Methods for detecting CSF tau species associated with stages and progression of Alzheimer's disease and uses thereof

Abstract

The present disclosure provides methods for quantifying and analyzing various CSF tau species and their use to measure pathological features and / or clinical symptoms of tauopathies, including determining time to Alzheimer's disease dementia, determining time from onset of dementia, staging Alzheimer's disease, guiding treatment decisions, and evaluating the clinical efficacy of certain therapeutic interventions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 140,230, filed January 21, 2021, U.S. Provisional Application No. 63 / 151,051, filed February 18, 2021, U.S. Provisional Application No. 63 / 170,185, filed April 2, 2021, U.S. Provisional Application No. 63 / 180,915, filed April 28, 2021, U.S. Provisional Application No. 63 / 187,697, filed May 12, 2021, and U.S. Provisional Application No. 63 / 213,006, filed June 21, 2021, each of which is incorporated by reference in its entirety herein.

[0002] government support This invention was made with government funding under awards AG046363 and AG032438 from the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing Reference This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy generated on January 20, 2022 is named "715616_ST25.txt" and is 8,936 bytes in size.

[0004] The present disclosure encompasses methods for quantifying and analyzing various CSF tau species and their use to measure pathological features and / or clinical symptoms of tauopathies, including primary tauopathies (e.g., MAPT, PSP, CBD) and secondary tauopathies (e.g., AD due to Aβ amyloidosis). [Background technology]

[0005] The microtubule-associated protein tau (MAPT or tau) plays an essential role in neuronal morphology and physiology. Tau has six distinct full-length protein isoforms and undergoes numerous potential post-translational modifications, including acetylation, glycosylation, and phosphorylation.

[0006] Accumulation of tau protein as insoluble aggregates in the brain is one of the hallmarks of Alzheimer's disease and other neurodegenerative disorders termed tauopathies. Intracellular tangles in the cerebral cortex are the defining pathological feature of Alzheimer's disease (AD) and correlate with the onset of clinical symptoms long after the appearance of extracellular amyloid-β (Aβ) plaques, which begin to appear up to 20 years before the onset of symptoms. Tau pathology propagates throughout brain regions and appears to spread by cell-to-cell propagation of specific pathological tau species in a prion-like manner, although the nature of these species (i.e., monomeric, oligomeric, and fibrillar species) and the process of spread are unknown.

[0007] In AD, soluble p-tau and non-phosphorylated tau are increased two-fold in the cerebrospinal fluid. These changes are proposed to reflect the effects of neuronal death (neurodegeneration) that passively releases tau and NFTs into the CSF. However, in other tauopathies with significant NFT pathology and neurodegeneration (e.g., progressive supranuclear palsy, frontotemporal lobar degeneration tau), the levels of soluble p-tau and total tau in the CSF are not increased. These observations suggest that Aβ may trigger processes that lead to AD-specific tauopathy, an idea that is supported by cell and animal models. This concept is further supported by the increased active production of soluble tau in the presence of amyloid plaques in humans.

[0008] Several mass spectrometry (MS) studies have suggested that the microtubule-binding region (MTBR) of tau is enriched in aggregates in AD brains. Furthermore, a series of cryo-electron microscopy (Cryo-EM) studies have demonstrated that the core structure of tau aggregates consists of subsegments of the MTBR domain, with specific conformations dependent on tauopathy. However, these studies used postmortem brain tissue, and little is known about the pathophysiology of the corresponding extracellular MTBR-containing tau species in biological samples such as CSF.

[0009] Although tau constitutes a hallmark of AD pathology and can be measured in aggregated or soluble forms, significant gaps remain in our understanding of how post-translational modifications and isoforms of this important neuronal protein lead to the development of NFTs and neurodegeneration in humans. For example, it is unclear what pathophysiological changes occur in tau during preclinical and clinical stages of AD. Therefore, it is unclear to what extent, if any, tau can be used to stage subjects prior to the onset of symptoms associated with AD to guide treatment decisions. Summary of the Invention [Means for solving the problem]

[0010] In one aspect, the disclosure encompasses a method of measuring time to onset of dementia in a subject by: (ai) measuring the phosphorylation occupancy of tau at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio, and optionally measuring MTBR-tau212, phosphorylation occupancy at residue T217 