while retaining one, several or all six non-human HVRs. Some framework residues may be replaced with corresponding residues from a non-human VL domain or VH domain (e.g., a non-human antibody from which the HVR residues are derived), for example, to restore or improve the specificity or affinity of the humanized antibody. The substantially human framework region has at least about 75% homology with a known human framework sequence (i.e., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity). HVRs may also be randomly mutated so that binding activity and affinity to the antigen are maintained or enhanced in the case of a fully human germline framework region or a substantially human framework region. As described above, the use of antibody fragments is sufficient for use in the methods of the present invention. Further, as used herein, the term "humanized antibody" refers to an antibody comprising a substantially human framework region, at least one HVR from a non-human antibody, and any constant region present therein is substantially human. Substantially human constant regions have at least about 90% (i.e., about 90%, about 95%, or about 99% sequence identity) with a known human constant sequence. Therefore, all portions of the humanized antibody, except possible HVRs, are substantially identical to corresponding one or more germline human immunoglobulin sequence pairs.

[0094] If necessary, the design of humanized immunoglobulins can be performed as follows or using similar methods familiar to those skilled in the art (e.g., see Almagro, et al. Front. Biosci. 2008, 13 (5): 1619-33). The mouse antibody variable region is aligned with the most similar human germline sequence (e.g., by using BLAST or similar algorithms). The CDR residues from the mouse antibody sequence are grafted into a similar human "receptor" germline. Subsequently, one or more positions (e.g., Vernier positions) near the CDR or within the framework can be restored to the original mouse amino acids to obtain a humanized antibody with similar binding affinity to the initial mouse antibody. Generally, several versions of humanized antibodies with different back mutations are generated, and the activity is tested empirically. The humanized antibody variant with the most similar properties to the parent mouse antibody and the least mouse framework restoration is selected as the final humanized antibody candidate.

[0095] The term "Aβ" refers to a peptide derived from a region in the carboxyl terminus of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Aβ that may have toxic effects: Aβ peptides are generally 37-43 amino acid sequences in length, although they may have truncations and modifications that change their overall size. They can exist in soluble and insoluble compartments intracellularly or extracellularly in monomeric, oligomeric and aggregated forms, and can be complexed with other proteins or molecules. The adverse or toxic effects of Aβ may be caused by any or all of the above-noted forms, as well as other forms not specifically described. For example, two such Aβ subtypes include Aβ40 and Aβ42; wherein the Aβ42 subtype is particularly fibrillogenic or insoluble and is associated with a disease state. The term "Aβ" generally refers to multiple Aβ species without distinguishing between individual Aβ species. Specific Aβ species are identified by the size of the peptide, such as Aβ42, Aβ40, Aβ38, etc.

[0096] As used herein, the term "Aβ42 / Aβ40 value" means the ratio of the amount of Aβ42 in a sample obtained from a subject compared to the amount of Aβ40 in the same sample.

[0097] "Aβ amyloidosis" is defined as clinically abnormal Aβ deposition in the brain. Subjects determined to have Aβ amyloidosis are referred to herein as "amyloid positive", while subjects determined to have no Aβ amyloidosis are referred to herein as "amyloid negative". There are recognized indicators of Aβ amyloidosis in the art. At the time of the present disclosure, Aβ amyloidosis is measured directly by amyloid imaging (e.g., PiB PET, fluorbetapir, or other imaging methods known in the art), or indirectly by reduced cerebrospinal fluid (CSF) Aβ42 or reduced CSF Aβ42 / 40 ratio. [11C]PIB-PET imaging with an average cortical binding potential (MCBP) score>0.18 is an indicator of Aβ amyloidosis, and the cerebrospinal fluid (CSF) Aβ42 concentration of about 1ng / ml measured by immunoprecipitation and mass spectrometry (IP / MS) is also an indicator of Aβ amyloidosis. Alternatively, a cutoff ratio of CSF Aβ42 / 40 that maximizes the accuracy of predicting amyloid positivity as determined by PIB-PET may be used. Values ​​such as these or other values ​​known in the art and / or used in the Examples may be used alone or in combination to clinically confirm Aβ amyloidosis. See, for example, 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, each of which is hereby incorporated by reference in its entirety. Subjects with Aβ amyloidosis may be symptomatic or may be asymptomatic, and symptomatic subjects may or may not meet the clinical criteria for a disease associated with Aβ amyloidosis. Non-limiting examples of symptoms associated with Aβ amyloidosis may include impaired cognitive function, behavioral changes, abnormal language function, mood disorders, epileptic seizures, dementia, and impaired nervous system structure or function. Diseases associated with Aβ amyloidosis include, but are not limited to, Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia, and inclusion body myositis. Subjects with Aβ amyloidosis are at an increased risk of developing diseases associated with Aβ amyloidosis.

[0098] "Clinical signs of Aβ amyloidosis" refers to measures of Aβ deposition known in the art. Clinical signs of Aβ amyloidosis may include, but are not limited to, Aβ deposition identified by amyloid imaging (e.g., PiB PET, frobetapib, or other imaging methods known in the art) or by reduced cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio. See, e.g., Klunk WE et al. Ann Neurol 55 (3) 2004, and Fagan AM et al. Ann Neurol 59 (3) 2006, each of which is hereby incorporated by reference in its entirety. Clinical signs of Aβ amyloidosis can also include measurements of Aβ metabolism, particularly measurements of Aβ42 metabolism alone or compared to measurements of metabolism of other Aβ variants (e.g., Aβ37, Aβ38, Aβ39, Aβ40, and / or total Aβ), as described in U.S. Pat. Nos. 14 / 366,831, 14 / 523,148, and 14 / 747,453, each of which is hereby incorporated by reference in its entirety. Additional 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 & Dementin, 2007, Vol. 7, pp. 280-292, each of which is hereby incorporated by reference in its entirety. Importantly, a subject with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition. However, a subject with clinical signs of Aβ amyloidosis is at increased risk for developing a disease associated with Aβ amyloidosis.

[0099] "Candidate for amyloid imaging" refers to a subject who has been identified by a clinician as an individual for whom amyloid imaging may be clinically necessary. As a non-limiting example, a candidate for amyloid imaging may be a subject with one or more clinical signs of Aβ amyloidosis, one or more Aβ plaque-associated symptoms, one or more CAA-associated symptoms, or a combination thereof. A clinician may recommend amyloid imaging for such a subject to guide his or her clinical care. As another non-limiting example, a candidate for amyloid imaging may be a potential participant (control subject or test subject) in a clinical trial for a disease associated with Aβ amyloidosis.

[0100] "Aβ plaque-associated symptoms" or "CAA-associated symptoms" refer to any symptoms caused by or associated with the formation of amyloid plaques or CAA, respectively, which are composed of regularly ordered fibril aggregates called amyloid fibrils. Exemplary Aβ plaque-associated symptoms may include, but are not limited to, neuronal degeneration, impaired cognitive function, impaired memory, behavioral changes, mood disorders, epileptic seizures, impaired structure or function of the nervous system, and increased risk of development or deterioration of Alzheimer's disease or CAA. Neuronal degeneration may include changes in neuronal structure (including molecular changes, such as intracellular accumulation of toxic proteins, protein aggregates, etc., and changes at the macroscopic level, 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, death of neurons, or any combination thereof. Impaired cognitive function may include, but is not limited to, difficulties with memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and organization. Behavioral changes may include, but are not limited to, physical or verbal aggression, impulsivity, decreased inhibition, apathy, decreased initiative, personality changes, alcohol, tobacco or drug abuse, and other addiction-related behaviors. Emotional disorders may include, but are not limited to, depression, anxiety, mania, irritability and emotional incontinence. Epileptic seizures may include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures and non-epileptic psychogenic seizures. Impaired nervous system structure or function may include, but are not limited to, hydrocephalus, Parkinson's disease, sleep disorders, psychosis, balance and coordination impairment. This may include motor impairment, such as monoparesis, hemiparesis, quadriplegia, ataxia, convulsions and tremors. This may also include sensory loss or disorder, including smell, touch, taste, vision and hearing. In addition, this may include autonomic nervous system impairment, such as bowel and bladder dysfunction, sexual dysfunction, blood pressure and temperature disorders. Finally, this can include hormonal impairment caused by dysfunction of the hypothalamus and pituitary glands, such as deficiencies and imbalances in growth hormone, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, gonadotropin-releasing hormone, prolactin, and numerous other hormones and regulators.

[0101] As used herein, the term "subject" refers to a mammal, preferably a human. Mammals include, but are not limited to, humans, primates, livestock, rodents, and pets. A subject may be awaiting medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment.

[0102] As used herein, the terms "control population," "normal population," or samples from "healthy" subjects refer to subjects or groups of subjects who are clinically determined to be free of tauopathy or Aβ amyloidosis, or clinical diseases associated with Aβ amyloidosis (including but not limited to Alzheimer's disease) based on qualitative or quantitative test results.

[0103] As used herein, the term "blood sample" refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. The blood sample may be whole blood, plasma or serum, although plasma is generally preferred.

[0104] As used herein, the term "isoform" refers to any of several different forms of the same protein variant that occur due to alternative splicing of the mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein, genetic variation, and somatic recombination. The terms "isoform" and "variant" are used interchangeably.

[0105] The term "tau" refers to a variety of subtypes encoded by the gene MAPT (or its homologue), and its species in vivo through C-terminal truncation, in vivo through N-terminal truncation, in vivo through post-translational modification or any combination thereof. As used herein, the terms "tau" and "tau protein" and "tau species" are used interchangeably. In many animals (including but not limited to humans, non-human primates, rodents, fish, cattle, frogs, goats and chickens), tau is encoded by the gene MAPT. In animals where the gene is not identified as MAPT, homologues can be identified by methods well known in the art.

[0106] In humans, there are six subtypes of tau, which are produced by alternative splicing of exons 2, 3 and 10 of MAPT. The length of these subtypes ranges from 352 to 441 amino acids. Exons 2 and 3 encode 29 amino acid insertions in each N-terminus (referred to as N), and the full-length human tau subtype may have two insertions (2N), one insertion (1N) or no insertion (0N). All full-length human tau subtypes also have repetitions of three microtubule binding domains (referred to as R). The inclusion of exon 10 at the C-terminus leads to the inclusion of the fourth microtubule binding domain encoded by exon 10. Therefore, the full-length human tau subtype may include repetitions of four microtubule binding domains (including exon 10: R1, R2, R3 and R4) or repetitions of three microtubule binding domains (excluding exon 10: R1, R3 and R4). Human tau may be post-translationally modified or not post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated and glycosylated. Human tau can also be processed in vivo at the C-terminus, at the N-terminus, or at the C-terminus and N-terminus with or without proteolytic processing. Accordingly, the term "human tau" includes 2N3R, 2N4R, 1N3R, 1N4R, ON3R and ON4R subtypes, and its C-terminal truncation in vivo, N-terminal truncation in vivo, post-translational modification in vivo or any combination thereof. Alternative splicing of genes encoding tau similarly occurs in other animals.

[0107] As used herein, the term "tau-441" refers to the longest human tau isoform (2N4R), which is 441 amino acids in length. The amino acid sequence of tau-441 is provided below: MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTP SLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIH HKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL(SEQ ID NO:1)

[0108] For this subtype, Fig.17 In the present invention, the N-terminus (N term), the middle domain, the MTBR and the C-terminus (C term) are identified. These regions will vary in a predictable manner for other tau subtypes (e.g., 2N3R, 1NR4, 1N3R, 0N4R and 0N3R). Accordingly, when the amino acid positions relative to tau-441 are identified, the technician will be able to determine the corresponding amino acid positions of other subtypes.

[0109] As used herein, the term "N-terminal tau" refers to a tau peptide or multiple tau peptides comprising two or more amino acids or all of the amino acids at the N-terminus of tau (eg, amino acids 1-103 of tau-441, etc.).

[0110] As used herein, the term "middle domain tau" refers to a tau peptide or multiple tau peptides comprising two or more amino acids or all of the amino acids of the middle domain of tau (eg, amino acids 104-243 of tau-441, etc.).

[0111] As used herein, the term "MTBR tau" refers to a tau peptide or multiple tau peptides comprising two or more amino acids or all of the amino acids of the microtubule binding region (MTBR) of tau (eg, amino acids 244-368 of tau-441, etc.).

[0112] As used herein, the term "long MTBR tau" refers to a tau peptide or multiple tau peptides that comprise two or more or all of amino acids 259-368 of tau-441.

[0113] As used herein, the term "very short MTBR-tau243" refers to a tau peptide or multiple tau peptides comprising two or more or all of amino acids 235-254 of tau-441.

[0114] As used herein, the term "C-terminal tau" refers to a tau peptide or multiple tau peptides comprising two or more amino acids or all of the amino acids at the C-terminus of tau (eg, amino acids 369-441 of tau-441, etc.).

[0115] "Endogenous cleavage fragments of tau" refer to peptide fragments of tau protein produced by in vivo proteolytic cleavage events. "Proteolytic fragments of tau" refer to peptide fragments of tau protein produced by in vitro proteolytic cleavage events. Cleaved fragments of tau peptides can be referred to herein by their amino acids numbered with reference to tau-441, wherein the first number indicates that the fragment contains the amino acids referred to and the second number indicates the last amino acid of the fragment. For example, "243-255N-deamidation" or "243-255 (dN)" or "243-255N@255N deamidation" refers to a cleavage fragment of tau including amino acids 243-255, wherein amino acid 255N is the end of the fragment and is post-translationally modified by deamidation at 255N (e.g., LQTAPVPMPDLK (dN) (SEQ ID NO: 10); also known as MTBR-tau243-255dN). Non-limiting examples of other cleavage fragments of the identified tau peptides include 243-257K@255N-deamidation (SEQ ID NO: 11); 243-253L (SEQ ID NO: 12); 243-252D (SEQ ID NO: 13); 243-258S@255N-deamidation (SEQ ID NO: 14); 243-256V (SEQ ID NO: 15); 243-256V@255N-deamidation (SEQ ID NO: 16; also referred to as 243-256VdN, 243-256V(dN), and 243-dN-256V); and 212-221 (SEQ ID NO: 17).

[0116] Diseases associated with tau deposition in the brain are referred to herein as "tauopathy". The term "tau deposition" comprises all forms of pathological tau deposition, including but not limited to tau aggregates in neurofibrillary tangles, neuropil filaments and dystrophic neurites. Tauopathies known in the art include but are not limited to progressive supranuclear palsy (PSP), dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and Parkinson's disease linked to chromosome 17, Lytico-Bodig disease, Guam Parkinson-dementia complex, tangle-dominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), frontotemporal degeneration (FTLD), Alzheimer's disease (AD) and frontotemporal dementia (FTD).

[0117] Tauopathies are classified by the preponderance of tau subtypes found in pathological tau deposition. Tauopathies with tau deposits consisting mainly of tau with three MTBRs are called "3R-taupathies". Pick's disease is a non-limiting example of 3R-taupathies. For the sake of illustration, the pathological tau deposits of some 3R-taupathies can be a mixture of 3R and 4R tau subtypes, in which 3R subtypes are dominant. Intracellular neurofibrillary tangles (i.e. tau deposits) in the brain of a subject suffering from Alzheimer's disease are generally considered to contain approximately equal amounts of both 3R and 4R subtypes. Tauopathies with tau deposits consisting mainly of tau with four MTBRs are called "4R-taupathies". PSP, CBD and AGD are non-limiting examples of 4R-taupathies, as are some forms of FTLD. Notably, pathological tau deposits in the brains of some subjects with genetically confirmed cases of FTLD, such as some V334M and R406W mutation carriers, showed a mixture of 3R and 4R isoforms.

[0118] Clinical signs of tauopathy can be tau aggregates in the brain, including but not limited to neurofibrillary tangles. Methods for detecting and quantifying tau aggregates in the brain are known in the art (e.g., 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) and other tau PET).

[0119] As used herein, the terms "treat", "treating" or "treatment" refer to the provision of medical care to a subject in need by a trained and licensed professional. Medical care can be diagnostic testing, therapeutic treatment and / or preventive or preventive measures. The purpose of therapeutic and preventive treatment is to prevent or slow down (mitigate) undesirable physiological changes or diseases / disorders. The beneficial or desired clinical results of therapeutic or preventive treatment include, but are not limited to, alleviation of symptoms, weakening of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonged survival compared to the expected survival if not receiving treatment. Those in need of treatment include those already suffering from a disease, condition or disorder, as well as those susceptible to a disease, condition or disorder, or those for which a disease, condition or disorder is to be prevented. Accordingly, a subject in need of treatment may or may not have any symptoms or clinical signs of the disease.

[0120] The phrase "tau therapy" refers collectively to any imaging agent, therapeutic treatment, and / or prophylactic or preventive measure that is contemplated for or used in a subject at risk of developing tau disease, or a subject clinically diagnosed with tau disease. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flumetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.). Non-limiting examples of therapeutic agents include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), γ-secretase inhibitors, β-secretase inhibitors, anti-Ab antibodies (including antigen-binding fragments, variants or derivatives thereof), anti-tau antibodies (including antigen-binding fragments, variants or derivatives thereof), stem cells, dietary supplements (e.g., lithium water, ω-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), antagonists of serotonin receptor 6, p38α MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, live vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylthionimium chloride (methylthionimium chloride), therapies to improve glycemic control (e.g., insulin, exenatide, liraglutide, pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, b-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT 1A and 1D receptor agonists, glutaminyl peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-γ agonists, tubulin modulators, calcium channel blockers, antihypertensive agents, statins, and any combination thereof.

[0121] 2. Composition

[0122] a. Anti-MTBR tau antibody

[0123] Provided herein is an anti-MTBR-tau antibody. The anti-MTBR tau antibodies disclosed herein may be described or specified according to the epitopes they recognize or bind to. The portion of the target polypeptide that specifically interacts with the antigen binding domain of the antibody is an "epitope". In addition, it should be noted that an "epitope" on MTBR tau may be a linear epitope or a conformational epitope, and in both cases may include non-polypeptide elements, for example, the epitope may include carbohydrate or lipid side chains. The term "affinity" refers to a measure of the strength of binding of a single epitope to the antigen binding site of an antibody. In one aspect, the antibodies disclosed herein bind to an MTBR tau epitope located within amino acids 244-368 of tau-441 (SEQ ID NO: 1).

[0124] As used herein, "anti-MTBR tau antibodies" refer to isolated antibodies that bind to human MTBR tau with an affinity constant or interaction affinity (KD) between about 0.1 pM and about 10 μM, preferably between about 0.1 pM and about 1 μM, more preferably between about 0.1 pM and about 100 nM. Methods for determining the affinity of antibodies for antigens are known in the art. Anti-MTBR tau antibodies useful herein include those suitable for administration to a subject in therapeutic amounts.

[0125] The anti-MTBR tau antibodies disclosed herein may also be described or specified according to their cross-reactivity. The term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen; it is a measure of the correlation between two different antigenic substances. Therefore, if an antibody binds to an epitope other than the epitope that induced its formation, the antibody is cross-reactive. Cross-reactive epitopes typically contain many of the same complementary structural features as the induced epitope, and in some cases, may actually match better than the original. For example, some antibodies have a certain degree of cross-reactivity because they bind to related but not identical epitopes, such as epitopes that have at least about 85%, at least about 90%, or at least about 95% identity (as calculated using methods known in the art) with a reference epitope. If the antibody does not bind to an epitope that has less than about 95%, less than about 90%, or less than about 85% identity with a reference epitope, it can be considered to have little or no cross-reactivity. If the antibody does not bind to any other analog, ortholog, or homolog of the epitope, it can be considered to be "highly specific" for a certain epitope.

[0126] Anti-MTBR tau antibodies may include one or more of the heavy chain and light chain variable regions of HJ34.11. Anti-MTBR tau antibodies may include one or more complementarity determining regions (CDRs) 1-3 of one or more heavy chain and light chain variable regions of HJ34.11. Anti-MTBR tau antibodies may include heavy chain variable region CDR1-3 (H1-3) and light chain variable region CDR1-3 (L1-3) of HJ34.11. In one example, the anti-MTBR tau antibody is HJ34.11 or an antigen-binding fragment thereof. Relative to tau-441, anti-MTBR tau antibodies may bind to an antigen contained within amino acids 226-264 of tau, and in particular amino acids 255-264. Relative to tau-441, anti-MTBR tau antibodies may bind to endogenous peptides of tau containing amino acids 243-254 of tau.

[0127] The sequences of the heavy chain and light chain variable regions of HJ34.11 are as follows. For the heavy chain and light chain variable region sequences provided herein, it is known in the art that such sequences contain signal peptides that can be removed during the post-translational processing of the antibody. References to light chain or heavy chain variable region sequences include sequences lacking the corresponding signal peptides.

[0128]

[0129] Anti-MTBR tau antibodies may include one or more of the heavy chain and light chain variable regions of HJ32.8. Anti-MTBR tau antibodies may include one or more complementarity determining regions (CDRs) 1-3 of one or more heavy chain and light chain variable regions of HJ32.8. Anti-MTBR tau antibodies may include heavy chain variable region CDR1-3 and light chain variable region CDR1-3 of HJ32.8. In one example, the anti-MTBR tau antibody is HJ32.8 or an antigen-binding fragment thereof. Relative to tau-441, anti-MTBR tau antibodies may bind to an antigen contained within amino acids 225-242 of tau, and in particular amino acids 225-235. Relative to tau-441, anti-MTBR tau antibodies may bind to endogenous peptides of tau containing amino acids 243-254 of tau.

[0130] The sequences of the heavy and light chain variable regions of HJ32.8 are shown below.

[0131]

[0132] Anti-MTBR tau antibodies may include one or more of the heavy chain and light chain variable regions of HJ32.11. Anti-MTBR tau antibodies may include one or more complementarity determining regions (CDRs) 1-3 of one or more heavy chain and light chain variable regions of HJ32.11. Anti-MTBR tau antibodies may include heavy chain variable region CDR1-3 and light chain variable region CDR1-3 of HJ32.11. In one example, the anti-MTBR tau antibody is HJ32.11 or an antigen-binding fragment thereof. Relative to tau-441, anti-MTBR antibodies may bind to an antigen contained within amino acids 225-242 of tau, and more particularly amino acids 235-242. Relative to tau-441, anti-MTBR tau antibodies may bind to endogenous peptides of tau containing amino acids 243-254 of tau. Relative to tau-441, the anti-MTBR tau antibody can bind to an endogenous peptide of tau containing amino acids 243-256 of tau, which can be MTBR-tau243-254, MTBR-tau243-256V, or MTBR-tau243-256V@255N deamidation.

[0133] The sequences of the heavy and light chain variable regions of HJ32.11 are shown below.

[0134]

[0135] Anti-MTBR tau antibodies may include one or more of the heavy chain and light chain variable regions of HJ34.8. Anti-MTBR tau antibodies may include one or more complementarity determining regions (CDRs) 1-3 of one or more heavy chain and light chain variable regions of HJ34.8. Anti-MTBR tau antibodies may include heavy chain variable region CDR1-3 and light chain variable region CDR1-3 of HJ34.8. In one example, the anti-MTBR tau antibody is HJ34.8 or an antigen-binding fragment thereof. Relative to tau-441, anti-MTBR tau antibodies may bind to an antigen contained within amino acids 226-264 of tau, and in particular amino acids 255-264. Relative to tau-441, anti-MTBR tau antibodies may bind to endogenous peptides of tau containing amino acids 243-254 of tau.

[0136] The sequences of the heavy and light chain variable regions of HJ34.8 are shown below.

[0137]

[0138] In an exemplary embodiment, an anti-MTBR tau antibody according to the present disclosure comprises a VL having one or more HVRs derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or a VH having one or more HVRs derived from SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. The HVRs derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 may be L1, L2, L3, or any combination thereof. The HVRs derived from SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs derived from SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 may further comprise a light chain variable region (VL) comprising one or more HVRs derived from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. HVR may be L1, L2, L3 or any combination thereof. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and / or a VH that is 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9. In each of the above embodiments, the anti-MTBR tau antibody may optionally comprise one or more constant regions, or a portion of a constant region, that is substantially human (i.e., has at least 90%, 95% or 99% sequence identity to a known human framework sequence). The present disclosure also includes nucleic acid sequences encoding SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 or 9, which can be readily determined by one skilled in the art and incorporated into a vector or other large DNA molecule (such as a chromosome) to express the antibodies of the present disclosure.

[0139] In each of the above embodiments, L1, L2, L3, H1, H2 and H3 can be determined as follows

[0140] (a) amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0141] (b) amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0142] (c) Antigenic contact points present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996));

[0143] (d) CDR1-IMGT (positions 27-38), CDR2-IMGT (positions 56-65) and CDR3-IMGT regions (positions 105-116 or 105-117), which are based on the IMGT unique numbering (Lefranc, "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains," The Immunologist, 1999, 7: 132-136; Lefranc et al., Nucleic Acids Research, 2009, 37 (Database issue): D1006-D1012; Ehrenmann et al., "Chapter 2: Standardized Sequence and Structure Analysis of Antibody Using IMGT," in Antibody Engineering, Vol. 2, Roland E. Kontermann and Stefan Dubel, eds., 2010, Springer-Verlag Berlin Heidelberg, doi:10.1007 / 978-3-642-01147-4; imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition), and

[0144] (e) A combination of (a), (b), (c) and / or (d) as defined below for various antibodies of the present disclosure.

[0145] In some embodiments, each of the above exemplary antibodies may also contain a variant Fc region.

[0146] b. Components of the composition

[0147] The present disclosure also provides pharmaceutical compositions comprising the antibodies described herein. Such compositions include an antibody as disclosed herein and at least one pharmaceutically acceptable excipient.

[0148] Pharmaceutically acceptable excipients may be diluents, binders, fillers, buffers, pH adjusters, disintegrants, dispersants, preservatives, lubricants, taste masking agents, flavoring agents or coloring agents. The amount and type of excipients used to form a pharmaceutical composition may be selected according to known pharmaceutical science principles.

[0149] (1) Diluent

[0150] In one embodiment, the excipient can be a diluent. The diluent can be compressible (i.e., plastically deformable) or abrasive brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethylcellulose, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphorylated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, maltitol (malitol), sorbitol, xylitol, maltodextrin and trehalose. Non-limiting examples of suitable abrasive brittle diluents include calcium hydrogen phosphate (anhydrous or dihydrate), tricalcium phosphate, calcium carbonate and magnesium carbonate.

[0151] (2) Adhesive

[0152] In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinyl pyrrolidone, cellulose, methyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohol, polyethylene glycol, polyol, carbohydrate, oligosaccharide, polypeptide, oligopeptide and combinations thereof.

[0153] (3) Fillers

[0154] In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. As non-limiting examples, the filler may be calcium sulfate (both calcium hydrogen phosphate and tricalcium sulfate), starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, calcium hydrogen phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

[0155] (4) Buffer

[0156] In yet another embodiment, the excipient may be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (eg, Tris buffered saline or phosphate buffered saline).

[0157] (5) pH adjuster

[0158] In various embodiments, the excipient may be a pH adjuster. As non-limiting examples, the pH adjuster may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0159] (6) Disintegrants

[0160] In another embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof; sweeteners; clays such as bentonite; microcrystalline cellulose; alginates; sodium starch glycolate; gums such as agar, guar gum, locust bean gum, karaya gum, pectin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0161] (7) Dispersant

[0162] In yet another embodiment, the excipient may be a dispersant or a dispersion enhancer. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinyl pyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

[0163] (8) Excipients

[0164] In another alternative embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include, but are not limited to, antioxidants such as BHA, BHT, vitamin A, vitamin C, vitamin E or retinol palmitate, citric acid, sodium citrate; chelating agents such as EDTA or EGTA; and antimicrobial agents such as parabens, chlorobutanol or phenol.

[0165] (9) Lubricant

[0166] In another embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silicon dioxide; and fats such as vegetable stearin, magnesium stearate or stearic acid.

[0167] (10) Odor-masking agents

[0168] In yet another embodiment, the excipient may be a taste masking agent. Taste masking materials include cellulose ethers; polyethylene glycol; polyvinyl alcohol; copolymers of polyvinyl alcohol and polyethylene glycol; monoglycerides or triglycerides; acrylic acid polymers; mixtures of acrylic acid polymers and cellulose ethers; cellulose acetate phthalate; and combinations thereof.

[0169] (11) Flavoring agents

[0170] In an alternative embodiment, the excipient may be a flavoring agent. The flavoring agent may be selected from synthetic flavor oils and flavoring aromatics and / or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.

[0171] (12) Colorants

[0172] In yet another embodiment, the excipient may be a colorant. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).

[0173] The weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

[0174] c. Administration form

[0175] The agents and compositions described herein may be formulated in any conventional manner using one or more pharmaceutically acceptable carriers or excipients, as described, for example, in Remington's Pharmaceutical Sciences (AR Gennaro ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such preparations will contain a therapeutically effective amount of a bioactive agent described herein, which may be in purified form, together with a suitable amount of the carrier to provide a form suitable for administration to a subject.

[0176] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject (such as a human). Therefore, a "formulation" may include a pharmaceutically acceptable excipient, including a diluent or carrier.

[0177] The term "pharmaceutically acceptable" as used herein can describe a substance or component that does not cause unacceptable loss of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those with monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF") or later versions, and components listed in the inactive ingredient search (Inactive Ingredient Search) online database continuously updated by FDA. Other available components not described in USP / NF etc. can also be used.

[0178] As used herein, the term "pharmaceutically acceptable excipient" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (AR Gennaro ed.), 21st edition, ISBN: 0781746736 (2005)). Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0179] A "stable" formulation or composition can refer to a composition that has sufficient stability to permit storage at a convenient temperature (e.g., between about 0°C and about 60°C) for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0180] The present disclosure includes pharmaceutical compositions comprising the anti-MTBR tau antibodies disclosed above to facilitate administration and promote stability of active agents. For example, the anti-MTBR tau antibodies of the present disclosure can be mixed with at least one pharmaceutically acceptable carrier or excipient to produce a pharmaceutical composition that can and effectively be administered (administered) to a living subject, such as a suitable subject (i.e., a "subject in need of treatment" or a "subject in need thereof"). Methods for preparing and administering the anti-MTBR tau antibodies disclosed herein to a subject in need thereof are well known or readily determined by those skilled in the art. The route of administration of the anti-MTBR tau antibodies can be, for example, peripherally, orally, parenterally, by inhalation or topically.

[0181] The pharmaceutical composition for effective administration is intentionally designed to be suitable for the selected mode of administration and uses pharmaceutically acceptable excipients, such as compatible carriers, dispersants, buffers, surfactants, preservatives, solubilizers, isotonic agents, stabilizers, etc., as appropriate.

[0182] Non-limiting examples of pharmaceutically acceptable carriers include physiological saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, lanolin, or combinations thereof.

[0183] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride will be included in the composition.

[0184] Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0185] The compositions disclosed herein may be frozen or lyophilized for storage and reconstituted in a suitable sterile vehicle prior to use.

[0186] In some embodiments, anti-MTBR tau antibodies can be formulated for parenteral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, etc., may also be present. Parenteral formulations may be single bolus doses, infusions before maintenance doses, or loading bolus doses. These compositions can be administered at specific fixed or variable intervals, such as once a day, or "as needed."

[0187] Certain pharmaceutical compositions as disclosed herein can be administered orally in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions can also be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, and / or other conventional solubilizing or dispersing agents.

[0188] The amount of anti-MTBR tau antibody to be combined with the carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The composition can be administered as a single dose, multiple doses, or by infusion over a given period of time. The dosage regimen can also be adjusted to provide the optimal desired response (e.g., a therapeutic or preventive response).

[0189] As mentioned above, the agents or compositions described herein can also be used in combination with other therapeutic agents. Thus, in addition to the therapies described herein, other therapies known to be effective in treating a disease, disorder or condition can also be provided to the subject.

[0190] The present disclosure includes pharmaceutical compositions comprising agents as disclosed above to facilitate administration and to promote the stability of the active agent. For example, the compounds of the present disclosure may be mixed with at least one pharmaceutically acceptable carrier or excipient to produce a pharmaceutical composition that can and effectively be administered (administered) to a living subject, such as administering a suitable subject (i.e., a "subject in need of treatment" or a "subject in need thereof"). For purposes of various aspects and embodiments of the present invention, the subject may be a human or any other animal.

[0191] 3. Methods for detecting Tau fragments

[0192] The present disclosure provides a method for detecting a fragment of tau in a biological sample. Detection may include confirming the presence of a fragment or quantifying the amount of a fragment. The fragment may be endogenously cut. In one example, the fragment of tau comprises one or more of the amino acids 212-254 of very short MTBR-tau243 or tau-441. The fragment of tau may indicate insoluble tau or tangles (e.g., neurofibrillary tangles) of aggregation associated with tauopathy. Detection and measurement may be performed by mass spectrometry or immunoassay. The biological sample may be obtained from a subject suffering from or suspected of suffering from tauopathy. Tauopathy may be AD.

[0193] The fragment of tau may comprise one or more of the peptides listed in Table 1, wherein the C-terminal amino acid refers to the last amino acid at the C-terminus of the peptide.

[0194] Table 1 Cleavage fragments of peptides

[0195] name sequence SEQ ID NO: 243-255N-deamidation LQTAPVPMPDLK(dN) 10 243-257K@255N-deamidation LQTAPVPMPDLK(dN)VK 11 243-253L LQTAPVPMPDL 12 243-252 DLQTAPVPMPD 13 243-258S@255N-Deamidation LQTAPVPMPDLK(dN)VKS 14 243-256V LQTAPVPMPDLKNV 15 243-256V@255N-Deamidation LQTAPVPMPDLK(dN)V 16 212-221 TPSLPTPPTR 17 243-257K LQTAPVPMPDLKNVK 18 243-255N LQTAPVPMPDLKN 19 243-254@244 deamidation LQTAPVPMPDLK 20 243-254 LQTAPVPMPDLK 21

[0196] In one example, the fragment of tau may include one or more of MTBR-tau243-256V, MTBR-tau243-256V(dN), and MTBR-tau212-221.

[0197] Detecting endogenous cleavage fragments of tau in a biological sample may include providing a biological sample and purifying cleavage fragments of tau from the biological sample. The cleavage fragments of tau may be performed without first exposing the cleavage fragments of tau to additional in vitro proteolytic cleavage. The cleavage fragments of tau may not be trypsin-cleaved tau peptides. Pre-clearing steps (e.g., protein precipitation and / or immunodepletion) before or after purification of the cleavage fragments of tau may not be performed. The cleavage fragments of tau may be further cleaved in vitro with a protease before or after purification. The protease may include one or more of trypsin, Lys-N endopeptidase, Lys-C endopeptidase, Asp-C endopeptidase, Arg-N endopeptidase, and Arg-C endopeptidase. In an example of detecting tau fragments in plasma, the protease includes an Arg-C endopeptidase.

[0198] The cleavage fragment samples of tau can be subjected to one or at least two steps of immunodepletion or immunoprecipitation. Each round of immunodepletion or immunoprecipitation can be performed using one or more anti-tau or anti-MTBR tau antibodies disclosed herein, or a combination thereof. After each immunodepletion or immunoprecipitation step, the purified tau fragments from one or more of the immunoprecipitated samples or immunodepleted samples can be further cleaved with one or more proteases disclosed herein.

[0199] Liquid chromatography-mass spectrometry (LC / MS) or immunoassays of samples containing purified tau cleavage fragments can be used to detect and measure the concentration (relative or absolute) of at least one tau cleavage fragment. One or more tau cleavage fragments can be used to detect and measure the amount of tau present in a biological sample, such as the amount of insoluble tau aggregates. The immunoassay may include contacting a sample containing purified tau cleavage fragments with an anti-MTBR tau antibody disclosed herein. In one example, the anti-MTBR tau antibody is capable of binding to MTBR-tau43 and comprises at least one of the light chain variable region and the heavy chain variable region of HJ32.11 or a sequence disclosed herein (e.g., H1-H3 and L1-L3 of HJ32.11). In another example, the anti-MTBR tau antibody is HJ32.11. In another example, the anti-tau antibody competitively inhibits HJ32.11 binding. In one example, the immunoassay is ELISA.

[0200] a. Detection of tau in biological samples

[0201] The method may include one or more of the following steps: (a) providing a biological sample selected from a blood sample or a CSF sample containing a cleavage fragment of tau (which may be endogenously cleaved); (b) purifying the cleavage fragment of tau from the biological sample without first exposing the cleavage fragment of tau to additional in vitro proteolytic cleavage; and (c) detecting the concentration of at least one purified cleavage fragment of tau. As used herein, detection may include one or more of confirming the presence, measuring, and quantifying. The blood sample disclosed herein may be a plasma sample.

[0202] In another example, the method includes one or more of the following: (a) providing a biological sample selected from a blood sample or a CSF sample containing endogenous cleavage fragments of tau; (b) purifying the cleavage fragments of tau from the biological sample, wherein before or after purification, the cleavage fragments of tau are optionally further cleaved in vitro with one or more proteases; and (c) detecting the concentration of at least one purified cleavage fragment of tau.

[0203] The method may include one or more of the following: (a) providing a biological sample selected from a blood sample or a CSF sample; (b) purifying cleavage fragments of tau from the biological sample; (c) optionally cleaving the purified cleavage fragments of tau with one or more proteases, and then optionally desalting the resulting cleavage products by solid phase extraction to obtain a sample comprising proteolytic peptides of tau; and (d) performing liquid chromatography-mass spectrometry on the sample comprising proteolytic peptides of tau to detect and measure the amount of at least one proteolytic peptide of the cleavage fragments of tau.

[0204] In another example, the method of the present disclosure includes reducing at least one tau peptide comprising one or more of N-terminal tau, middle domain tau, and long MTBR tau in a biological sample by affinity depletion or immunoprecipitation. In one example, the peptide comprises N-terminal tau, middle domain tau, and long MTBR tau. The tau peptide comprising N-terminal tau can be contacted with an antibody that binds to an antigen within N-terminal tau, and the antibody can be HJ8.5, HJ8.7, or both. The tau peptide comprising middle domain tau can be contacted with an antibody that binds to an antigen within middle domain tau, and the antibody can be Tau1. The tau peptide comprising the MTBR of tau can be contacted with an antibody that binds to an antigen within the MTBR, and the antibody can be HJ34.8. The tau peptide comprising the very short MTBR-tau243 can be contacted with an antibody that binds to an antigen within the very short MTBR-tau243, and the antibody can be HJ32.11. The antibodies disclosed herein can be attached to a solid matrix, which can include a plate, a bead, or a column. In one example, the antibody is attached to a bead for use in immunodepletion or immunoprecipitation. For use in immunodepletion, immunoprecipitation, or immunodetection, the antibody can be attached to a plate or a bead.

[0205] A biological sample containing endogenous cleavage fragments of tau is contacted with one or more or all of HJ8.5, HJ.8.7 and Tau1. In one example, the biological sample is contacted with Tau1, HJ8.5 and HJ8.7. In another, the biological sample is contacted with Tau1, HJ8.5, HJ8.7 and HJ34.8. Samples depleted of tau peptides using the above antibodies can be proteolytically cleaved in vitro using one or more proteases disclosed herein, and the resulting in vitro cleaved tau fragments (proteolytic fragments) can be contacted with antibodies (such as HJ32.11) that bind to very short MTBR-tau243. In one example, the protease comprises an Arg-C endopeptidase.

[0206] The biological sample may be a CSF sample or a plasma sample. The biological sample may further comprise an isotope-labeled tau internal standard. The method may include enriching tau cleavage fragments by a method comprising purifying one or more tau cleavage fragments from an affinity-depleted sample. The enriched tau cleavage fragments may be enriched in very short MTBR-tau243 peptides. Very short MTBR-tau243 peptides may be detected and / or measured using an immunoassay (which may include the use of HJ32.11). Samples containing enriched very short MTBR-tau243 may be proteolytically cleaved with a protease. The resulting cleavage may be desalted by solid phase extraction to obtain a sample containing tau peptides. Tau peptides may be detected by performing liquid chromatography-mass spectrometry (LC / MS) or immunoassay on a sample containing tau peptides to detect and measure the amount of at least one peptide of the cleavage fragments of tau.

[0207] Although not required, in each of the above methods, before or after purification of the cleaved fragments of tau, removal of additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant may be performed.

[0208] (1) Detection of tau peptide in CSF

[0209] The present invention provides a method for detecting tau peptide in a CSF sample. The tau peptide may be a very short MTBR-tau243 peptide. The method may include contacting a CSF sample from a subject with an anti-tau antibody. In one example, an internal standard is added to the CSF sample. The internal standard may be 15 N0N3R-tau and 15 One or both of N 2N4R-tau. Anti-tau antibodies may include one or more or all of HJ8.5, HJ8.7 and Tau1. Anti-tau antibodies may also include HJ34.8. Antibodies that competitively inhibit the above antibodies (e.g., N-terminal tau, middle domain tau, and long MTBR tau) can be used in the methods disclosed herein. As described herein, the antibodies can be attached to a solid matrix, which can be beads. The sample can then be subjected to affinity depletion (immunodepletion) or immunoprecipitation to form a first immunoprecipitate containing immunoprecipitated tau peptides. Immunodepleted samples can be enriched in one or more of very short MTBR-tau243, MTBR-tau243-254, MTBR-tau243-256V, MTBR-tau243-256V (dN), and MTBR-tau212-221.

[0210] The immunoprecipitated tau peptides can be cleaved by proteolytic cleavage in vitro by contacting the tau peptides in the sample with a protease disclosed herein (which may include trypsin and may further include a Lys-C endopeptidase). In one example, the protease does not include an Arg-C endopeptidase. In the resulting sample, tau proteins that may indicate amyloid plaques, such as p-tau, can be detected. P-tau can be one or more of p-tau181, p-tau205, p-tau217, and p-tau231.

[0211] The immunodepleted sample can be contacted with an antibody capable of binding to very short MTBR-tau43, which can be HJ32.11. In one example, an internal standard is added to the immunodepleted sample. The internal standard can be 13 C 15 N 2N4R-tau. The immunodepleted sample can then be subjected to immunoprecipitation to form a second immunoprecipitate. The very short MTBR-tau243 in the second immunoprecipitate can be detected and / or measured in an immunoassay, the immunoassay comprising contacting the very short MTBR-tau243 with an anti-tau antibody, the antibody being HJ32.11. The second immunoprecipitate can also be subjected to in vitro proteolytic cleavage by contacting the tau peptide in the second immunoprecipitate with one or more proteases disclosed herein (which may include trypsin). The proteolytic peptides in the second immunoprecipitate may include one or more of MTBR-tau243-254, MTBR-tau243-256V, MTBR-tau243-256V (dN) and MTBR-tau212-221, which can be detected and / or measured using LC / MS. Before performing LC / MS, the second immunoprecipitate can be desalted.

[0212] b. Detection of tau peptide in plasma

[0213] The present invention provides a method for detecting tau peptides in plasma samples. The tau peptide may be one or more of very short MTBR-tau243, MTBR-tau243-254, MTBR-tau243-256V, MTBR-tau243-256V (dN) and MTBR-tau212-221. In one example, the peptide is MTBR-tau243-256V (dN). The method may include contacting a plasma sample from a subject with an anti-tau antibody. In one example, an internal standard is added to a CSF sample. The internal standard may be 15N 2N4R-tau. The anti-tau antibodies may include one or more or all of HJ8.5, HJ8.7, and Tau1. The anti-tau antibodies may also include HJ34.8. The sample may then be subjected to affinity depletion or immunoprecipitation to form a first immunoprecipitate comprising immunoprecipitated tau peptides, and an immunodepleted sample that may be enriched for very short MTBR-tau243.

[0214] The immunoprecipitated tau peptides can be cleaved by proteolytic cleavage in vitro by contacting the tau peptides in the sample with a protease disclosed herein (which may include trypsin). In one example, the protease does not include Arg-C endopeptidase. In the resulting sample, tau proteins that may indicate amyloid plaques, such as p-tau, can be detected. P-tau can be one or more of p-tau181, p-tau217, and p-tau231.

[0215] The immunodepleted sample can be contacted with an antibody capable of binding to very short MTBR-tau43, which can be HJ32.11. In one example, an internal standard is added to the immunodepleted sample. The internal standard can be 13 N 15 N 2N4R-tau and 15 One or more of N0N3R-tau. Then the immunodepleted sample can be subjected to immunoprecipitation to form a second immunoprecipitate. The very short MTBR-tau243 in the second immunoprecipitate can be detected and / or measured in an immunoassay, and the immunoassay includes contacting very short MTBR-tau243 with an anti-tau antibody, and the antibody can be HJ32.11. The second immunoprecipitate can also be cut by contacting the tau peptide in the second immunoprecipitate with one or more proteases including Arg-C endopeptidase and proteolytic cleavage outside the receptor. Protease can further include other proteases disclosed herein. The Tau peptide in the second immunoprecipitate can include one or more of MTBR-tau243-254, MTBR-tau243-256V, MTBR-tau243-256 (dN) and MTBR-tau212-221, each of which can be detected and / or measured using LC / MS. In particular, one or more of MTBR-tau243-256V(dN) and MTBR-tau212-221 may be detected and / or measured. Prior to LC / MS, the second immunoprecipitate may be desalted.

[0216] In another example, a plasma sample is contacted with HJ32.11. The plasma sample may be supplemented with an internal reference disclosed herein. The contacted sample may be subjected to immunoprecipitation. The very short MTBR-tau243 in the resulting immunoprecipitate may be detected and / or measured in an immunoassay, comprising contacting the immunoprecipitate with an anti-tau antibody (such as HJ32.11) that can bind to very short MTBR-tau243. The resulting immunoprecipitate sample may also be subjected to in vitro proteolytic cleavage with one or more proteolytic enzymes including Arg-C endopeptidase. Proteolytically cleaved tau peptides (which may include one or more of MTBR-tau243-256, MTBR-tau243-256 (dN) and MTBR-tau212-221) may be detected and / or measured using LC / MS. In particular, one or more of MTBR-tau243-256(dN) and MTBR-tau212-221 may be detected and / or measured. Prior to LC / MS, the second immunoprecipitate may be desalted.

[0217] c. Detection of tau peptides other than the very short MTBR-tau243

[0218] The present disclosure further contemplates that in each of the above methods, the presence / absence of one or more proteins in a biological sample is determined, and / or the concentration of one or more other proteins in the biological sample is measured. In some embodiments, one or more proteins may be proteins depleted from a biological sample before tau purification. For example, in certain embodiments, N-terminal tau and / or middle domain tau species may be identified and / or quantified separately from tau species (e.g., MTBR tau, C-terminal tau) quantified by the methods disclosed herein. In some embodiments, in addition to the cleavage fragments of tau, post-translational modifications such as phosphorylation of tau at specific residues may also be measured and quantified. Alternatively or in addition, Aβ (e.g., Aβ42 / 40), ApoE, or any other target protein may be identified and / or quantified by: processing a portion of a biological sample in parallel, depleting the target protein from a biological sample before being used in the methods disclosed herein, or depleting the target protein from a biological sample during the sample processing steps disclosed herein.

[0219] The biological sample, a suitable internal standard, and the steps of purifying tau, optionally depleting one or more proteins, optionally cleaving the purified tau with a protease, and detecting the cleaved fragments of tau are described in more detail below.

[0220] d. Biological samples

[0221] Suitable biological samples include blood samples or cerebrospinal fluid (CSF) samples obtained from subjects. In some embodiments, subjects are human. Human subjects may be waiting for medical care or treatment, may be under medical care or treatment, or may have received medical care or treatment. In various embodiments, human subjects may be healthy subjects, subjects under the risk of developing neurodegenerative diseases, subjects with signs and / or symptoms of neurodegenerative diseases, or subjects diagnosed with neurodegenerative diseases. In further embodiments, neurodegenerative diseases may be tau diseases. In a particular example, tau diseases may be Alzheimer's disease (AD), progressive supranuclear palsy (PSP), cortical basal degeneration (CBD) or frontotemporal lobar degeneration (FTLD). In other embodiments, subjects are laboratory animals. In another embodiment, subjects are laboratory animals genetically engineered to express human tau and optionally one or more other human proteins (such as human A β, human ApoE, etc.).

[0222] CSF can be obtained by lumbar puncture with or without an indwelling CSF catheter. Multiple blood or CSF samples collected from the subject at the same time can be merged. Blood can be collected by venous puncture with or without an intravenous catheter, or by finger puncture (or its equivalent). Once collected, blood or CSF samples can be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cell debris; additives designed for stabilization and preservation of samples before analytical testing, etc.). Blood or CSF samples can be used immediately, or can be frozen and stored indefinitely. Before being used for the methods disclosed herein, if necessary or desired, biological samples can also be modified to include protease inhibitors, isotope-labeled internal standards, detergents and chaotropes, and / or optionally depleted of other analytes (e.g., protein peptides, metabolites).

[0223] The size of the sample used can and will vary depending on the sample type, the health status of the subject from which the sample is obtained, and the analyte (other than tau) to be analyzed. The CSF sample volume can be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In a specific example, the size of the sample can be about 0.05 mL to about 1 mL CSF. The plasma sample volume can be about 0.01 mL to about 20 mL.

[0224] In some embodiments, a single sample is obtained from a subject. Alternatively, a sample can be obtained from a subject over time. Therefore, more than one sample can be collected from a subject over time. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more samples can be collected from a subject over time. When more than one sample is collected from a subject over time, samples can be collected every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days. In some embodiments, sample collection is spaced apart by 1 month, spaced apart by 3 months, spaced apart by 6 months, spaced apart by 1 year, spaced apart by 2 years, spaced apart by 5 years, spaced apart by 10 years, spaced apart by 20 years or more.

[0225] e. Isotope-labeled internal tau standard

[0226] Isotopically labeled tau can be used as an internal standard to account for variability throughout sample processing and optionally calculate absolute concentrations. Typically, an isotopically labeled internal tau standard is added prior to significant sample processing, and it can be added more than once if desired.

[0227] A plurality of isotope-labeled internal tau standards are described herein. All have heavy isotope labels incorporated into at least one amino acid residue. One or more full-length isoforms can be used. Alternatively or in addition, tau isoforms and / or peptide fragments of tau with post-translational modifications can also be used, as known in the art. Generally speaking, the amino acid residues of the incorporated label should increase the quality of the peptide without affecting its chemical properties, and the mass shift caused by the presence of the isotope label must be sufficient to enable mass spectrometry to distinguish internal standards (IS) from endogenous tau analyte signals. As shown herein, suitable heavy isotope labels include, but are not limited to 2H, 13C and 15N. Generally, an internal standard of about 1-10ng is usually sufficient.

[0228] f. Purification of tau

[0229] Another step of the method disclosed herein includes purifying tau, particularly endogenous cleavage and proteolytic cleavage fragments of tau. In some examples, the cleavage fragments of tau are N-terminal-independent, mid-domain-independent and / or C-terminal-independent. The purified tau can be partially purified or completely purified.

[0230] In some embodiments, the method of the present disclosure includes purifying tau by affinity purification. Affinity purification refers to a method for enriching a target protein by the specific binding properties of a target protein to a molecule. Generally, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support such as beads, resins, tissue culture plates, etc. The immobilization of the ligand on the solid support may also occur after the ligand-protein interaction occurs. Suitable ligands include antibodies, aptamers and other epitope binding agents. Purifying the cleavage fragments of tau by affinity purification includes contacting a sample containing endogenous cleavage fragments tau with a suitable immobilized ligand, one or more washing steps, and eluting the cleavage fragments tau from the immobilized ligand.

[0231] In some embodiments, the methods of the present disclosure include purifying the cleaved fragment tau by affinity purification using at least one epitope binding agent, which specifically binds to an epitope within amino acids 225-258 (including endpoints) of tau-441, or within amino acids 235-258 (including endpoints) of tau-441, or within amino acids 235-242 (including endpoints) (or within similarly defined regions of other full-length isoforms). In various embodiments, one, two, three or more epitope binding agents may be used. When two or more epitope binding agents are used, they may be used sequentially or simultaneously. Non-limiting examples of suitable epitope binding agents are disclosed herein. In various embodiments, one, two, three or more epitope binding agents may be used. When two or more epitope binding agents are used, they may be used sequentially or simultaneously.

[0232] In each of the above embodiments, the epitope binding agent may include an antibody or an aptamer. In some embodiments, the epitope binding agent that specifically binds to an epitope within the cleavage fragment tau is HJ32.11, or an epitope binding agent that binds to the same epitope as HJ32.11 and / or competitively inhibits HJ32.11.

[0233] In some embodiments, the methods of the present disclosure include purifying tau by solid phase extraction. Purifying tau by solid phase extraction includes contacting a sample containing tau with a solid phase containing an adsorbent to which tau is adsorbed, one or more washing steps, and eluting tau from the adsorbent. Suitable adsorbents include reversed phase adsorbents. Suitable reversed phase adsorbents are known in the art and include, but are not limited to, alkyl bonded silica, aryl bonded silica, styrene / divinylbenzene materials, N-vinylpyrrolidone / divinylbenzene materials. In an exemplary embodiment, the reversed phase material is a polymer comprising N-vinylpyrrolidone and divinylbenzene or a polymer comprising styrene and divinylbenzene. In an exemplary embodiment, the adsorbent is Oasis HLB (Waters). Prior to contact with a supernatant containing tau, the adsorbent is generally pretreated according to the manufacturer's instructions or as known in the art (e.g., with a water-miscible organic solvent and then with a buffer containing a mobile phase). In addition, the supernatant may optionally be acidified because some reversed phase materials retain ionized analytes more strongly than others. The use of volatile components in the mobile phase and elution is preferred because they promote sample drying. In an exemplary embodiment, the washing step may include the use of a liquid phase comprising about 0.05% v / v trifluoroacetic acid (TFA) to about 1% v / v TFA or its equivalent. In some examples, the washing may be with a liquid phase comprising about 0.05% v / v to about 0.5% v / v TFA or about 0.05% v / v to about 0.1% v / v TFA. In some examples, the washing may be with a liquid phase comprising about 0.1% v / v to about 1.0% v / v TFA or about 0.1% v / v to about 0.5% v / v TFA. The bound tau is then eluted with a liquid phase comprising about 20% v / v to about 50% v / v acetonitrile (ACN) or its equivalent. In some examples, tau can be eluted with a liquid phase comprising about 20% v / v to about 40% v / v ACN, or about 20% v / v to about 30% v / v ACN. In some examples, tau can be eluted with a liquid phase comprising about 30% v / v to about 50% v / v ACN, or about 30% v / v to about 40% v / v ACN. The eluate can be dried by methods known in the art, such as vacuum drying (e.g., speed-vac), lyophilization, evaporation under a stream of nitrogen, etc.

[0234] g. Depletion of one or more proteins

[0235] The method of the present disclosure may include a step in which one or more proteins are depleted from a sample. The term "depletion" means a reduction in amount or quantity. Accordingly, a sample depleted of protein may have any protein amount that is measurably less than the amount in the initial sample, including no protein amount.

[0236] Protein can be depleted from a sample by a method specifically targeting one or more proteins, such as by affinity depletion, solid phase extraction, or other methods known in the art. Where it is desired to perform downstream analysis of a protein or multiple proteins (e.g., identification, quantification, analysis, etc. of post-translational modifications), the targeted depletion of the protein can be used. For example, after the affinity depletion of Aβ with a suitable epitope binding agent, Aβ peptides can be identified and quantified by methods known in the art. As another non-limiting example, after the affinity depletion of apolipoprotein E (ApoE) and the identification of ApoE subtypes, the state of ApoE can be determined by methods known in the art. Targeted depletion can also be used to separate other proteins for subsequent analysis, including but not limited to apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, interleukin, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, NFL, prion protein, pNFH and DJ-1. Targeted depletion of certain tau proteins is also used herein to enrich other tau proteins and / or eliminate proteins that confound mass spectrometry analysis. For example, in certain embodiments of the present disclosure, N-terminal tau proteins, mid-domain tau proteins, and / or long MTBR-tau proteins are depleted from the sample prior to further sample processing for analysis. Downstream analysis of depleted tau proteins may or may not be present, but the methods of the present disclosure contemplate both options.

[0237] In some embodiments, targeted depletion can occur by affinity depletion. Affinity depletion refers to a method of depleting a target protein from a sample by the specific binding properties of the target protein to a molecule. Generally, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support such as beads, resins, tissue culture plates, etc. The immobilization of the ligand on the solid support can also occur after the ligand-protein interaction occurs. Suitable ligands include antibodies, aptamers and other epitope binding agents. The molecule can also be a polymer or other materials that selectively adsorb the target protein. As a non-limiting example, polyhydroxymethylene (e.g., PHM-L LIPOSORB, Sigma Aldrich) substituted by fat oxethylated alcohol can be used to selectively adsorb lipoproteins (including ApoE) from serum. Two or more affinity depletion agents can be combined to deplete a variety of proteins sequentially or simultaneously.

[0238] In some embodiments, the methods of the present disclosure include affinity depletion of one or more proteins from a sample using at least one epitope binding agent that specifically binds to an epitope within amino acids 1 to 243 (inclusive) of tau-441 (or within a similarly defined region of the ON or 1N subtype). 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.

[0239] In some embodiments, the methods of the present disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within the N-terminus of tau (e.g., amino acids 1 to 103 (inclusive) of tau-441), and an epitope binding agent that specifically binds to an epitope within the middle domain of tau (e.g., amino acids 104 to 243 (inclusive) of tau-441). The epitope binding agents may be used sequentially or simultaneously.

[0240] In some embodiments, the methods of the present disclosure include affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1 to 35 (inclusive) of tau-441 and an epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of tau-441 (or within a similarly defined region of the 0N or 1N subtype). The epitope binding agents may be used sequentially or simultaneously.

[0241] In some embodiments, the methods of the present disclosure include using an epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive of endpoints) of tau-441 (or within a similar defined region of the 0N or 1N subtype); an epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive of endpoints) of tau-441 (or within a similar defined region of the 0N or 1N subtype); and an epitope binding agent that specifically binds to an epitope of amyloid β, affinity depleting one or more proteins from a sample. The epitope binding agents may be used sequentially or simultaneously.

[0242] In some embodiments, the methods of the present disclosure include using an epitope binding agent that specifically binds to an epitope within amino acids 1 to 35 (inclusive) of tau-441 (or within a similar defined region of the 0N or 1N subtype); an epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of tau-441 (or within a similar defined region of the 0N or 1N subtype); and an epitope binding agent that specifically binds to an epitope of amyloid β, affinity depleting one or more proteins from a sample. The epitope binding agents may be used sequentially or simultaneously.

[0243] In some embodiments, the methods of the present disclosure include affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (inclusive) of tau-441 (or within a similarly defined region of the 0N or 1N subtype); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0244] In some embodiments, the methods of the present disclosure include affinity depletion of one or more proteins from a sample using an epitope-binding agent that specifically binds to an epitope within amino acids 1 to 35 (inclusive) of tau-441 (or within a similarly defined region of the 0N or 1N subtype); and an epitope-binding agent that specifically binds to an epitope of amyloid β. The epitope-binding agents may be used sequentially or simultaneously.

[0245] In some embodiments, the methods of the present disclosure include affinity depletion of one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 104 to 243 (inclusive) of tau-441 (or within a similarly defined region of the 0N or 1N subtype); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents may be used sequentially or simultaneously.

[0246] In some embodiments, the methods of the present disclosure comprise affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 260 to 441 (inclusive) of tau-441.

[0247] In each of the above embodiments, the epitope binding agent may include an antibody or an aptamer. In some embodiments, the epitope binding agent that specifically binds to amyloid β is HJ5.1, or an epitope binding agent that binds to the same epitope as HJ5.1 and / or competitively inhibits HJ5.1. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 1 to 103 (including endpoints) of tau-441 is HJ8.5, or an epitope binding agent that binds to the same epitope as HJ8.5 and / or competitively inhibits HJ8.5. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 104 to 221 (including endpoints) of tau-441 is Tau1, or an epitope binding agent that binds to the same epitope as Tau1 and / or competitively inhibits Tau1. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 260 to 441 (inclusive) of tau-441 is HJ34.8, or is an epitope binding agent that binds to the same epitope as HJ34.8 and / or competitively inhibits HJ34.8. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 260 to 441 (including endpoints) of tau-441 is HJ34.8, or an epitope binding agent that binds to the same epitope as 77G7, RD3, RD4, UCB1017, or PT76, is an epitope binding agent that binds to the same epitope as HJ34.8 and / or competitively inhibits 77G7, RD3, RD4, UCB1017, or PT76 described in Vandermeeren et al., J Alzheimers Dis, 2018, 65: 265-281, and an epitope binding agent of antibodies E2814 and 7G6 described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, and other epitope binding agents that specifically bind to the same epitope as those antibodies. Methods for identifying an epitope to which an antibody specifically binds, and assays for evaluating competitive inhibition between two antibodies are known in the art.

[0248] Alternatively, proteins can be depleted from a sample by more general methods, such as ultrafiltration or protein precipitation with acids, organic solvents or salts. Generally speaking, these methods are used to reliably reduce high abundance and high molecular weight proteins, which in turn enrich for low molecular weight and / or low abundance proteins and peptides (e.g., tau, Aβ, etc.).

[0249] In some embodiments, protein can be depleted from a sample by precipitation. In short, precipitation includes adding a precipitant to the sample and mixing thoroughly, incubating the sample with the precipitant to precipitate the protein, and separating the precipitated protein by centrifugation or filtration. The resulting supernatant can then be used for downstream applications. The amount of the required reagent can be experimentally determined by methods known in the art. Suitable precipitants include perchloric acid, trichloroacetic acid, acetonitrile, methanol, etc. In an exemplary embodiment, protein is depleted from a sample by acid precipitation. In another embodiment, protein is depleted from a sample by acid precipitation using perchloric acid.

[0250] As a non-limiting example, protein can be depleted from a sample by acid precipitation using perchloric acid. As used herein, unless otherwise indicated, "perchloric acid" refers to 70% perchloric acid. In some embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 15% v / v. In other embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 10% v / v. In other embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 15% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 10% v / v.

[0251] In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 5% v / v.

[0252] 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 fully mixed (e.g., by a vortex mixer) and kept at low temperatures generally for about 10 minutes or longer, to promote precipitation. For example, the sample can keep about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes or about 30 minutes to about 60 minutes. In other examples, the sample can keep about 15 minutes to about 45 minutes or about 30 minutes to about 45 minutes. In other examples, the sample can keep about 15 minutes to about 30 minutes or about 20 minutes to about 40 minutes. In other examples, the sample keeps about 30 minutes. The sample is then centrifuged at low temperature to pellet the precipitated protein and transfer the supernatant (i.e., the acid-soluble fraction) containing soluble tau to a fresh container. As used in the above context, "low temperature" refers to a temperature of 10°C or less. 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 narrower temperature range may be preferred, such as about 3°C ​​to about 5°C, or even about 4°C. In certain embodiments, low temperature can be achieved by placing the sample on ice.

[0253] Two or more methods from one or both of the above methods can be combined to deplete multiple proteins sequentially or simultaneously. For example, one or more proteins can be selectively depleted (targeted depletion), and then high abundance / molecular weight proteins are depleted. Alternatively, high abundance / molecular weight proteins can be first depleted, and then targeted depletion of one or more proteins. In another alternative, high abundance / molecular weight proteins can be first depleted, 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 those targeted in the first round. Other iterations are readily apparent to the technician.

[0254] h. Cleavage of purified tau with proteases

[0255] Another step of the method disclosed herein optionally includes cleaving the purified tau with a protease. Cleavage of purified tau with a protease includes contacting a sample containing purified tau with a protease under conditions suitable for digesting tau. When affinity purification is used, digestion may occur after eluting tau from the immobilized ligand or when tau is bound. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C, Asp-C, Arg-N, and Arg-C. In order to detect very short MTBR-tau243, the protease includes Arg-C. The resulting cleavage product is a composition comprising proteolytic peptides of tau. When the protease is trypsin, the resulting cleavage product comprises tryptic peptides of tau. After proteolytic cleavage, the resulting cleavage product can be desalted by solid phase extraction.

[0256] i. Detection and quantification

[0257] Another step of the method disclosed herein includes detecting the amount of cleaved fragments of tau from the processed biological sample. All suitable methods for detecting an amount of tau protein are considered within the scope of the present disclosure. The method for detecting and quantifying tau protein is described in detail below.

[0258] (1) LC-MS

[0259] The steps of detecting and quantifying include performing liquid chromatography-mass spectrometry (LC-MS) on a sample containing tau peptides to detect and measure the concentration of at least one tau peptide. Therefore, in practice, the disclosed method uses one or more tau peptides to detect and measure the amount of tau protein present in a biological sample.

[0260] Proteolytic peptides of tau indicating the presence of endogenous cleavage fragments of tau may include one or more of the peptides listed in Table 1. When alternative enzymes are used for digestion, the resulting proteolytic peptides may be slightly different, but can be easily determined by a person of ordinary skill in the art. Without wishing to be bound by theory, it is believed that the variation in the amount of cleavage fragments of tau peptides between two biological samples of the same type reflects the differences in the cleavage fragments of tau that constitute these biological samples. As disclosed herein, the amounts of certain proteolytic peptides of cleavage fragments of tau and the ratios of certain proteolytic peptides of tau can provide clinically meaningful information to guide treatment decisions. Therefore, methods that allow detection and quantification of cleavage fragments of tau have utility in the diagnosis and treatment of many neurodegenerative diseases.

[0261] The proteolytic peptides of tau can be separated by a liquid chromatography system connected to a high-resolution mass spectrometer interface. Suitable LC-MS systems may include <1.0 mm ID columns and use a flow rate of less than about 100 μl / min. In a preferred embodiment, a nanoflow LC-MS system (e.g., about 50-100 μm ID columns and <1 μL / min, preferably about 100-800 nL / min, more preferably about 200-600 nL / min flow rate) is used. In an exemplary embodiment, the LC-MS system may include a 0.05 mM ID column and use a flow rate of about 400 nL / min.

[0262] Tandem mass spectrometry can be used to improve resolution, as is known in the art, or techniques can be improved to achieve the resolution of tandem mass spectrometry with a single mass analyzer. Suitable types of mass spectrometers are known in the art. These include, but are not limited to, quadrupole, time-of-flight, ion trap, and orbital trap, as well as hybrid mass spectrometers that combine different types of mass analyzers into one architecture (e.g., Orbitrap Fusion Mass Spectrometer). TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, Orbitrap Tribrid TM Eclipse TM Mass spectrometer, Q Exactive mass spectrometer, each from ThermoFisherScientific). In an exemplary embodiment, the LC-MS system may include a TM Tribrid TM Mass spectrometer, Orbitrap Fusion TM Lumos TM Mass spectrometer, OrbitrapTribrid TM Eclipse TM A mass spectrometer with a mass spectrometer, or a mass spectrometer with similar or improved ion focusing and ion transmittance (ion-transparency) at the quadrupole. Suitable mass spectrometry protocols can be developed by optimizing the number of ions collected before analysis (e.g., using the AGC setting of the orbital trap) and / or the injection time. In an exemplary embodiment, the mass spectrometry protocol outlined in the examples is used.

[0263] (2) Immunoassay

[0264] In another embodiment, the cleavage fragments of tau can be measured and quantified by immunoassay. In a specific embodiment, ELISA is used to detect and quantify the cleavage fragments of tau.

[0265] Methods for assessing protein expression using epitope binding agent-based methods are known in the art, and all suitable methods known to those skilled in the art for assessing protein expression are contemplated to be within the scope of the present disclosure.

[0266] Therefore, in some embodiments, the method for evaluating the amount of tau protein is a method based on an epitope binding agent. Generally, the method for evaluating the amount of protein expression based on an epitope binding agent includes contacting a sample containing a polypeptide with an epitope binding agent specific for the polypeptide under conditions that effectively allow the epitope binding agent to form a complex with the polypeptide. The method based on the epitope binding agent can occur in a solution, or the epitope binding agent or sample can be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtiter plates, test tubes, beads, resins and other polymers.

[0267] As will be appreciated by those skilled in the art, epitope binding agents can be attached to a substrate in a variety of ways. The epitope binding agent can be first synthesized and then attached to the substrate, or can be synthesized directly on the substrate. The substrate and epitope binding agent can be derivatized with chemical functional groups to carry out subsequent attachment of the two. For example, the substrate can be derivatized with chemical functional groups, including but not limited to amino, carboxyl, oxo or thiol groups. Using these functional groups, the epitope binding agent can be attached directly using the functional groups or indirectly using a linker.

[0268] Epitope binding agents may also be non-covalently attached to a substrate. For example, a biotinylated epitope binding agent may be prepared that covalently binds to a surface coated with streptavidin to produce attachment. Alternatively, epitope binding agents may be synthesized on a surface using techniques such as photopolymerization and photolithography. Additional methods for attaching epitope binding agents to solid surfaces and methods for synthesizing biomolecules on substrates are well known in the art, i.e., VLSIPS technology from Affymetrix (e.g., see U.S. Patent No. 6,566,495, and Rockett and Dix, Xenobiotica 30 (2): 155-177, both of which are hereby incorporated by reference in their entirety).

[0269] The sample is contacted with the epitope binding agent under effective conditions for a period of time sufficient to allow the formation of a complex, typically including adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough to allow the epitope binding agent to bind to any antigen present. Thereafter, the complex will be washed and the complex can be detected by any method well known in the art. Methods for detecting epitope binding agent-polypeptide complexes are generally based on the detection of a label or marker. The term "label" as used herein refers to any substance attached to an epitope binding agent or other matrix material, wherein the substance can be detected by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescent quenchers, colored molecules, radioisotopes, scintillants, biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals and enzymes (including alkaline phosphatase, peroxidase and luciferase). Methods for detecting epitope binding agent-polypeptide complexes based on the detection of labels or markers are well known in the art.

[0270] In some embodiments, the method based on epitope binding agent is an immunoassay. Immunoassays can be run in many different forms. Generally speaking, immunoassays can be divided into two categories: competitive immunoassays and non-competitive immunoassays. In competitive immunoassays, unlabeled analytes in the sample compete with labeled analytes for binding antibodies. Unbound analytes are washed away and the bound analytes are measured. In non-competitive immunoassays, antibodies are labeled instead of analytes. Non-competitive immunoassays can use one antibody (e.g., labeled capture antibody) or more than one antibody (e.g., at least one unlabeled capture antibody and at least one labeled "capping" or detection antibody). Suitable labels are as described above.

[0271] In one embodiment, the epitope binding agent method is an immunoassay. In another embodiment, the epitope binding agent method is selected from enzyme-linked immunosorbent assay (ELISA), fluorescence-based assay, dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), radioassay, multiple immunoassay and flow-encoded microsphere assay (cytometric bead assay, CBA). In some embodiments, the epitope binding agent-based method is an enzyme-linked immunosorbent assay (ELISA). In other embodiments, the epitope binding agent-based method is a radioimmunoassay. In other embodiments, the epitope binding agent-based method is an immunoblot or protein blot. In an alternative embodiment, the epitope binding agent-based method is an array. In another embodiment, the epitope binding agent-based method is flow cytometry. In different embodiments, the epitope binding agent-based method is immunohistochemistry (IHC). IHC uses antibodies to detect and quantify antigens in intact tissue samples. Tissue samples can be fresh frozen and / or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for research by IHC. Methods of preparing tissue blocks for study by IHC and methods of performing IHC are well known in the art.

[0272] 4. Use of Tau cleavage fragment measurement

[0273] The present disclosure also includes the measurement of the cleavage fragment of tau in the blood or CSF as the biomarker of the pathological characteristics and / or clinical symptoms of tau disease for diagnosis, staging, selection of treatment suitable for a given disease stage and modification of a given treatment regimen (e.g., changing dosage, converting to different drugs or treatment modes, etc.). Pathological characteristics can be an aspect of tau pathology (e.g., the amount of tau deposition, the presence / absence of post-translational modifications, the amount of post-translational modifications, etc.). Alternatively, or in addition to tau deposition, pathological characteristics can be tau-independent. For example, when tau disease is Alzheimer's disease, amyloid beta (Aβ) deposition in the brain or cerebral arteries. Clinical symptoms can be dementia as measured by clinically verified instruments (e.g., MMSE, CDR-SB, etc.), or any other clinical symptoms related to tau disease.

[0274] Accordingly, in one aspect, the disclosure provides a method for measuring tauopathy-related pathology in a subject, the method being included in a biological sample obtained from a subject, such as a blood sample or a CSF sample, quantifying the cleavage fragments of one or more tau, wherein the amount of the cleavage fragments of the tau quantified is a representative of tauopathy-related pathology in the subject's brain. Tauopathy may be 3R-tauopathy, mixed 3R / 4R-tauopathy or 4R-tauopathy. The pathology associated with the disease may be tau deposition, tau post-translational modification, amyloid plaques in the brain and / or cerebral arteries, or other pathological features known in the art. The subject may have or not have the clinical symptoms of tauopathy. The cleavage fragments of the tau quantified may comprise the amino acid sequences in one or more Tables 1. In a further embodiment, quantify the cleavage fragments of two or more tau.

[0275] In another aspect, the disclosure provides a method for diagnosing tau disease in a subject, the method being included in a biological sample obtained from the subject, such as a blood sample or a CSF sample, quantifying the cleavage fragments of one or more tau, and when the cleavage fragments of the tau quantified are about 1.5σ or more, diagnosing tau disease, wherein σ is the standard deviation defined by the normal distribution measured in the control population of amyloid negative by measuring the Aβ42 / 40 measured by PET imaging and / or CSF without clinical signs or symptoms of tau disease. Tau disease may be 3R-tau disease, mixed 3R / 4R-tau disease or 4R-tau disease. The subject may have or not have the clinical symptoms of the disease. The cleavage fragments of the tau quantified may include the amino acid sequences in one or more Tables 1. In a further embodiment, quantify the cleavage fragments of two or more tau.

[0276] In another aspect, the disclosure provides a method for measuring tau disease stability in a subject, the method being included in the first biological sample obtained from the subject, quantifying the cleavage fragments of one or more tau, and then quantifying the cleavage fragments of one or more tau in the second biological sample obtained from the same subject at a later time (e.g., weeks, months, or years later), and calculating the difference between the cleavage fragments of the tau quantified between the samples, wherein the cleavage fragments of the tau quantified in the second sample are statistically significantly increased to indicate disease progression, and the cleavage fragments of the tau quantified in the second sample are statistically significantly reduced to indicate disease improvement, and no change indicates that the disease is stable. Tau disease can be 3R-tau disease, mixed 3R / 4R-tau disease or 4R-tau disease. Subjects may have or do not have the clinical symptoms of the disease, and may be receiving or not receiving tau therapy. In some instances, one or more tau therapies are administered to a subject in the time period between the collection of the first biological sample and the second biological sample, and the measure of disease stability is the effectiveness of tau therapy or the indication of its lack. In preferred embodiments, the cleavage fragments of tau that are quantified comprise one or more of the amino acid sequences in Table 1. In further embodiments, two or more cleavage fragments of tau are quantified.

[0277] In another aspect, the present disclosure provides a method for treating a subject with tauopathy, the method comprising quantifying cleavage fragments of tau in a biological sample obtained from the subject, such as a blood sample or a CSF sample; and providing a tau therapy to the subject to improve a measurement of a disease-related pathology or clinical symptom, wherein the subject has a quantified cleavage fragment of tau that is at least 1 standard deviation above or below the mean of a control population, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least 2 standard deviations (i.e., differing by 1σ, 1.3σ, 1.5σ, or 1.5σ, respectively, wherein σ is a measure of the difference in tau cleavage fragments in the absence of clinical signs or symptoms of tauopathy). The standard deviation of the normal distribution defined by the Aβ42 / 40 measurement in the negative control population of amyloid protein measured by PET imaging and / or CSF. In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean value), in some embodiments, the degree of change above or below the mean value can be used as a standard for treating a subject. Tau disease can be 3R-tau disease, mixed 3R / 4R-tau disease or 4R-tau disease. The measurement of disease-related pathology can be tau deposition as measured by PET imaging, tau post-translational modification as measured by mass spectrometry or other suitable methods, brain or brain as measured by PET imaging. Amyloid plaques in arteries, amyloid plaques as measured by Aβ42 / 40 in CSF, or other pathological features known in the art. Clinical symptoms may be dementia as measured by clinically validated instruments (e.g., MMSE, CDR-SB, etc.), or other clinical symptoms of 3R-tau disease and 4R-tau disease known in the art. In a preferred embodiment, the quantified tau cleavage fragments comprise one or more of the amino acid sequences in Table 1. In a further embodiment, two or more tau cleavage fragments are quantified. Many tau therapies target specific pathophysiological changes. For example, Aβ targeted therapies are typically designed to reduce Aβ production, antagonize Anti-Aβ aggregation or increase brain Aβ clearance; tau targeted therapy is generally designed to change tau phosphorylation pattern, antagonize tau aggregation (generally antagonize tau or antagonize specific tau subtypes), or increase NFT clearance; a variety of therapies are designed to reduce CNS inflammation or brain insulin resistance, etc. However, not all tauopathies share the same pathophysiological changes. Therefore, by administering these various tau therapies to subjects correctly identified as having tau pathology, including determining the stage of the disease in which the subject is located, thereby more effectively changing tau phosphorylation pattern, antagonizing tau aggregation, or increasing NFT clearance based on the specific pathological state of the subject, their efficacy can be improved.

[0278] As disclosed herein, tau species containing MTBR-tau243 (reference tau-441) are widely present in biological fluids such as CSF and blood, although not all tau species containing MTBR-tau243 show disease-specific correlation. The present disclosure provides tau cleavage fragments containing MTBR-tau243 that are directly associated with Tau-PET status. Therefore, detecting these tau cleavage fragments in biological fluids provides a sensitive and specific method for measuring tau pathology. Suitable biological samples are described herein. Determining these tau cleavage fragments includes purifying them from biological samples. Suitable methods for purifying tau cleavage fragments are described herein, as are epitope binding agents.

[0279] In each of the above aspects, suitable cleavage fragments of tu may include, but are not limited to, cleavage fragments of tau comprising or consisting of one or more amino acid sequences in Table 1 or a combination thereof.

[0280] The exemplary use of tau cleavage fragments can be used to illustrate the various aspects discussed above, but this discussion does not limit the scope of the present invention. The cleavage fragments of tau are described in detail in the embodiments. Generally speaking, these tau fragments contain MTBR-tau243 and are endogenously cleaved at their C-terminal ends. Measuring the amount of endogenous cleavage fragments of tau means measuring the amount of tau species of this particular group in a given sample. As shown in the examples, the increase in the amount of cleavage fragments of blood and / or CSF tau summarizes the direct measure of tau deposition in the brain associated with Alzheimer's disease (AD). In other words, the amount of cleavage fragments of blood and / or CSFtau is a representative of AD-related pathology (e.g., tau deposition in the brain). Therefore, these amounts can be used to measure AD-related pathology to determine the tau state of the subject, as well as to diagnose the AD stage in the subject, etc. The amount of cleavage fragments of blood and / or CSFtau also summarizes the changes measured during the clinical stage of AD, such as defined by the results of tau-PET tests. Therefore, the amount of tau cleavage fragments can also help to diagnose and stage AD in subjects across the entire disease spectrum (e.g., preclinical to clinical). After diagnosing and / or staging the disease, the subject can then be provided with treatment to reduce or prevent any further increase in the amount of tau cleavage fragments in the blood and / or CSF, and / or reduce or prevent any further increase in another clinical sign or symptom of AD. The choice of treatment can be further guided by knowledge of the specific disease stage, which is informed by the amount of tau cleavage fragments - for example, therapies designed to prevent Aβ deposition, reverse Aβ deposition, prevent tau deposition, reverse tau deposition, and improve clinical signs of disease will be used for subjects with different but potentially overlapping amounts of tau cleavage fragments.

[0281] In a specific embodiment, the present disclosure provides a method for measuring Alzheimer's disease (AD)-related pathology in a subject, the method comprising providing a CSF or blood sample obtained from the subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; and quantifying the cleavage fragments of tau in the sample. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of the cleavage fragments of tau, or their ratio, is representative of AD-related pathology in the subject's brain.

[0282] In another specific embodiment, the present disclosure provides a method for measuring Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, the method comprising providing a CSF or blood sample obtained from the subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; and quantifying the cleavage fragments of tau in the sample. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of the cleavage fragments of tau is representative of AD-associated tau deposition in the brain of the subject.

[0283] In another specific embodiment, the present disclosure provides a method for diagnosing Alzheimer's disease, the method comprising providing a CSF or blood sample obtained from a subject, and quantifying the cleavage fragments of tau in the sample. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof. In some embodiments, the method comprises quantifying p-tau (e.g., tau181, p-tau205, p-tau217, and p-tau231), Aβ (e.g., Aβ42 / 40), or a combination thereof. In some embodiments, the subject is diagnosed with Alzheimer's disease when the quantified cleavage fragments of tau differ from the mean of a control population by about 1.5σ or more, where σ is the standard deviation defined by a normal distribution measured in a control population that is amyloid negative in the absence of clinical signs or symptoms of tauopathy and as measured by PET imaging (e.g., by PiB-PET SUVR as described in Ann Neurol 2016;80:379-387) and / or Aβ42 / 40 measurement in CSF (e.g., a cutoff value of CSF Aβ42 / 40 calculated by PiB-PET SUVR (Ann Neurol 2016;80:379-387) that maximizes sensitivity %+specificity %).

[0284] In another specific embodiment, the present disclosure provides a method for measuring the progression of Alzheimer's disease (AD) in a subject, the method comprising providing a first CSF or blood sample and a second CSF or blood sample, wherein each sample is obtained from a single subject, and each sample is purified for cleavage fragments of tau; and for each sample, the cleavage fragments of tau are quantified. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof. In some embodiments, the method comprises p-tau or Aβ, or a combination thereof. The method further comprises calculating the difference between the cleavage fragments of tau quantified in the second sample and the first sample, wherein a statistically significant increase in the cleavage fragments of tau quantified in the second sample indicates the progression of Alzheimer's disease in the subject.

[0285] In another specific embodiment, the present disclosure provides a method for measuring tau deposition in the brain of a subject, the method comprising providing a CSF or blood sample obtained from the subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; and quantifying the cleavage fragments of tau in the sample. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of cleavage fragments of tau or their ratio is representative of tau deposition in the brain of the subject.

[0286] The following specific embodiments relate to methods comprising methods of measuring cleavage fragments of tau in a biological sample can be found in Section 2, which is incorporated herein by reference.

[0287] In each of the aforementioned methods, additional biomarkers may be detected and measured to assist in staging and / or determining the pathology of the subject and / or the disease progression of the subject. For example, the method may include detecting the amount of Aβ. In some embodiments, determining the Aβ42 / 40 value assists in measuring the specific abnormality of amyloid plaques. Deviations from the Aβ42 / 40 values ​​of the normal control population without amyloid pathology begin to appear about 20 years before the onset of symptoms and prove to be a highly sensitive measure of these changes. In addition to or in place of Aβ measurement, each of the aforementioned methods may include detecting the amount of tau phosphorylation at a specific residue. In some embodiments, determining the phosphorylation of tau at 217, 181, 231, 205, 153, 111, 208 and / or any combination thereof assists in measuring the specific abnormality of amyloid plaques. Aβ42 / 40 changes first, followed by p-tau217 / 181 / 231 shortly thereafter, and these are associated with amyloid plaques. Then about 10 years later, p-tau205 increases and is associated with both Aβ plaques and tau tangles, similar to total tau (N-terminal to middle domain tau). In some embodiments, the composite value of ptau phosphorylation and Aβ42 / 40 values ​​can be measured and determined. For example, pT217×Aβ42 / 40 values ​​are determined to help measure specific abnormalities of amyloid plaques, and are found to be highly sensitive and specific. Finally, about 20 years after the Aβ42 / 40 abnormality, the cleavage fragments of tau as disclosed herein increase and are only associated with tau pathology (rather than amyloid pathology). Therefore, when measured and combined with the methods disclosed herein, each marker provides a more accurate ability to determine the stage, pathology and / or disease progression of a subject, which was not available before the present disclosure. Aβ42 / 40 is a measure of pre-abnormal amyloid plaques; Aβ42 / 40 and ptau217% abnormal are measures of abnormal amyloid plaques, ptau205 is a measure of amyloid plaques plus neurodegeneration; and cleaved fragments of tau as disclosed herein are a measure of tau tangles and clinical onset of dementia.

[0288] In any of the above embodiments, alternatively or in addition to the above, other markers such as total tau measurements, ratios calculated from measured phosphorylation levels, or ratios calculated from measured phosphorylation levels and total tau can be used. Mathematical operations other than ratios can also be used. For example, examples use cleavage fragments, Aβ values, and / or site-specific tau phosphorylation values ​​of tau as disclosed herein that can be combined with other known biomarkers (e.g., MAPT status, APOEε4 status, age, sex, cognitive test scores, functional test scores, etc.) for various statistical models (e.g., linear regression, LME curves, LOESS curves, etc.). The selection of measurement and the selection of mathematical operations can be optimized to maximize the specificity of the method. For example, diagnostic accuracy can be evaluated by the area under the ROC curve, and in some embodiments, 0.7 or greater ROC AUC values ​​are set as thresholds (e.g., 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.).

[0289] The brain amyloid plaques of people are routinely measured by amyloid-positron emission tomography (PET). For example, 11C-Pittsburgh compound B (PiB) PET imaging of cortical Aβ plaques is generally used to detect Aβ plaque pathology. The standard uptake value ratio (SUVR) of cortical PiB-PET reliably identifies significant cortical Aβ plaques, and is used to divide subjects into PIB positive (SUVR≥1.25) or negative (SUVR<1.25). Accordingly, in the above embodiments, the control population without brain amyloid plaques as measured by PET imaging can refer to the subject population of cortical PiB-PET SUVR<1.25. Other values ​​(such as average cortical binding potential) of PiB binding or analysis of target areas other than cortical areas can also be used to divide subjects into PIB positive or negative. Other PET imaging agents can also be used.

[0290] In another specific embodiment, the present disclosure provides a method for treating a subject in need or a method for selecting a therapeutic agent for a subject in need, the method comprising providing a CSF or blood sample obtained from a subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; and quantifying the cleavage fragments of tau in the sample. In some embodiments, the cleavage fragments of tau include one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of cleavage fragments of tau or their ratio is a representative of tau deposition in the subject's brain. In some embodiments, when the amount of cleavage fragments of tau differs from the mean value of a healthy control population by about 1.5σ or more, a therapeutic agent is administered to the subject, wherein σ is a standard deviation defined by a normal distribution measured in a control population that is negative for amyloid protein in the absence of clinical signs or symptoms of tau disease and as measured by PET imaging and / or Aβ42 / 40 in CSF, and wherein the amount of quantified cleavage fragments of tau is a representative of tau pathology in the subject's brain. In some embodiments, administering a treatment to a subject to alter tau pathology will alter or stabilize the amount of quantified cleavage fragments of tau.

[0291] In another specific embodiment, the present disclosure provides a method for treating a subject in need or a method for selecting a therapeutic agent for a subject in need, the method comprising providing a CSF or blood sample obtained from a subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; quantifying the cleavage fragments of tau in the sample; and administering treatment to the subject to alter tau pathology, wherein the amount of cleavage fragments of tau differs from the mean of a healthy control population by about 1.5σ or more, wherein σ is a standard deviation defined by a normal distribution measured in a control population that is negative for amyloid protein in the absence of clinical signs or symptoms of tau disease and as measured by PET imaging and / or Aβ42 / 40 measurement in CSF, and wherein the amount of quantified cleavage fragments of tau is representative of tau pathology in the subject's brain. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of cleavage fragments of tau or their ratio is representative of tau deposition in the subject's brain.

[0292] Treatment may include one or more of the following: lencanezumab, donetumab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, berenezumab, dasatinib + quercetin, E2814, epothilone D, gonelizumab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, sirenetumab, TPI 287. Tigrexib, tipranezumab, zagteneizumab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors, and live tau vaccines.

[0293] In some embodiments, the treatment is a pharmaceutical composition comprising: a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, an antidepressant (e.g., a selective serotonin reuptake inhibitor, an atypical antidepressant, an aminoketone, a selective serotonin and norepinephrine reuptake inhibitor, a tricyclic antidepressant, etc.), a γ-secretase inhibitor, a β-secretase inhibitor, an anti-Aβ antibody (including an antigen-binding fragment, variant, or derivative thereof), an anti-tau antibody (including an antigen-binding fragment, variant, or derivative thereof), an anti-TREM2 antibody (including an antigen-binding fragment, variant, or derivative thereof), a TREM2 agonist, stem cells, a dietary supplement (e.g., lithium water, omega-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin, and grape seed extract drugs, etc.), antagonists of serotonin receptor 6, p38α MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, live vaccines (e.g. CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g. TRx0237, methylthiocarbamide chloride, etc.), therapies to improve blood sugar control (e.g. insulin, exenatide, liraglutide, pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT 1A and 1D receptor agonists, glutaminyl peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-γ agonists, tubulin regulators, calcium channel blockers, antihypertensive agents, statins and any combination thereof. In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors may inhibit one thousand and one amino acid kinases (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments, the treatment is a pharmaceutical composition comprising a tau targeted therapy, which includes but is not limited to an active pharmaceutical ingredient that changes the tau phosphorylation pattern, antagonizes tau aggregation, or increases the clearance of pathological tau subtypes and / or aggregates.In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a γ-secretase inhibitor, a β-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau aggregation inhibitor.

[0294] In some embodiments, when the detected Aβ42 / 40 value deviates from the average value of the healthy control population and optionally pTau217 / tau217, ptau205 / tau205, pTau181, pTau 231 and / or the cleavage fragments disclosed herein do not significantly deviate from the average value of the healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent is selected to prevent the increase of amyloid deposition. In an exemplary embodiment, the detected Aβ42 / 40 value deviates significantly from the average value below the healthy control population. In some embodiments, when the detected Aβ42 / 40 value and pTau217 / tau217 (or pTau217×Aβ42 / 40 composite value) deviate significantly from the average value of the healthy control population and optionally ptau205 / tau205 and / or the cleavage fragments disclosed herein do not significantly deviate from the average value of the healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent is selected to prevent the increase of amyloid deposition and / or reduce the existing plaque load of the subject. In an exemplary embodiment, the detected Aβ42 / 40 value deviates significantly from the average value below the healthy control population, the detected pTau217 / tau217 is higher than the average value of the healthy control population and / or the pTau217×Aβ42 / 40 composite value is higher than the average value of the healthy control population. In some embodiments, when the detected Aβ42 / 40 value, pTau217 / tau217 (or pTau217×Aβ42 / 40 composite value) and pTau205 / tau205 value deviate significantly from the average value of the healthy control population and optionally the cleavage fragment of the present disclosure does not deviate significantly from the average value of the healthy control population, the subject is administered a therapeutic agent or a therapeutic agent is selected to prevent the increase of amyloid deposition and / or reduce the existing plaque load of the subject and / or treat or prevent neurodegeneration and / or prevent Tau tangles or related pathology. In an exemplary embodiment, the detected Aβ42 / 40 value deviates significantly from the average value of a healthy control population, the detected pTau217 / tau217 is higher than the average value of a healthy control population and / or the pTau217×Aβ42 / 40 composite value is higher than the average value of a healthy control population and / or the pTau205 / tau205 value is higher than the average value of a healthy control population. In some embodiments, when the detected Aβ42 / 40 value, pTau217 / tau217 (or pTau217×Aβ42 / 40 composite value), pTau205 / tau205 and the cleavage fragments of the present disclosure deviate significantly from the average value of a healthy control population, a therapeutic agent is administered to the subject or a therapeutic agent is selected to prevent an increase in amyloid deposition and / or reduce an existing plaque load in the subject and / or treat or prevent neurodegeneration and / or prevent an increase in tau tangles and / or reduce an existing tangle or related pathology in the subject.In an exemplary embodiment, the detected Aβ42 / 40 value deviates significantly from the average value below that of a healthy control population, the detected pTau217 / tau217 is higher than the average value of a healthy control population and / or the pTau217×Aβ42 / 40 composite value is higher than the average value of a healthy control population and / or the pTau205 / tau205 value is higher than the average value of a healthy control population and / or the value of the cleavage fragment of the present disclosure is higher than the average value of a healthy control population.

[0295] In each of the above embodiments, the pharmaceutical composition administered to the subject may include an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, flubitaban, flobetapine, flumetazol, radionuclide-labeled antibodies, etc.).

[0296] Another aspect of the present disclosure is a method for selecting a subject to enter a clinical trial, in particular a clinical trial of Aβ or tau therapy (provided that all other criteria for the clinical trial have been met). In one embodiment, the method for selecting a subject to enter a clinical trial may include (a) providing a CSF or blood sample obtained from a subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; (b) quantifying the cleavage fragments of tau in the sample; and (c) when the value of the cleavage fragment of tau is approximately the same as that of a healthy control population and the subject's Aβ42 / 40 value is lower than the average value of a healthy control population, selecting the subject to enter a clinical trial of Aβ therapy. In some embodiments, the cleavage fragments of tau comprise one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of cleavage fragments of tau or their ratio is representative of tau deposition in the subject's brain.

[0297] In another embodiment, a method for selecting a subject to enter a clinical trial may include (a) providing a CSF or blood sample obtained from a subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; (b) quantifying the cleavage fragments of tau in the sample; and (c) excluding the subject from entering a clinical trial for Aβ therapy when the value of the cleavage fragments of tau is higher than the average value of a healthy control population and the Aβ42 / 40 value of the subject is approximately the same as or lower than the average value of a healthy control population. In some embodiments, the cleavage fragments of tau include one or more peptides having an amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of the cleavage fragments of tau or their ratio is representative of tau deposition in the subject's brain.

[0298] In another embodiment, a method for selecting a subject to enter a clinical trial, in particular a clinical trial of tau therapy (provided that all other criteria for the clinical trial have been met). In one embodiment, the method for selecting a subject to enter a clinical trial may include (a) providing a CSF or blood sample obtained from a subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; (b) quantifying the cleavage fragments of tau in the sample; and (c) when the value of the cleavage fragment of tau is higher than the average value of a healthy control population and optionally when the subject's Aβ42 / 40 value is approximately the same as or lower than the average value of a healthy control population, selecting the subject to enter a clinical trial of tau therapy. In some embodiments, the cleavage fragments of tau comprise one or more peptides comprising the amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of cleavage fragments of tau or their ratio is representative of tau deposition in the subject's brain.

[0299] In another embodiment, a method for selecting a subject to enter a clinical trial, in particular a clinical trial of tau therapy (provided that all other criteria for the clinical trial have been met). In one embodiment, the method for selecting a subject to enter a clinical trial may include (a) providing a CSF or blood sample obtained from a subject, wherein the CSF or blood sample is purified for cleavage fragments of tau; (b) quantifying the cleavage fragments of tau in the sample; and (c) excluding the subject from entering the clinical trial of tau therapy when the value of the cleavage fragments of tau is approximately the same as the average value of the healthy control population. In some embodiments, the cleavage fragments of tau comprise one or more peptides comprising the amino acid sequence in Table 1 (wherein the C-terminal amino acid represents the last amino acid on the C-terminus of the peptide) or a combination thereof, wherein the amount of cleavage fragments of tau or their ratio is representative of tau deposition in the subject's brain. The phrase "a control population without brain amyloid plaques as measured by PET imaging" is defined above.

[0300] In any of the above embodiments, alternatively or in addition to the measurement of the cleavage fragment of tau as disclosed herein, site-specific tau phosphorylation (optionally together with the measurement of total tau), the ratio calculated from the measured phosphorylation level or the ratio calculated from the measured phosphorylation level and total tau can be used. The ratio calculated from the measured phosphorylation level can be the ratio between pT181 and pT205, pT217 and pT205 or pT181 and pT217. The ratio calculated from the measured phosphorylation level and total tau can be the ratio between pT181 and total tau, p-T205 and total tau or pT217 and total tau. Mathematical operations other than ratios can also be used. For example, examples combine site-specific tau phosphorylation values ​​with other known biomarkers (e.g., APOEε4 status, age, gender, cognitive test scores, functional test scores, etc.) for various statistical models (e.g., linear regression, LME curves, LOESS curves, etc.).

[0301] The methods disclosed herein can greatly help the clinical trial design of AD and tau therapy. Many clinical trials are designed to test the efficacy of imaging agents or therapeutic agents that target specific pathophysiological changes that occur before the onset of AD symptoms. As described in Section III above, by administering the agent to a subject with certain site-specific tau phosphorylation levels as measured by the method disclosed and illustrated herein, the efficacy of these different agents can be improved. Similarly, clinical trials that select subjects with symptoms of Aβ pathology or only tau pathology will also benefit from being able to accurately distinguish the pathology of the participants to determine whether the efficacy is related to a specific disease state. Accordingly, before selecting subjects to enter clinical trials, especially before entering the treatment group of clinical trials, measuring tau phosphorylation levels as described herein can produce smaller tests and / or improved results. In some cases, the methods described herein can be developed and used as companion diagnostics for therapeutic agents.

[0302] In each of the above embodiments, the subject may be enrolled in the treatment group of the clinical trial. "Treatment" is defined above. The subject enrolled in the treatment group of the clinical trial may be administered the pharmaceutical composition. In some embodiments, the pharmaceutical composition may include an imaging agent. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, flubivac, flobetapine, flumetazol, radionuclide labeled antibodies, etc.). Alternatively, the pharmaceutical composition may include an active pharmaceutical ingredient. Non-limiting examples of active pharmaceutical ingredients include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), γ-secretase inhibitors, β-secretase inhibitors, anti-Aβ antibodies (including antigen-binding fragments, variants or derivatives thereof), anti-tau antibodies (including antigen-binding fragments, variants or derivatives thereof), stem cells, dietary supplements (e.g., lithium water, omega-3 fatty acids with lipoic acid, long-chain triglycerides, genistein, resveratrol, curcumin and grape seed extract, etc.), antagonists of serotonin receptor 6, p38αMA PK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, live vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylthiocarbamide chloride, etc.), therapies to improve blood sugar control (e.g., insulin, exenatide, liraglutide, pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma-1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT 1A and 1D receptor agonists, glutaminyl peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulators, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-γ agonists, tubulin regulators, calcium channel blockers, antihypertensive agents, statins and any combination thereof. In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors may inhibit one thousand and one amino acid kinases (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, a phosphatase activator may increase the activity of protein phosphatase 2A.

[0303] In each of the above embodiments, the subject may be symptomatic or may be asymptomatic. "Asymptomatic subject" as used herein refers to a subject that does not show any signs or symptoms of tauopathy. Alternatively, the subject may show signs or symptoms (such as memory loss, misplacing items, mood or behavior changes, etc.) but does not show cognitive or functional impairment sufficient for clinical diagnosis. Symptomatic or asymptomatic subjects may have Aβ amyloidosis; however, prior knowledge of Aβ amyloidosis is not necessary for treatment. In still further embodiments, the subject may suffer from AD. In any of the above embodiments, the subject may carry one of the genetic mutations known to cause hereditary tauopathy. In alternative embodiments, the subject may not carry the genetic mutations known to cause hereditary tauopathy.

[0304] 5. Kit

[0305] Kits are also provided. Such kits may include the agents or compositions described herein, and in certain embodiments, may include instructions for administering or measuring endogenous fragments of tau. Such kits may promote the performance of the methods described herein. When provided as a kit, the different components of the composition may be packaged in separate containers and mixed just before use. Components include, but are not limited to, systems, assays, epitope binding agents, reagents, internal standards, or software. If desired, such separate packaging of components may be present in a packaging or dispenser device, which may include one or more unit dosage forms containing the composition. For example, the packaging may include metal or plastic foil, such as blister packs. In some cases, such separate packaging of components may also allow long-term storage without losing the activity of the components.

[0306] The kit may also include reagents in separate containers, such as sterile water or saline to be added to the separately packaged freeze-dried active components. For example, a sealed glass ampoule may contain freeze-dried components, and in a separate ampoule, sterile water, sterile saline or sterile may be contained, each of which is packaged under a neutral non-reactive gas such as nitrogen. The ampoule may be made of any suitable material, such as glass, an organic polymer, such as polycarbonate, polystyrene, ceramic, metal or any other material generally used to hold the reagent. Other examples of suitable containers include bottles that may be made of materials similar to ampoules, and envelopes that may be composed of an interior of a foil lining such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, etc. The container may have a sterile inlet, such as a bottle with a stopper that may be pierced by a hypodermic needle. Other containers may have two compartments separated by a film that can be easily removed, which allows the components to mix after the film is removed. The removable film may be glass, plastic, rubber, etc.

[0307] In certain embodiments, the kit may be provided with instructional materials. The instructions may be printed on paper or other substrates, and / or may be provided as an electronically readable medium or video. The detailed instructions may not be physically associated with the kit; alternatively, the user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0308] As described herein, a control sample or reference sample can be a sample from a healthy subject or from a randomized subject group. A reference value can be used instead of a control or reference sample, which is previously obtained from a healthy subject or a healthy subject group. A control sample or reference sample can also be a sample or spiked sample with a known amount of a detectable compound.

[0309] The methods and algorithms of the present invention may be encapsulated in a controller or processor. In addition, the methods and algorithms of the present invention may be embodied as one or more computer-implemented methods for executing such computer-implemented one or more methods, and may also be embodied in the form of a tangible or non-temporary computer-readable storage medium containing a computer program or other machine-readable instructions (hereinafter referred to as a "computer program"), wherein when the computer program is loaded into a computer or other processor (hereinafter referred to as a "computer") and / or executed by a computer, the computer becomes a device for practicing the one or more methods. Storage media containing such computer programs include, for example, floppy disks and magnetic disks, compact disks (CD)-ROMs (whether writable or not), DVD digital disks, RAM and ROM memories, computer hard drives and backup drives, external hard drives, "thumb" drives, and any other storage media readable by a computer. The one or more methods may also be embodied in the form of a computer program, for example, whether stored in a storage medium or transmitted via a transmission medium such as an electrical conductor, an optical fiber or other optical conductor, or transmitted by electromagnetic radiation, wherein when the computer program is loaded into a computer and / or executed by a computer, the computer becomes a device for practicing the one or more methods. The one or more methods may be implemented on a general-purpose microprocessor or on a digital processor specifically configured to practice the one or more processes. When a general-purpose microprocessor is used, the computer program code configures the circuits of the microprocessor to create a specific logic circuit arrangement. Computer-readable storage media include media that can be read by the computer itself or another machine that reads computer instructions to provide these instructions to the computer to control its operation. For example, such a machine may include a machine for reading the above-mentioned storage medium.

[0310] The following examples are included to illustrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques found by the inventors to function well in the practice of the present invention. However, based on the present disclosure, it will be appreciated by those skilled in the art that changes may be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Therefore, all matters set forth or shown in the accompanying drawings should be interpreted as illustrative and not restrictive.

[0311] Example

[0312] The present invention has several aspects, which are illustrated by the following non-limiting examples.

[0313] Example 1

[0314] CSF MTBR-tau243 biomarker of tau tangle pathology

[0315] Given the growing interest in tau-targeted therapeutics for Alzheimer's disease (AD), reliable and specific biomarkers of insoluble aggregated tau are urgently needed to understand AD pathophysiology and assess treatment efficacy. 1 Positron emission tomography (PET) with radioligands that bind to the fibrillar form of tau reflects the burden of insoluble AD-specific tau aggregates in the brain, including neurofibrillary tangles (NFTs) and neuropil filaments 2-6 Tau-PET imaging studies show that insoluble tau aggregates are strongly associated with cognitive decline even during the early presymptomatic stages of AD 7 , and tau-PET is the most accurate prognostic marker for AD available today. 8 . However, PET imaging is extremely expensive and requires a complex infrastructure, which reduces its use to only highly specialized centers. In contrast, fluid biomarkers are cheaper and more clinically accessible. The most widely used fluid biomarkers of tau are N-terminal or mid-domain total tau (t-tau) and phosphorylated tau species resulting from cleavage near residue 224 of tau 9,10 , including tau phosphorylated at residues 181, 217, and 231 (p-tau181, p-tau217, and p-tau231) 11-17 However, these biomarkers were strongly associated with increased amyloid plaque burden, exceeding that of insoluble tau aggregates. 18-20 For example, plasma and CSF concentrations of these p-tau species are increased in preclinical AD for many years before widespread insoluble tau aggregates are observed in the neocortex. 21-24Further, recent clinical trials have demonstrated that CSF or plasma concentrations of t-tau, p-tau181, and p-tau217 are significantly reduced in response to anti-amyloid passive immunotherapy, which significantly removes amyloid plaques. 25-28 Neuropathological and imaging studies have also reported strong associations between these fluid biomarkers and amyloid plaques. 19,20,29 Additionally, animal studies have found that CSF t-tau and p-tau are increased in mouse models with amyloid β (Aβ) pathology, even when aggregated tau pathology is not observed. 23,30-32 Taken together, these findings suggest that plasma and CSF concentrations of N-terminal to mid-domain t-tau and p-tau do not directly represent insoluble tau aggregates but instead reflect a response to amyloid plaque pathology. Therefore, there are currently no fluid biomarkers that specifically reflect AD-associated tau pathology.

[0316] Therefore, in this study, we evaluated new CSF biomarkers of insoluble tau aggregates. Importantly, tau species containing microtubule binding regions (MTBR-tau) are the major component of insoluble tau aggregates in the brain. 33-37 However, these fragments have been poorly studied as candidate biomarkers. In an initial study with a small sample size (n=35) of controls and AD patients, we show preliminary results that MTBR-tau is present in human CSF and that a specific MTBR-tau species containing residue 243 (MTBR-tau243) is strongly associated with tau-PET and disease progression. 33 Here, we extend these results to two large independent sporadic AD cohorts: the Swedish BioFINDER-2 study and the Charles F. and Joanne Knight Alzheimer's Disease Research Center (Knight ADRC), which span the entire AD continuum with available amyloid PET and tau PET images. In this study, we compared MTBR-tau243 with other CSF phospho-tau measures, including p-tau181, p-tau205, p-tau217, and p-tau231 phosphorylation occupancy (i.e., p-tau to total tau %), which have also been reported as biomarkers summarizing AD pathology. 21,29) and we showed that MTBR-tau243 was the fluid biomarker that correlated most strongly with tau-PET. We also investigated the proportion of CSF biomarker level changes explained by amyloid PET and tau-PET pathology measures. We then assessed longitudinal CSF biomarker changes to investigate their rates of change based on the presence or absence of amyloid and tau pathology to show which ones increase with amyloid relative to tau pathology. Finally, we evaluated whether prediction of continuous AD-related measures could be improved by a combination of multiple biomarkers and found that MTBR-tau243, together with p-tau205, was able to best predict tau-PET measures and cognitive impairment.

[0317] result

[0318] Participant characteristics

[0319] The BioFINDER-2 cohort included 448 individuals, most of whom had cognitive impairment (281, 63%): 81 cognitively intact Aβ negative (CU-), 79 cognitively intact Aβ positive (CU+), 90 Aβ positive with mild cognitive impairment (MCI+), 102 Aβ positive with AD dementia (AD+), and 96 with other dementia (non-AD) (Table 2). The mean age was 70.9±8.4 years (mean±SD), 221 (49.3%) were female, and 258 (57.6%) were APOEε4 carriers. The Knight ADRC cohort included 219 individuals, most of whom were cognitively intact (171, 78%): 83 CU-, 88 CU+, 35 very mild AD, and 13 AD+. The mean age was 71.2±6.6 years, 112 (51.1%) were female, and 96 (43.8%) were APOEε4 carriers (Table 3). CSF biomarkers were measured in the BioFINDER-2 and Knight ADRC cohorts, including MTBR-tau243 concentrations, and phosphorylation occupancy at different tau residues (pT181 / T181, pT205 / T205, pT217 / T217, and pT231 / T231 percentages). Phosphorylation occupancy represents the percentage of soluble tau phosphorylated at a certain amino acid position (see Methods for details), which is a more specific measure of phosphorylation that is not confounded by the total tau concentration and is superior to the corresponding pTau concentration in predicting abnormal Aβ status. 29,38 .exist Figure 6In Tables 4 and 5 we compared CSF levels of all biomarkers across all diagnostic groups in the BioFINDER-2 cohort. We observed that MTBR-tau243 concentrations were not increased in other non-AD tauopathies, such as progressive supranuclear palsy (PSP) or frontotemporal dementia (FTD), thus suggesting a high specificity for AD-related tau. Further, we did not observe any significant differences between MTBR-tau243 concentrations in CU+ compared to CU-. Notably, we found two outliers with very high MTBR-tau243 levels (one in CU- and the other in FTD) to be MAPT R406W mutation carriers who were amyloid negative but significantly tau-PET positive (in Figure 6 Specified in ).

[0320] Table 4 shows the association between CSF biomarkers and Aβ-PET and tau-PET. Linear regression models were used to obtain the association between CSF biomarkers adjusted for age and sex and amyloid PET (Centiloid) and tau-PET (SUVR) of Braak I-IV ROI. Amyloid-positive participants were selected based on the previously validated cutoff value of CSF Aβ42 / 40 (CSF Aβ42 / 40<0.08 in BioFINDER-2 and CSF Aβ42 / 40<0.0673 in Knight ADRC). P comparisons (p comparisons) (pcomp.) were calculated using bootstrapping (n=500) from adjusted β to evaluate the difference between the strongest association (reference) and each other CSF biomarker. Significant p comparisons (<0.05) suggest that the association is weak. Abbreviations: Aβ, amyloid beta; CI, confidence interval; CSF, cerebrospinal fluid; MTBR, microtubule-binding region; PET, positron emission tomography; SUVR, standardized uptake value ratio.

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

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[0337]

[0338]

[0339]

[0340]

[0341] Associations between CSF markers and amyloid or tau measures

[0342] The association of CSF MTBR-tau243, pT181 / T181, pT205 / T205, pT217 / T217, and pT231 / T231 with measures of amyloid-PET and tau-PET pathology was evaluated using linear regression models adjusted for age and sex. All participants were compared, except for the subset that was only amyloid-positive, to distinguish the effects of amyloid pathology from those of tau pathology ( Figure 1A -B). Phosphorylation occupancy at T217 (pT217 / T217) was the CSF measure most strongly associated with amyloid PET (BioFINDER-2: β [95% CI] = 0.81 [0.74, 0.88]; Knight ADRC: β = 0.87 [0.79, 0.95]; all p < 0.001, Figure 1Aand Table 4). There was a significant difference in the mean ± SD between all participants (BioFINDER-2: β = 0.85 [0.80, 0.90]; Knight ADRC: β = 0.76 [0.65, 0.87]; all p < 0.001) and amyloid-positive participants (BioFINDER-2: β = 0.84 [0.77, 0.91]; Knight ADRC: β = 0.76 [0.63, 0.89]; all p < 0.001, Figure 1B MTBR-tau243 concentrations were the CSF measure most strongly associated with tau-PET in all participants and in amyloid-positive participants (BioFINDER-2: β = 0.76 [0.69, 0.84]; Knight ADRC: β = 0.58 [0.43, 0.73]; Table 4). Notably, CSF MTBR-tau243 concentrations were significantly more strongly associated with tau-PET when compared with pT217 / T217 (BioFINDER-2: β = 0.77 [0.71, 0.83]; Knight ADRC: β = 0.61 [0.49, 0.73]). Fig. 7A Scatter plots of the association of all CSF tau biomarkers with amyloid PET and tau-PET in both cohorts are shown in ​-B and8A-B, respectively.

[0343] We also investigated the correlation of CSF tau measures with CSF Aβ42 / 40. Among CSF tau measures, pT217 / T217 was most strongly correlated with CSF Aβ42 / 40 (BioFINDER-2: β [95% CI] = -0.80 [-0.86, -0.74]; Knight ADRC: β = -0.88 [-0.95, -0.81]; all p < 0.001, Fig. 12A -B and Table 8). In contrast, MTBR-tau243 concentrations showed a significantly lower association with CSF Aβ42 / 40 compared with pT217 / T217 (BioFINDER-2: β = -0.63 [-0.70, -0.55]; Knight ADRC: β = -0.59 [-0.71, -0.47]; all p < 0.001, Fig. 12A -B and Table 8).

[0344] The correlation of CSF tau measures with tau-PET signals in different Braak regions (entorhinal [Braak I], temporal lobe [Braak III-IV], and neocortex [Braak V-VI]) was also investigated as an additional analysis. Comparison of the amyloid-positive only groups confirmed that CSF MTBR-tau243 had the highest correlation with all Braak regions (BioFINDER-2: β = 0.85, 0.84, and 0.76 for each Braak region; Knight ADRC: β = 0.83, 0.84, and 0.76 for each Braak region, respectively; all p < 0.001, Fig.9A -C and Table 9).

[0345] Biomarker changes explained by amyloid and tau pathology

[0346] Next, we assessed the proportion of CSF biomarker variation explained by amyloid and tau pathology. In our models, including CSF biomarker levels as outcomes and both amyloid PET and tau-PET as predictors, we controlled for age and sex. In the BioFINDER-2 cohort, the variance in CSF pT217 / T217 levels was significantly better explained by Aβ pathology as assessed by amyloid PET relative to tau (Aβ: partial R 2 [pR 2 ]=0.57,74.7% R 2 Relative to tau: pR 2 =0.19, 24.7% R 2 , p<0.001, Figure 2A and Table 5). CSF pT231 / T231 (Aβ: pR 2 =0.46, 76.0% R 2 ;tau: pR 2 =0.02, 3.3% R 2 , p<0.001) and pT181 / T181 (Aβ: pR 2 =0.32, 55.7% R 2 ;tau: pR 2 =0.13, 22.0% R 2 , p = 0.006) was also significantly better explained by Aβ pathology. In contrast, the variance in CSF MTBR-tau243 concentrations was significantly better explained by tau pathology (Aβ: pR 2 =0.14, 22.3% R 2 ;tau: pR 2 =0.38, 60.6% R 2, p<0.001). Tau and amyloid contributed similarly to CSF ​​pT205 / T205, and the difference between them was not significant (Aβ: pR 2 =0.29, 45.4% R 2 ;tau: pR 2 =0.25, 39.7% R 2 , p=0.657).

[0347] Similar trends were observed in the Knight ADRC cohort, although a greater proportion of the variance was explained by Aβ pathology for all CSF biomarkers, most likely because this cohort included relatively few individuals with significant tau pathology (only n = 36 [16.4%] were tau-PET positive). CSF pT217 / T217 (Aβ: pR 2 =0.51, 75.1% R 2 ;tau: pR 2 =0.14, 19.9% ​​R 2 , p<0.001), pT181 / T181 (Aβ: pR 2 =0.25, 70.1% R 2 ;tau: pR 2 =0.02,4.9% R 2 , p<0.001) and pT231 / T231 (Aβ: pR 2 =0.31, 68.8% R 2 ;tau: pR 2 =0.04, 8.6% R 2 , p<0.001) was better explained by Aβ pathology. In contrast, tau pathology was the main contributor to explaining the variance in CSF MTBR-tau243 levels (Aβ: pR 2 =0.09, 16.0% R 2 ;tau: pR 2 =0.36, 66.7% R 2 , p<0.001). Notably, pT205 / T205 levels were similarly explained by both tau and amyloid (Aβ: pR 2 =0.27, 45.2% R 2 ;tau: pR 2 =0.27, 45.4% R 2 , p = 0.990, Figure 2B and Table 5).

[0348] Because amyloid PET was not performed in the dementia patients in BioFINDER-2, the analyses were repeated separately in both cohorts, with all participants using CSF Aβ42 / 40 rather than amyloid PET as a measure of Aβ pathology ( Fig.13A -B and Table 5). CSF pT217 / T217 levels were slightly, but significantly better explained by CSF Aβ42 / 40 levels relative to tau-PET (BioFINDER-2: 66.6% R 2 vs. 56.5%, p=0.044; Knight ADRC: 87.6% R 2 =1.5 (BioFINDER-2: 69.8% vs. 19.1%, p<0.001; Knight ADRC: 84.0% vs. 7.0%, p<0.001) and pT181 / T181 (BioFINDER-2: 52.9% vs. 33.7%, p=0.014; Knight ADRC: 82.8% vs. 3.5%, p<0.001). In these models, tau pathology remained the main factor explaining the variance in MTBR-tau243 levels (BioFINDER-2: 21.4% vs. 75.1%, p<0.001; Knight ADRC: 33.5% vs. 63.8%, p=0.014). For pT205 / T205, the major contributor was tau pathology (BioFINDER-2: 19.6% vs. 66.8%, p<0.001), although the difference was not significant in Knight ADRC (36.5% vs. 57.6%, p=0.125).

[0349] Longitudinal changes in CSF biomarkers

[0350] Longitudinal data from the BioFINDER-2 cohort were used to examine changes in CSF biomarkers stratified by amyloid (A) and tau (T) pathology status (positive: +, negative: -). The characteristics of the 220 participants with longitudinal CSF measurements are described in Table 10. When the interaction with time was significant, a linear mixed model was used to compare the CSF longitudinal trajectories between the groups (i.e., A- / T-, A+ / T-, and A+ / T+) using a post hoc paired Wilcoxon test. Amyloid positivity versus negativity was derived from CSF Aβ42 / 40 levels and tau was dichotomized based on tau-PET measurements. The trajectories of individuals and groups over time are shown in Fig.10The rates of increase of CSF pT217 / T217, pT181 / T181, and pT231 / T231 were greatest in the A+T- group, and lower in the A+T+ group, suggesting that the rates of increase of these biomarkers stabilize in the later stages of the disease when tau pathology increases the most ( Figure 3 ). In contrast, the rates of increase in CSF pT205 / T205 and MTBR-tau243 were greatest in the A+T+ group, corresponding to increases in tau pathology. Importantly, CSF MTBR-tau243 increased faster in the A+T+ group than in the A+T- group (vs. AT-: Cohen's d = 1.48, p < 0.001; vs. A+T-: Cohen's d = 1.13, p < 0.001), while the rates of increase in pT205 / T205 in the A+T+ and A+T- groups were not significantly different (Cohen's d = 0.08, p = 0.788, Figure 3 and Table 11). This suggests that CSF MTBR-tau243 best reflects AD progression in tau-PET positive individuals.

[0351] As a sensitivity analysis, we repeated the analysis using amyloid PET instead of CSF Aβ42 / 40, using the previously validated threshold 39 We found that the longitudinal trajectories of all CSF biomarkers were replicated, with rates of change increasing with progression of A / T status for pT205 / T205 and especially MTBR-tau243, and the remaining biomarkers having the highest rates of change in the A+T- state ( Fig.14 and Table 11).

[0352] Association of CSF and PET biomarkers with MMSE scores

[0353] We evaluated CSF and PET biomarkers with a common clinical assessment of dementia, the Mini-Mental State Examination (MMSE), using linear regression models adjusted for age, sex, and years of education. 40(which was evaluated in both cohorts). MTBR-tau243 was the CSF biomarker most strongly associated with MMSE scores in all participants (BioFINDER-2: β = -0.65 [-0.74, -0.57]; KnightADRC: β = -0.54 [-0.67, -0.42], all p < 0.001) and amyloid-positive participants (BioFINDER-2: β = -0.56 [-0.66, -0.46]; Knight ADRC: β = -0.54 [-0.69, -0.39], all p < 0.001, Figure 4 and Table 12). These associations were significantly stronger than those for pT217 / T217, as assessed by bootstrapping, for both all participants (BioFINDER-2: β = -0.60 [-0.69, -0.52], p comparison for β = 0.001; Knight ADRC: β = -0.40 [-0.53, -0.26], p comparison for β = 0.003) and amyloid-positive participants (BioFINDER-2: β = -0.48 [-0.59, -0.38], p comparison = 0.002; Knight ADRC: β = -0.36 [-0.52, -0.19], p comparison = 0.001). However, tau-PET was more strongly associated with MMSE than any CSF biomarker (BioFINDER-2: β = -0.73 [-0.79, -0.64], p = 0.036; Knight ADRC: β = -0.64 [-0.74, -0.53], p < 0.001). Scatter plots for each CSF biomarker are shown in Fig.11A -B.

[0354] AD-related measures and best predictors of cognitive function

[0355] Finally, we aimed to determine whether combinations of CSF biomarkers could be used as accurate quantitative surrogates for amyloid PET, tau-PET, or cognitive measures. We first assessed the variance explained by each individual biomarker for each outcome. Next, we used the least absolute shrinkage and selection operator (LASSO) procedure to select which CSF biomarker combination best fits each outcome, and then we compared this new model with models from individual biomarkers.

[0356] CSF pT217 / T21 is the best single biomarker for predicting amyloid PET (BioFINDER-2: R 2 =0.73, AICc=404.5; Knight ADRC: R 2=0.73, AICc=265.2, Figure 5 and Table 13). Based on LASSO regression, we found that combining CSF pT217 / T217 with pT205 / T205 and Aβ42 / 40 significantly improved the prediction of amyloid PET in both cohorts (BioFINDER-2: R 2 =0.77, AICc=370.2, F=20.974, p<0.001; Knight ADRC: R 2 =0.73, AICc=261.7, F=5.266, p=0.006, Table 6).

[0357] MTBR-tau243 was the best single biomarker predicting tau-PET (BioFINDER-2: R 2 =0.68, AICc=715.6; Knight ADRC: R 2 =0.51, AICc=363.2, Figure 5 and Table 13). The best model combining MTBR-tau243 and pT205 / T205 significantly improved the prediction of tau-PET amount in both cohorts (BioFINDER-2: R 2 =0.75, AICc=614.1, F=116.49, p<0.001; Knight ADRC: R 2 =0.58, AICc=339.5, F=30.268, p<0.001, Figure 5 and Table 6).

[0358] MTBR-tau243 was the best single biomarker predicting MMSE score (BioFINDER-2: R 2 =0.42, AICc=790.3; Knight ADRC: R 2 =0.30, AICc=423.0, Figure 5 and Table 13), and the prediction improved when pT205 / T205 was increased (BioFINDER-2: R 2 =0.48, AICc=754.6, F=27.693, p<0.001; Knight ADRC: R 2 =0.34, AICc=415.2, Figure 5 and Table 6). Tau-PET predicted MMSE scores better than the combination of CSF biomarkers, but the difference was not statistically significant (BioFINDER-2: R 2 =0.52, AICc=724.9, z=0.900, p=0.184; Knight ADRC: R 2=0.44, AICc=385.8, z=-1.405, p=0.080, Figure 5). Results for amyloid-positive participants only confirmed similar results ( Figure 5A -B and Table 12 and Table 6).

[0359] discuss:

[0360] In this study, we found that in two large independent sporadic AD cohorts, the new CSF biomarker MTBR-tau243 was strongly associated with tau pathology and minimally associated with Aβ pathology. We also found that the correlation between CSFMTBR-tau243 and cognitive measures was significantly higher than that of phosphorylated tau measures (e.g., pT217 / T217 and pT181 / T181), suggesting its potential utility in clinical settings. Further, we found that CSF MTBR-tau243 was the biomarker with the greatest rate of increase in participants who were already positive for both amyloid and tau pathology, suggesting that CSF MTBR-tau243 best reflects disease progression in the late stages. We further extended these findings by combining CSF MTBR-tau243 with phosphorylated tau measures to predict Aβ pathology, tau pathology, and cognitive measures in the continuous course of AD. We found that CSF MTBR-tau243 in combination with pT205 / T205 accurately predicted continuous tau-PET measures and had similar predictive accuracy for cognitive measures as tau-PET. Based on these results, our study suggests that CSF MTBR-tau243 may be a viable alternative to tau-PET for use as a prescreening tool or tau pathology endpoint surrogate for clinical trials and also as an accurate diagnostic measure of tau pathology.

[0361] Our first goal was to characterize MTBR-tau243 concentrations and compare them with four phosphorylated tau measures by observing their association with Aβ and tau pathology measured by PET. Notably, MTBR-tau243 was the tau biomarker that was confirmed to have the highest correlation with tau-PET and the lowest correlation with amyloid PET not only in the total group, but also in the Aβ-positive group. This suggests that MTBR-tau243 is a biomarker that specifically reflects aggregated tau pathology independent of amyloid pathology. Although pT217 / T217 also correlated well with tau-PET, there was a nonlinear relationship and a significant increase in pT217 / T217 before the tau-PET pathology increased, and it stabilized once the tau-PET threshold was exceeded. This may indicate that pT217 / T217 is primarily associated with tau pathology through its quantitative relationship with the amount of Aβ pathology. This is further supported by the observation that a significantly greater proportion of pT217 / T217 level changes were explained by Aβ pathology than tau when both amyloid PET and tau-PET measures were included in the model. Interestingly, although pT205 / T205 levels demonstrated a high correlation with tau-PET, they also showed the second highest correlation with amyloid PET, second only to pT217 / T217. In the combined model, both amyloid PET and tau-PET explained a similar proportion of pT205 / T205 level changes, suggesting that it is an intermediate biomarker affected by both Aβ and tau pathology. Regarding other p-tau measures, pT181 / T181 and pT231 / T231 were highly correlated with amyloid PET, while the correlation with tau-PET was significantly lower than that of the other three CSFtau biomarkers, suggesting that they mainly reflect Aβ pathology. These results are consistent with several recent studies suggesting that p-tau181, p-tau217, and p-tau231 may be more relevant to amyloid pathology than tau. 13,14,17,21,22,41-43 , more closely associated with amyloid-PET than with tau-PET 18,23,44 , or actual amyloid pathology in autopsy studies 45,46 Finally, we found that MTBR-tau243 was specifically increased in two amyloid-negative MAPT R406W mutation carriers with high tau-PET binding. The tau pathology in MAPT R406W mutation carriers is known to be similar to AD tau pathology. 47,48 , and is responsive to AD tau-PET tracers 4,49-51, which further supports our finding that MTBR-tau243 is a specific biomarker for AD-like tau pathology.

[0362] Changes in longitudinal CSF biomarkers were also studied to understand how these biomarkers change at different stages of the disease. Most notably, of the five CSF tau biomarkers, only MTBR-tau243 showed a significant increase in the rate of change between the A+T- and A+T+ groups, suggesting that it allows longitudinal disease tracking during the disease stage characterized by neocortical tau aggregates (which mainly appear in the symptomatic phase of AD). On the other hand, there was no significant difference in the rate of change of pT205 / T205 between A+T- and A+T+, although it was still confirmed to be a positive rate of change in this late stage, suggesting a lower but still significant increase after tau deposition. Interestingly, for the other phospho-tau measures (pT181 / T181, pT217 / T217, and pT231 / T231), there was a clear increase in the rate of change during the transition from AT- to A+T-, which is consistent with previous reports showing that phospho-tau (particularly p-tau217) is the best marker for disease monitoring during the very early (preclinical) stages of the disease. 52 Interestingly, we found here that there was neither a significant increase nor a significant decrease in the rate of change of phosphorylated tau occupancy during the transition from A+T- to A+T+, consistent with previous reports. 21 These results suggest that the rate of change of these phosphorylated tau measures may plateau or decline in late disease stages when insoluble tau aggregates are deposited in the neocortex, indicating that they are not longitudinally consistent and that the classic p-tau measures are not direct measures of AD tau pathology. 53 In summary, the findings from CSF p-tau measures are consistent with previous clinical observational studies and preclinical mouse models. 22,32 , where these biomarkers appear to be driven by Aβ pathology. These results further support the recent proposal to revise the A / T / (N) criteria system, where any p-tau biomarker can be used as a tau(T) marker. 54 .

[0363] As a relevant question for clinical practice, we also investigated the relationship between these CSF biomarkers and cognitive measures. As expected by the observed association with tau pathology, MTBR-tau243 was the measure most strongly associated with MMSE, a cognitive test frequently used in clinical settings. Notably, this association was not significantly different from tau-PET, thus supporting the idea that CSF MTBR-tau243 may be a viable alternative to tau-PET for clinical purposes. Although pT205 / T205 was less correlated with MMSE than MTBR-tau243, pT205 / T205 was also well correlated with MMSE and was not significantly different from tau-PET. In contrast, other CSF biomarkers such as pT217 / T217 or pT181 / T181 showed significantly lower associations.

[0364] An unmet need is to determine not only who has amyloid or tau pathology, but also whether the symptoms are due to these pathologies. Since tau pathology is most highly correlated with cognitive and clinical impairment, an important question is how well CSF biomarkers can predict tau pathology or cognitive impairment. We next examined whether combining CSF biomarkers would improve the prediction of Aβ or tau pathology or cognitive measures. We evaluated the predictive performance of the combination of these CSFtau biomarkers for amyloid PET load, tau-PET load, and MMSE. Based on a data-driven approach, we observed that the combination of pT205 / T205, Aβ42 / 40, and pT217 / T217 was best suited for predicting continuous measures of amyloid PET and significantly improved the performance of any single measure. We also found that the combination of MTBR-tau243 and pT205 / T205 improved the prediction of tau-PET load in a single model compared to any other single fluid biomarker. The fact that such high predictive accuracy for both amyloid PET imaging and tau-PET imaging can be achieved with CSF biomarkers suggests that CSF assays may be an alternative to PET measures, which are costly and have limited accessibility. Notably, MTBR-tau243 and pT205 / T205 were also the best combination for predicting cognitive measures (i.e., MMSE), suggesting potential clinical utility of this biomarker combination in predicting not only tau pathology but also cognitive impairment. For wider use, it will be critical to convert these biomarkers to blood-based biomarkers.

[0365] The main advantage of this study is that we have replicated our key findings in two large independent groups representing different types of populations and used different PET tracers, and we have also prospectively measured the collected samples and predefined outcome measures. Although further research is needed in more diverse and generalized populations to implement our findings clinically, it is important to emphasize that BioFINDER-2 participants were recruited continuously from secondary ClinicalMemory in Sweden. Therefore, the group is a representative of Swedish memory clinic patients (memory clinical patients) and includes both AD and non-AD dementia patients. Limitations include that, although similar trends are shown, the trend amplitudes between the two groups are different in some analyses. Potential reasons include that the BioFINDER-2 group includes more tau-PET positive participants with AD dementia than the Knight ADRC group, and more participants in the advanced stage of the disease, which can affect the results of tau-PET and MMSE. Another limitation is that relatively few participants with AD dementia in the BioFINDER-2 group underwent amyloid PET scanning according to the study design, although all of these participants had CSF Aβ42 / 40. Therefore, we used CSF Aβ42 / 40 instead of amyloid PET as a marker of Aβ pathology in sensitivity analyses and confirmed that this limitation did not affect the overall results and interpretation. Further, we acknowledge that our measures of Aβ and tau pathology are only surrogate biomarkers and not actual measures of pathology, but both amyloid PET and tau-PET markers have been validated as measures of neuropathology for insoluble Aβ and tau aggregates, respectively. 2,3,6,55-58 Future studies using animal models and measures of neuropathology will be important to further validate the results presented here.

[0366] Together, these findings confirm that CSF MTBR-tau243 specifically reflects changes in aggregated tau pathology occurring in later stages of AD progression and is associated with clinical and cognitive symptoms. Therefore, it may be important when defining AD pathology and in future versions of the commonly used A / T / (N) criteria for AD. 53, we propose that MTBR-tau243 should replace the commonly used p-tau measurement as a fluid biomarker representing insoluble tau aggregate pathology (T). Therefore, MTBR-tau243 can be used to evaluate AD tauopathy and track the effect of drug treatment independently of the amyloid effect. The combination of CSF MTBR-tau243 and pT205 / T205 is almost equivalent to tau-PET measurement, and predicts MMSE almost as accurately as tau-PET, which shows the clinical utility of the biomarker group containing MTBR-tau243. Compared with biomarkers that are often abnormal in cognitively normal elderly individuals and are changed by amyloidosis, CSF MTBR-tau243 makes it possible to confirm tau pathology and provide higher certainty that cognitive symptoms are due to AD, as proposed by the latest clinical AD criteria that require biomarker evidence of both amyloid and tau pathology to diagnose AD with a high probability. 59 These findings may help improve the diagnostic accuracy of biomarkers for AD and aid in the development of new strategies for AD treatment.

[0367] method:

[0368] Participants

[0369] Included participants from the following two cohorts: Swedish BioFINDER-2 (NCT03174938) at Lund University (Lund, Sweden) 13 and Knight ADRC from Washington University (Port St. Louis, USA). The BioFINDER-2 cohort included cognitively intact participants (recruited as cognitively normal controls or patients with subjective cognitive decline [SCD]), patients with MCI, patients with AD dementia, and patients with non-AD neurodegenerative diseases. University Hospital and Hospital of Recruitment. Details on recruitment, exclusion, and inclusion criteria have been described previously. 13 All participants underwent lumbar puncture for CSF sampling at baseline and at follow-up two years later. Participants underwent cognitive testing, including the MMSE. The Knight ADRC cohort consisted of community-dwelling volunteers enrolled in the Memory and Aging Study at Washington University in St. Louis. All Knight ADRC participants underwent a comprehensive clinical evaluation, which included a detailed interview with a collateral source, a neurological examination of the participant, clinical dementia, and a 3-year follow-up. (CDR) 60 and Mini-Mental State Examination (MMSE) 40Individuals with a CDR of 0.5 or greater were considered to have a dementia syndrome, and the possible etiology of the dementia syndrome was determined by the clinician based on the clinical features according to standard criteria and methods. 61 .

[0370] In the BioFINDER-2 cohort, participants were divided into CU Aβ negative or positive (CU- and CU+, respectively), MCI patients Aβ positive (MCI+), AD dementia patients Aβ positive (AD+), or non-AD neurodegenerative patients, regardless of their Aβ status. Two participants (one in CU- and the other in non-AD) were carriers of the MAPT R406W mutation, Aβ negative and tau positive. In the Knight ADRC cohort, participants were divided into CU with CDR=0 Aβ negative or positive (CU- and CU+, respectively), very mild AD patients with CDR=0.5 Aβ positive, and AD dementia patients with CDR≥1 Aβ positive (AD+). In accordance with the research framework of the National Institute on Aging-Alzheimer's Association, study patients with SCD and cognitively healthy controls were considered the CU group. 5 All participants gave written informed consent, and ethical approval was granted by the Regional Ethical Committee in Lund, Sweden and the Washington University Human Research Protection Office, respectively.

[0371] Anti-tau antibody generation

[0372] Antibodies HJ32.11 and HJ34.8 were generated by immunizing tau knockout mice (Jacksonlabs) with keyhole limpet hemocyanin (KLH) fused to amino acids 225-242 of tau (to generate antibody HJ32.11) or KLH fused to amino acids 226-264 (to generate antibody HJ34.8). Splenocytes from immunized mice were fused with P3 hybridoma cells and amplified. Clones were screened by direct ELISA.

[0373] CSF measurements

[0374] Measurement of CSF tau species, including p-tau and MTBR-tau243, was performed at the University of Washington in two cohorts using a recently developed immunoprecipitation / mass spectrometry (IP / MS) approach. We developed two new monoclonal antibodies to immunopurify CSF MTBR-tau243: HJ32.11, which binds near residue 243, and HJ34.8, which binds near residue 260. The procedure for CSF tau analysis is described in Figure 16. Phosphorylated and non-phosphorylated peptides were measured in the same injection and the percentage of phosphorylation occupancy was calculated as p-tau / t-tau% 21 To calculate the phosphorylation percentage.

[0375] Additionally, CSF Aβ42 / 40 levels were used to assess Aβ positivity in both cohorts. In the BioFINDER-2 cohort, CSF levels of Aβ42 / 40 were measured as explained previously. 13 A threshold of 0.080 based on Gaussian mixture modeling determined Aβ-positive 39 In the Knight ADRC cohort, CSF Aβ42 / 40 levels were measured as explained previously. 62 The threshold value (0.0673) had the greatest combined sensitivity and specificity in distinguishing amyloid PET status.

[0376] Imaging acquisition and quantification

[0377] In the BioFINDER-2 cohort, amyloid PET and tau PET acquisition methods have been previously reported 13 In short, use [ 18 F] flumetinol to obtain amyloid PET, and the use of [ 18 F]RO948 to obtain tau-PET. Of note, due to the study design, most AD patients in BioFINDER-2 did not undergo amyloid PET. In the KnightADRC cohort, as explained previously, participants used [ 18 F] Frobetapil ([ 18 F]AV45) or [ 11 C]PiB for amyloid PET and the use of [ 18 F]Flutociclib ([ 18 F]flortaucipir)([ 18 F]AV1451) for tau-PET 33Amyloid PET was measured in the neocortex meta-ROI using cerebellar gray matter as a reference region. Centiloid was calculated in the BioFINDER-2 cohort using the Computational Analysis of PET (CapAIBL) pipeline from AIBL. 63 For tau-PET, SUVR was calculated using the inferior cerebellar cortex as a reference region, and the primary analysis was performed using a combination from the temporal lobe meta-ROI (Braak I-IV) to capture the regions most affected by tau. In supplementary analyses, we also quantified tau-PET in regions with early (Braak I), intermediate (Braak III-IV), and late (Braak V-VI) tau deposition. 64 In all cases, Tau positivity was based on tau-PET assessment. In the Braak I-IV region, the positivity cutoff was set to SUVR>1.32 in both the BioFINDER-2 and Knight ADRC cohorts. 65,66 .

[0378] Cognitive testing

[0379] The MMSE was used as a measure of global cognition in both groups.

[0380] Statistical analysis

[0381] Differences in CSF biomarker levels between diagnostic groups were tested using ANCOVA adjusted for age and sex. Post hoc analyses were performed using the Tuckey test. Linear regression models were used to evaluate the association between amyloid PET and tau-PET (independent variables) and each CSF biomarker (dependent variable) after adjusting for age and sex. For cognition, we additionally used educational status as a covariate in the linear regression models. Distributions of beta differences were constructed using a bootstrap method (n=500) using the boot package and used to infer significance, with all standardized betas compared to the highest for each outcome and group. The proportion of changes in CSF levels of amyloid and tau measures was evaluated using a linear regression model using both amyloid and tau as predictors, CSF levels as outcomes, and age and sex as covariates. We calculated partial R for amyloid and tau using the rsq package. 2 , the original value and the total R 2Percentage. This is used as a measure of the variance ratio explained by amyloid and tau. Next, the prediction of amyloid and tau continuous measurements was evaluated with a linear regression model (wherein amyloid PET and tau-PET measurements were used as results in independent models and single CSF biomarkers as predictors). A basic model was also created, in which only covariates (age and gender) were used as predictors. In addition, a minimalist model was also constructed to independently best predict each of these measurements (reaching the highest accuracy when the number of predictors was small) for each group. For this purpose, a LASSO regression model (glmnet package) was used, which initially included all CSF biomarkers and covariates. Only those predictors selected by LASSO regression and having a significant contribution (p<0.1) in the model were finally included in the minimalist model. A similar method was used to predict cognition (MMSE in two groups and CDR in Knight ADRC), and educational status was additionally included as a covariate. In these cases, we compared the minimalist model with a model that only included tau-PET as a predictor. The F test was used to compare nested models (including the same subset of predictors). When comparing models with different predictors, we used the Vuong test using the nonnest2 package. Finally, the CSF longitudinal changes of baseline amyloid and tau status were evaluated in the BioFINDER-2 group. The slope of a single participant was calculated using a linear regression model to calculate the difference in rate of change (average percentage change) and compare them between groups using the Kruskal-Wallis test. Further, we used the lme4 package to create group trajectories with a linear mixed model for visualization. Here, CSF biomarkers were used as results, the interaction between time and baseline amyloid and tau status was used as a predictor, and the main effects of age and gender were used as covariates, using random intercepts and fixed time slopes (because the number of time points was small). CSF and amyloid PET and tau-PET measurements were logarithmically transformed in linear regression analysis. Bilateral P values ​​<0.05 were considered statistically significant. R version 4.1.0 was used for all statistical analyses.

[0382] Example 2

[0383] MTBR-tau243 is a fluid Tau biomarker specific for Tau pathology

[0384] Two-step immunoprecipitation and / or immunodepletion method for detecting cleaved fragments of MTBR-tau243

[0385] A two-step method with immunodepletion and immunoprecipitation was developed to isolate the cleaved fragment of MTBR-tau243. The method was tested in CSF and plasma.

[0386] MTBR-tau 243 detection in CSF

[0387] An exemplary method for detecting MTBR-tau243 in CSF is provided in Example 1. Fig.18A Two methods of immunodepletion and / or immunoprecipitation are shown. One method (Design 1) used Tau1 / HJ8.5 / HJ8.7 antibodies in the first affinity depletion step. The second method (Design 2) used Tau1 / HJ8.5 / HJ8.7 antibodies in the first step. For all antibodies, 3mg / gram agarose beads were generated. For Tau1 immunoprecipitation (IP), 1.1ug antibody / sample was used. For HJ8.5 and HJ8.7 IP, 2.3ug antibody / sample was used. For HJ32.11 and HJ34.8 IP, 11.25ug antibody / sample was used. Fig.18A As shown, NP40, guanidine, 1 ng 15 A premix of N-2N4R-tau (internal standard) and anti-tau antibody beads Tau1 / HJ8.5 / HJ8.7 (or Tau1 / HJ8.5 / HJ8.7 / HJ34.8). The premix was added to a CSF sample from a subject and incubated at room temperature for 2 hours for affinity depletion (immunodepletion) or immunoprecipitation of MTBR-tau243, producing a first immunodepletion precipitate including antibody beads and a CSF solution. The first immunodepletion precipitate was washed and subjected to trypsin for trypsin digestion. The digested sample was subjected to desalting by C18 toptip (Glygen) and analyzed by LC / MS. In the second immunoprecipitation step, the remaining CSF sample was mixed with 2 ng of 13 N 15 N-2N4R-tau (internal standard), 2 ng 15 The second mixture of N-ON3R-tau (internal standard) and HJ32.11 antibody beads was mixed and incubated at room temperature for 2 hours, and the affinity of MTBR-tau243 was exhausted (immune exhaustion) or immunoprecipitation, and the second immunoexhaustion precipitate including HJ32.11 antibody beads was produced. The second immunoexhaustion precipitate was washed and subjected to trypsin for trypsin digestion. The digested sample was desalted by C18 toptip (Glygen) and analyzed by LC / MS. Fig.18C MTBR-tau243 peptide affinity is shown for HJ8.5, HJ8.7, HJ34.8, and HJ32.11 antibodies.

[0388] like Fig.18B As shown, NP40, guanidine, 1.25 ng 15N-2N4R-tau (internal standard), 1.25 ng 15 N-0N3R-tau (internal standard) and anti-tau antibody beads Tau1 / HJ8.5 / HJ8.7 / HJ34.8 premix. The premix was added to 450 μL CSF samples from the subjects and incubated at room temperature for 2 hours for affinity depletion (immunodepletion) or immunoprecipitation of MTBR-tau243, producing a first immunodepletion precipitate and CSF solution including antibody beads. The first immunodepletion precipitate was washed and subjected to trypsin digestion, which is a form of proteolytic cleavage. The digested sample was desalted by C18 toptip (Glygen) and analyzed by LC / MS. In the second immunoprecipitation step, the remaining CSF sample was mixed with 1 ng of 13 N 15 The second mixture of N-2N4R-tau (internal standard) and HJ32.11 antibody beads was mixed and incubated at room temperature for 2 hours for affinity depletion (immuno-depletion) or immunoprecipitation of MTBR-tau243 to produce a second immuno-depletion precipitate including HJ32.11 antibody beads. The second immuno-depletion precipitate was washed and subjected to trypsin for trypsin digestion. The digested sample was desalted by C18 toptip (Glygen) and analyzed by LC / MS.

[0389] Fig.19 Different MTBR-tau fragments isolated by HJ34.8 antibody beads in the first immunodepletion step and by anti-tau antibody HJ32.11 beads in the second immunoprecipitation step are shown. Long MTBR-tau243 fragments showed affinity for the HJ38.8 antibody and exhibited reduced association with AD tau tangles. Notably, very short MTBR-tau243 fragments showed affinity for the HJ32.11 antibody and exhibited high association with tau tangles.

[0390] The amount of cleaved MTBR fragments precipitated by each antibody during immunoprecipitation and / or immunodepletion is shown in Fig. 20A -B. Fig. 20A The HJ34.8 antibody was shown to precipitate additional cleaved MTBR-tau243 fragments in the first immunodepletion step. Fig. 20B Confirmation of cleavage of the MTBR-tau243 fragment by HJ34.8 immunodepletion.

[0391] Both immunoprecipitation and / or immunodepletion methods (with or without HJ34.8 antibody in the first immunodepletion step) were validated by correlating the results obtained using chemical extraction (CX) to isolate MTBR-tau243 fragments. Fig.21A-B shows the correlation between the amount of MTBR-tau243 detected by chemical extraction and each of the two immunoprecipitation and / or immunodepletion methods. ( Fig.21A ) Correlation of immunoprecipitation with Tau1 / HJ8.5 / HJ8.7 and / or immunodepletion of MTBR-tau243 with chemical extraction. Fig. 22B ) Correlation of immunoprecipitation with Tau1 / HJ8.5 / HJ8.7 / HJ34.8 and / or immunodepletion of MTBR-tau243 with chemical extraction. Fig.21A As shown, tau species precipitated in the absence of HJ34.8 did not correlate well with CSF MTBR-tau243 prepared by the chemical extraction method. The Spearman correlation was 0.8791. When the HJ34.8 antibody was used, the correlation was improved to 0.9576 using the second method.

[0392] Fig.22A -B shows the correlation between the amount of MTBR-tau243 detected by two immunoprecipitation and / or immunodepletion methods and Tau PET SUVR for 54 subjects. ( Fig.22A ) Correlation of MTBR-tau243 and Tau PET SUVR using Tau1 / HJ8.5 / HJ8.7 antibodies. ( Fig. 22B ) Correlation of MTBR-tau243 and Tau PET SUVR using Tau1 / HJ8.5 / HJ8.7 / HJ34.8 antibodies. Fig.22A As shown, the tau species precipitated by HJ32.11 did not correlate well with tau-PET. The Spearman correlation was 0.5308. Using the second method in the case of the HJ34.8 antibody improved the correlation to 0.7532. The improved correlation shows the criticality of the depletion using HJ34.8 in the first immunodepletion step. The tau species precipitated by HJ34.8 did not correlate with tau-PET, so depletion was performed in the first step. The second immunoprecipitation using HJ32.11 showed better correlation with tau-PET.

[0393] Fig.23A -D shows that MTBR-tau243 is highly correlated with Tau-PET in both all and amyloid only (+) populations, but has a lower association with amyloid PET relative to pT217. Data were collected from 222 subjects. pT205 is an intermediate feature that summarizes both amyloid and tau pathology (between MTBR-tau243 and pT217). Fig.24A-C shows that MTBR-tau243 maintained its discriminatory ability to identify tau-PET positivity even in the amyloid-positive subset as well as in all participants. These data suggest that cleavage fragments of tau containing MTBR-tau243 are directly associated with Tau-PET status.

[0394] Example 3

[0395] MTBR-tau 243 detection in plasma compared with CSF

[0396] This example describes a method for detecting MTBR-tau243 fragments such as MTBR-tau243-256V and MTBR-tau243-256V(dN) in plasma versus CSF and demonstrates that these fragments can be used to detect tangles. Fig.25 As shown, NP40, guanidine, 1 ng 15 A premix of N-2N4R-tau (internal standard) and anti-tau antibody beads Tau1 / HJ8.5 / HJ8.7 (or Tau1 / HJ8.5 / HJ8.7 / HJ34.8). The premix was added to 950 μL of plasma sample and incubated at room temperature for 2 hours for affinity depletion (immunodepletion) or immunoprecipitation of MTBR-tau243, producing a first immunodepletion precipitate including antibody beads and a plasma solution. The first immunodepletion precipitate was washed and subjected to trypsin digestion. The digested sample was desalted by C18 toptip (Glygen) and analyzed by LC / MS. The remaining plasma sample was mixed with 2 ng of 13 N 15 N-2N4R-tau (internal standard), 2 ng 15 The second mixture of N-0N3R-tau (internal standard) and 30 μ L of HJ32.11 antibody beads was mixed and incubated at room temperature for 2 hours, and the affinity of MTBR-tau243 was exhausted (immune exhaustion) or immunoprecipitation, and the second immunoexhaustion precipitate including HJ32.11 antibody beads was produced. The second immunoexhaustion precipitate was washed and subjected to trypsin for trypsin digestion. The digested sample was desalted by C18 toptip (Glygen) and analyzed by LC / MS.

[0397] MTBR-tau243 relative to Tau-PET status obtained from CSF and plasma from subjects is shown in Fig.26A -B. Fig.26A -B shows that the cleavage fragments of tau containing MTBR-tau243 in CSF are associated with Tau-PET status (low, medium, high) ( Fig.26A ), but not in plasma ( Fig.26B ).like Fig.26B As shown, the two-step immunodepletion and / or immunoprecipitation method except in CSF ( Fig.26A ) could not distinguish between low, medium and high Tau-PET alone. A two-step immunoprecipitation method using trypsin digestion in plasma did not correlate with Tau-PET status.

[0398] Detection of MTBR-tau243 in plasma versus CSF and use of ArgC endopeptidase

[0399] As mentioned above, the two-step immunodepletion and / or immunoprecipitation approach using trypsin digestion in plasma did not correlate with Tau-PET status. Fig. 27 Shown is a protocol for determining the feasibility of detecting MTBR-tau243 species in both CSF and plasma, using tau extracted from brain. Fig.28 The MTBR-tau243 peptide isolated and detected from the soluble fraction of brain containing tau using the above method was shown to include multiple lysine residues near the MTBR-tau243 region, which helps explain why MTBR-tau243 is not detectable in plasma. To address this issue, it was hypothesized that digestion of the MTBR-tau peptide with ArgC endopeptidase could be used to screen for naturally occurring cleavage sites. An exemplary method for detecting MTBR-tau243 in CSF is shown in Fig.29 A premix of NP40, guanidine, protease inhibitors, and HJ32.11 antibody beads was prepared. 450 μL of CSF samples with low, medium, and high tau PETs were incubated with the premix for 2 hours. The HJ32.11 antibody beads were removed by immunoprecipitation. The immunoprecipitated samples were washed, digested with ArgC for 2 hours, desalted, and the resulting solution was analyzed by LC-MS. Table 14 shows the CSF sample demographics used to identify endogenous MTBR-tau243 species working as alternative tau-PETs.

[0400] The cleaved peptide fragments detected by LC-MS are listed in Table 1. Fig. 30A -B shows MTBR-tau243-256V (SEQ ID NO: 15) in CSF ( Fig. 30A ) and MTBR-tau 243-256 (SEQ ID NO: 16) with @255N-deamidation ( Fig. 30B ) Correlation with various Tau-PET levels. In CSF samples containing high Tau-PET, both MTBR-tau243-256 and MTBR-tau243-256 species with @255N-deamidation increased dramatically.

[0401] Fig.31A-B shows MTBR-tau243-255 (SEQ ID NO: 19) in CSF ( Fig.31A ) and MTBR-tau 243-255 with @255N-deamidation (SEQ ID NO: 10) ( Fig.31B ) Correlation with various Tau-PET levels. MTBR-tau243-255 species with @255N-deamidation showed better specificity for low, medium and high Tau-PET compared to non-deamidated fragments.

[0402] Fig.32A -B shows MTBR-tau243-257K (SEQ ID NO: 18) in CSF ( Fig.32A ) and MTBR-tau 243-257K with @255N-desamidation (SEQ ID NO: 11) ( Fig.32B ) Correlation with various Tau-PET levels. MTBR-tau243-257K species with @255N-deamidation showed better specificity for low, medium and high Tau-PET compared to non-deamidated fragments.

[0403] Fig.33A -B shows MTBR-tau243-252D (SEQ ID NO: 13) in CSF ( Fig.33A ) and MTBR-tau 243-258S with @255N-deamidation (SEQ ID NO: 14) ( Fig.33B ) Correlation with various Tau-PET levels. MTBR-tau243-258S species with @255N-deamidation showed better specificity for low, medium and high Tau-PET compared to non-deamidated fragments.

[0404] Fig.34A -B shows MTBR-tau243-253L (SEQ ID NO: 12) in CSF ( Fig.34A ) and MTBR-tau 243-254K with @244-deamidation (SEQ ID NO: 14) ( Fig.34B ) and Tau-PET. Both categories leveled off from medium to high Tau-PET.

[0405] Compared with the CSF results, Fig.35A -B shows MTBR-tau243-253L (SEQ ID NO: 12) (FIG.35A) and MTBR-tau243-256V with @244-deamidation (SEQ ID NO: 20) in plasma ( Fig.35B) with various Tau-PET levels. The MTBR-tau243-254 species did not correspond to Tau-PET. In contrast, the MTBR-tau243-256V fragment correlated well with increasing Tau-PET levels.

[0406] Detection of tau peptide in plasma

[0407] The one-step immunoprecipitation method described herein was applied to plasma collected from a subject. Fig.36A Figures of MTBR-tau243-254 (SEQ ID NO: 21) and MTBR-TAU243-256V (SEQ ID NO: 15) are combined. Of note, 243-256V@255N-deamidation (SEQ ID NO: 16) was only detected for high Tau-PET and not for low and medium Tau-PET ( Fig.36B ). Fig.37A -F shows LC / MS data corresponding to the levels of MTBR-tau243-256V@255N-deamidation (SEQ ID NO: 16) as a function of Tau-PET state.

[0408] To detect deamidated fragments such as 243-256V@255N-deamid (SEQ ID 16), the immunoprecipitation method using ArgC was further modified. Fig.38 and 39 Two exemplary methods are shown. Fig.38 In the method shown, about 1 mL of plasma is collected and placed in a single tube or multiple tubes. The tube is placed in a centrifuge and centrifuged at 20,000G for 30 minutes. 600-800 μL of supernatant is collected. 50 μL of NP40 / guanidine / protease inhibitor and 60 μL of HJ32.11 antibody (50%, 1×) are added to the supernatant and incubated at room temperature for 2 hours. The sample is immunoprecipitated to remove the HJ32.11 antibody from the supernatant. The HJ32.11 antibody is washed and vortexed 4 times with TEABC buffer. 40 μL of ArgC endopeptidase and 4 μL of activation buffer containing DTT are added to the washed mixture and incubated at 37°C for 2 hours to digest the sample. Next, the mixture is purified by passing it through the OASIS-HLB purification system. The system is balanced with 0% acetonitrile. The digested sample is injected into the OASIS-HLB purification system. The digested sample is washed 3 times with 10% acetonitrile and eluted with 20% acetonitrile solution. The separated samples were collected and dried using speed-vacuum drying. The dried samples were resuspended and injected into the Eclipse TM In LC / MS.

[0409] exist Fig.39In the method shown, plasma (1800 μL) was mixed with 20 μL of a solution containing 0.05 fmol / μL of each of N-deamidated stable isotope labeled 243-256 (MTBR-tau243-dN) and 243-256 (MTBR-tau243) as internal standards. Then, the tau species were immunoprecipitated with antibody HJ32.11 at room temperature for 2 hours. All immunoprecipitated tau species were digested with Arg-C and desalted by OASIS-HLB (Waters). The resulting tau peptides were analyzed by a nanoAcquity ultra-high performance LC system (Waters) coupled to an Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operating in parallel reaction monitoring mode.

[0410] Fig.40A -C shows the comparison of 243-256V@255N-deamidated (SEQ ID NO: 16) obtained from plasma with MTBR-tau243 from CSF (using chemical extraction) ( Fig.40A ), Tau-PET and plasma 243-256V@255N-deamidation ( Fig.40B ) and Tau-PET and CSF MTBR-tau243 ( Fig.40C ). A modified immunoprecipitation method using ArgC digestion was able to detect 243-256V@255N-deamidation (SEQ ID NO: 16) in plasma. It was found that control subjects (amyloid negative) did not show any peak of 243-256V@255N-deamidation (SEQ ID NO: 16). This endogenous species is very specific for tau pathology, so if Fig.40C As shown, control participants obtained zero values. Notably, plasma and CSF 243-256V@255N-deamidation (SEQ ID NO: 16) were highly correlated with Tau-PET status ( Fig.40A -B).

[0411] A summary of endogenously cleaved peptides, their detection in CSF and / or plasma, and ability to stage Alzheimer's disease is provided in Table 19.

[0412] Plasma MTBR-tau243-256 (dN) for AD staging

[0413] Fig.41The amino acid sequence of the region near the 243-256V@255N-deamidated (SEQ ID NO: 16) peptide and how various anti-tau antibodies bind to this region are shown. This figure helps explain how using HJ32.11 and HJ34.8 in the assay in plasma provides meaningful information about the presence of tangles compared to CSF. Fig.42 A hypothesis is shown as to why plasma 243-256V@255N-deamidated (SEQ ID NO: 16) is dramatically increased in plasma (CDR≥1) after AD onset compared to MTBR-tau243-254 in CSF.

[0414] like Fig.43A As shown in Figure 2C, 243-256V@255N-deamidation in plasma indicates the Alzheimer's disease stage of the subject. Fig.43A CSF pT217 / T217 plateau was shown when the Clinical Dementia Rating (CDR) score was 0.5 or higher. Fig.43B CSF MTBR-tau243 was shown to increase steadily with increasing CDR scores. Notably, Figure 43C shows that plasma MTBR-tau243-256(dN) levels increased dramatically when the CDR score was greater than 0.5. When the CDR score was greater than or equal to 1, MTBR-tau243-256V@255N-deamidation levels increased dramatically.

[0415] Example 4

[0416] MTBR-tau 212-221 correlates with Tau-PET levels in plasma

[0417] Reference Fig.19 , Fig.19 Different MTBR-tau fragments isolated by HJ34.8 antibody beads in the first immunodepletion step and by anti-tau antibody HJ32.11 beads in the second immunoprecipitation step are shown. The very short MTBR-tau243 fragment showed affinity for the HJ32.11 antibody and exhibited high association with tau tangles. Notably, the MTBR-tau212-221 fragment also showed affinity for the HJ32.11 antibody.

[0418] This example shows that the MTBR-tau212-221 fragment is also highly associated with tau tangles. Fig.44 and Table 16 show that MTBR-tau212-221 was highly correlated with MTBR-tau243 in plasma samples obtained from the Knight ADRC cohort. Fig.45and Table 17 show that MTBR-tau212-221 was highly correlated with MTBR-tau243 in plasma samples obtained from the BioFINDER-2 cohort.

[0419] References

[0420] The entire disclosures of the following references, as well as any other references disclosed herein, are incorporated herein by reference.

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Claims

1. An isolated anti-MTBR tau antibody comprising: (a) L1, L2 and L3 from the light chain variable region of the sequence shown in SEQ ID NO:6, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:7; (b) L1, L2 and L3 from the light chain variable region sequence shown in SEQ ID NO: 2, and H1, H2 and H3 from the heavy chain variable region sequence shown in SEQ ID NO: 3; (c) L1, L2 and L3 from the light chain variable region of the sequence shown in SEQ ID NO:4, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:5; or (d) L1, L2 and L3 from the light chain variable region of the sequence shown in SEQ ID NO:8, and H1, H2 and H3 from the heavy chain variable region of the sequence shown in SEQ ID NO:

9.

2. The isolated anti-MTBR tau antibody of claim 1, comprising (a) a light chain variable region from the sequence shown in SEQ ID NO: 6, and a heavy chain variable region from the sequence shown in SEQ ID NO: 7; (b) a light chain variable region from the sequence shown in SEQ ID NO: 2, and a heavy chain variable region from the sequence shown in SEQ ID NO: 3; (c) a light chain variable region from the sequence shown in SEQ ID NO:4, and a heavy chain variable region from the sequence shown in SEQ ID NO:5; or (d) the light chain variable region from the sequence shown in SEQ ID NO:8, and the heavy chain variable region from the sequence shown in SEQ ID NO:

9.

3. The isolated antibody of claim 1, wherein the antibody is a monoclonal antibody or an antibody fragment. The isolated antibody according to claim 3 , wherein the antibody is a monoclonal antibody.

5. A method for detecting insoluble tau aggregates in a plasma sample, comprising: (a) purifying endogenous cleavage fragments of tau by contacting the plasma sample with an epitope binding agent that specifically binds to an epitope within amino acids 235-242 of SEQ ID NO: 1, without first contacting the endogenous cleavage fragments of tau with a protease in vitro; (b) contacting the purified endogenous cleavage fragment of tau with Arg-C endopeptidase to obtain a proteolytic MTBR-tau243 peptide comprising amino acids 243-254 of SEQ ID NO: 1; and (c) detecting the proteolytic MTBR-tau243 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or immunoassay, wherein detection of the proteolytic MTBR-tau243 peptide indicates insoluble tau aggregates in the sample. The method of claim 5 , wherein the purification comprises immunoprecipitation.

7. The method of claim 5, wherein the proteolytic MTBR-tau243 peptide comprises one or more of MTBR-tau243-254, MTBR-tau243-256V, MTBR-tau243-256V(dN) and MTBR-tau212-221.

8. The method of claim 5, wherein detecting the proteolytic MTBR-tau243 peptide comprises performing an immunoassay or liquid chromatography-mass spectrometry.

9. The method of claim 5, wherein the epitope binding agent is an anti-tau antibody comprising a light chain variable region from the sequence shown in SEQ ID NO:6 and a heavy chain variable region from the sequence shown in SEQ ID NO:

7.

10. The method of claim 9, wherein the anti-tau antibody is HJ32.

11.

11. The method of claim 5, wherein the solution comprising the proteolytic MTBR-tau243 peptide is desalted by solid phase extraction prior to step (c).

12. The method of claim 5, further comprising one or more of the following: detecting and quantifying one or more of amyloid β, N-terminal tau, mid-domain tau, post-translational modifications of tau, and ApoE isoforms in a biological or CSF sample.

13. The method according to claim 12, wherein quantifying amyloid β comprises quantifying Aβ42 / 40 value or quantifying post-translational modification of tau, wherein the post-translational modification of tau comprises one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111 and phosphorylated tau208.

14. A method for detecting insoluble tau aggregates in a cerebrospinal fluid (CSF) sample, comprising (a) affinity depleting the CSF sample by contacting the sample with an affinity depletion agent, the affinity depletion agent comprising one or more epitope binding agents that each bind to one of N-terminal tau, middle domain tau, and long MTBR tau, but not to an antigen within amino acids 235-256 of SEQ ID NO: 1, wherein the CSF sample comprises cleavage fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, middle domain tau, and long MTBR tau, and wherein the enriched sample is enriched for endogenous cleavage fragments of tau comprising amino acids 235-256 of SEQ ID NO: 1 (endogenous MTBR-tau243 peptide); (b) immunoprecipitating the enriched sample by contacting the enriched sample with an immunoprecipitant, the immunoprecipitant comprising an epitope binding agent that binds to an endogenous MTBR-tau243 peptide to obtain a purified sample; (c) contacting the endogenous MTBR-tau243 peptide in the purified sample with a protease to obtain a sample containing proteolytically hydrolyzed MTBR-tau243 peptide; and (d) detecting the proteolytic MTBR-tau243 peptide by performing liquid chromatography-mass spectrometry (LC / MS) or an immunoassay, wherein detection of the proteolytic MTBR-tau243 peptide is indicative of insoluble tau aggregates in the CSF sample.

15. The method of claim 14, wherein the sample comprising proteolytic MTBR-tau243 peptides is desalted prior to step (d).

16. The method of claim 14, wherein the affinity depleting agent comprises: an epitope binder that specifically binds to an epitope within amino acids 1 to 243 of SEQ ID NO: 1; a first epitope binder that specifically binds to an epitope within amino acids 1 to 103 of tau-441, and a second epitope binder that specifically binds to an epitope within amino acids 104 to 243 of SEQ ID NO: 1; or, a first epitope binder, a second epitope binder, and a third epitope binder that specifically binds to an epitope within amino acids 257-264 of SEQ ID NO:

1.

17. The method of claim 16, wherein the first epitope binding agent is HJ8.5, the second epitope binding agent is one or more of Tau1 and HJ8.7, and the third epitope binding agent is HJ34.

8.

18. The method of claim 14, wherein the affinity depleting agent comprises Tau1, HJ8.5, and HJ8.

7.

19. The method of claim 18, wherein the affinity depleting agent further comprises HJ34.

8.

20. The method of claim 17, wherein the immunoprecipitant comprises HJ32.

11.

21. The method of claim 14, wherein the proteolytically cleaved MTBR-tau243 peptide comprises a cleaved tau fragment, wherein the cleaved tau fragment comprises one or more of MTBR-tau243-254, MTBR-tau243-255(dN), MTBR-tau243-257K(dN), MTBR-tau243-256V, MTBR-tau243-256V(dN), MTBR-tau243-258S, and MTBR-tau212-221.

22. The method of claim 21, wherein the proteolytic MTBR-tau243 peptide comprises one or more of MTBR-tau243-256V and MTBR-tau243-256V(dN).

23. The method of claim 14, wherein the protease comprises trypsin.

24. The method of claim 14, wherein the CSF sample comprises an internal standard.

25. The method of claim 24, wherein the internal standard comprises 15 N-0N3R-tau and 15 N-2N4R-tau.

26. The method of claim 14, wherein the purified sample comprises an internal standard.

27. The method of claim 26, wherein the internal standard comprises 13 C 15 One or more of N-2N4R-tau.

28. The method of claim 14, further comprising detecting and / or quantifying amyloid beta, detecting and / or quantifying N-terminal tau, detecting and / or quantifying mid-domain tau, detecting and / or quantifying post-translational modifications of tau, or identifying ApoE isoforms in a biological or CSF sample.

29. The method of claim 28, wherein quantifying amyloid β comprises quantifying Aβ42 / 40 value and / or quantifying post-translational modification of tau, wherein the post-translational modification of tau comprises one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111, and phosphorylated tau208.

30. A method for detecting insoluble tau aggregates in a plasma sample, comprising (a) affinity depleting a plasma sample by contacting the sample with an affinity depleting agent comprising one or more epitope binding agents that each bind to one of N-terminal tau, middle domain tau, and long MTBR tau, but not to an antigen within amino acids 235-256 of SEQ ID NO: 1, wherein the plasma sample comprises cleavage fragments of tau, to obtain a depleted sample and an enriched sample, wherein the depleted sample comprises N-terminal tau, middle domain tau, and long MTBR tau, and wherein the enriched sample is enriched for endogenous cleavage fragments of tau comprising amino acids 235-254 of SEQ ID NO: 1 (endogenous MTBR-tau243 peptide); (b) immunoprecipitating the enriched sample by contacting the enriched sample with an immunoprecipitant, the immunoprecipitant comprising an epitope binding agent that binds to an endogenous MTBR-tau243 peptide to obtain a purified sample; (c) contacting the endogenous MTBR-tau243 peptide with a protease including Arg-C endopeptidase to obtain a sample comprising proteolytically hydrolyzed MTBR-tau243 peptide; and (d) detecting the proteolytic MTBR-tau243 peptides by performing liquid chromatography-mass spectrometry (LC / MS) or immunoassay, wherein detection of one or more of MTBR-tau243-256V, MTBR-tau243-256V(dN) and MTBR-tau212-221 indicates insoluble tau aggregates in the plasma sample.

31. The method of claim 30, wherein the sample comprising proteolytic MTBR-tau243 peptides is desalted prior to step (d).

32. The method of claim 30, wherein the affinity depleting agent comprises: an epitope binder that specifically binds to an epitope within amino acids 1 to 243 of SEQ ID NO: 1; a first epitope binder that specifically binds to an epitope within amino acids 1 to 103 of tau-441, and a second epitope binder that specifically binds to an epitope within amino acids 104 to 243 of SEQ ID NO: 1; or, a first epitope binder, a second epitope binder, and a third epitope binder that specifically binds to an epitope within amino acids 257-264 of SEQ ID NO:

1.

33. The method of claim 32, wherein the first epitope binding agent is HJ8.5, the second epitope binding agent is one or more of Tau1 and HJ8.7, and the third epitope binding agent is HJ34.

8.

34. The method of claim 30, wherein the affinity depleting agent comprises Tau1, HJ8.5, and HJ8.

7.

35. The method of claim 34, wherein the affinity depleting agent further comprises HJ34.

8.

36. The method of claim 30, wherein the immunoprecipitant comprises HJ32.

11.

37. The method of claim 30, wherein the plasma sample comprises an internal standard.

38. The method of claim 37, wherein the internal standard comprises 15 N-2N4R-tau.

39. The method of claim 30, wherein the purified sample comprises an internal standard.

40. The method of claim 39, wherein the internal standard comprises 13 N 15 N-2N4R-tau and 15 One or more of N-0N3R-tau.

41. The method of claim 30, further comprising detecting and / or quantifying amyloid beta, detecting and / or quantifying N-terminal tau, detecting and / or quantifying mid-domain tau, detecting and / or quantifying post-translational modifications of tau, or identifying ApoE isoforms in a biological or CSF sample.

42. The method of claim 41, wherein quantifying amyloid β comprises quantifying Aβ42 / 40 value and / or quantifying post-translational modification of tau, wherein the post-translational modification of tau comprises one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111, and phosphorylated tau208.

43. A method for detecting Alzheimer's disease (AD)-associated pathology in a subject, comprising detecting insoluble tau aggregates in a CSF or plasma sample according to the method of any one of claims 5-42, wherein the CSF or plasma sample is from the subject, and wherein detection of insoluble aggregates of tau indicates AD-associated pathology in the subject.

44. The method of claim 43, wherein the AD-related pathology is tau deposition in the subject's brain.

45. The method of claim 43 or 44, wherein the AD-related pathology is amyloid beta deposition in the brain or cerebral arteries of the subject.

46. ​​A method for detecting Alzheimer's disease (AD)-associated tau deposition in the brain of a subject, comprising detecting insoluble tau aggregates in a CSF or plasma sample according to the method of any one of claims 5-42, wherein the CSF or plasma sample is from the subject, and wherein detection of insoluble aggregates of tau indicates AD-associated tau deposition in the brain of the subject.

47. A method for diagnosing Alzheimer's disease in a subject, comprising (a) detecting insoluble tau aggregates in a CSF or plasma sample according to the method of any one of claims 5-42, wherein the CSF or plasma sample is from the subject; and (b) diagnosing Alzheimer's disease when the amount of proteolytic MTBR-tau243 peptide detected is about 1.5σ or more above the mean of a healthy control population, where σ is the standard deviation defined by a normal distribution measured in an amyloid-negative control population as measured by PET imaging and / or Aβ42 / 40 measurement in CSF.

48. A method of measuring progression of Alzheimer's disease (AD) in a subject, comprising: (a) detecting insoluble tau aggregates in a first and a second CSF or plasma sample according to the method of any one of claims 5-29; and (b) calculating the difference between the amount of proteolytic MTBR-tau243 peptide in the second sample and the first sample, wherein a statistically significant increase in the amount of proteolytic MTBR-tau243 peptide in the second sample compared to the first sample indicates progression of Alzheimer's disease in the subject.

49. The method of any one of claims 46-48, wherein the subject is amyloid negative.

50. The method of claim 49, wherein the subject does not have dementia.

51. The method of claim 49, wherein the subject suffers from dementia.

52. The method of any one of claims 46-48, wherein the subject is amyloid positive.

53. The method of claim 52, wherein the subject does not have dementia.

54. The method of claim 52, wherein the subject suffers from dementia.

55. The method of claim 49 or 52, wherein the subject has a Clinical Dementia Rating (CDR) score of 0.5 to 1.

0.

56. The method of claim 49 or 52, wherein the subject has a CDR score of >1.0 to 2.0 (moderate AD).

57. The method of claim 49 or 52, wherein the subject has a CDR score of >2.

0.

58. The method of any one of claims 46-57, further comprising quantifying amyloid beta, quantifying N-terminal tau, quantifying mid-domain tau, quantifying post-translational modifications of tau, or identifying ApoE isoforms in a biological or CSF sample.

59. The method of claim 58, wherein quantifying amyloid beta comprises quantifying Ab42 / 40 value, and quantifying post-translational modification of tau comprises quantifying one or more of phosphorylated tau217, phosphorylated tau205, phosphorylated tau181, phosphorylated tau153, phosphorylated tau111, and phosphorylated tau208.

60. A method for treating tauopathy in a subject in need thereof, comprising (a) detecting insoluble tau aggregates in a CSF or plasma sample according to the method of any one of claims 5-46, wherein the CSF or plasma sample is from the subject; and (b) administering to the subject a treatment that alters tau pathology, wherein the amount of proteolytically cleaved MTBR-tau243 peptide detected is about 1.5σ or more above the mean for a healthy control population, wherein σ is the standard deviation defined by a normal distribution measured in a control population that is free of clinical signs or symptoms of tauopathy and is amyloid negative as measured by PET imaging and / or Aβ42 / 40 measurement in CSF, and wherein the amount of proteolytically cleaved MTBR-tau243 peptide is indicative of tauopathy.

61. A method for treating tauopathy in a subject in need thereof, comprising administering to the subject a treatment that alters tau pathology, wherein the subject has been identified as having an amount of proteolytically cleaved MTBR-tau243 peptide detected according to the method described in any one of claims 5-46, wherein the amount is about 1.5σ or more higher than the mean for a healthy control population, wherein σ is the standard deviation defined by a normal distribution measured in a control population that is amyloid-negative in the absence of clinical signs or symptoms of tauopathy and as measured by PET imaging and / or Aβ42 / 40 measurements in CSF, and wherein the amount of proteolytically cleaved MTBR-tau243 peptide indicates tauopathy.

62. The method of claim 60 or 61, wherein the treatment alters or stabilizes the amount of proteolytically cleaved MTBR-tau243 peptide detected.

63. The method of any one of claims 60-62, wherein the treatment is selected from lencanezumab, donetumab, AADvac1, ACI-3024, ACI-35, APNmAb005, ASN51, AZP2006, BIIB076, BIIB080, BIIB113, berenezumab, dasatinib + quercetin, E2814, epothilone D, gonelizumab, JNJ-63733657, LMTM, LY3372689, Lu AF87908, MK-2214, NIO752, OLX-07010, PNT001, PRX005, RG7345, Rember TM, sirenetumab, TPI 287. Tigrexib, tipranezumab, zagteneizumab, anti-tau monoclonal antibodies, anti-tau antisense oligonucleotides, anti-tau small interfering RNA, tau production inhibitors, and live tau vaccines.

64. The method of claim 63, wherein the treatment is selected from anti-Aβ antibodies, anti-tau antibodies, anti-TREM2 antibodies, TREM2 agonists, γ-secretase inhibitors, β-secretase inhibitors, kinase inhibitors, phosphatase activators, vaccines, and tau protein aggregation inhibitors.

65. The method of claim 64, wherein the kinase inhibitor is an inhibitor of one thousand and one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5, or Fyn.

66. The method of claim 64, wherein the phosphatase activator increases the activity of protein phosphatase 2A.

67. The method of claim 64, wherein the vaccine is CAD106 or AF20513.

68. The method of claim 64, wherein the anti-A[beta] antibody is aducanumab or another anti-amyloid antibody that removes plaques.

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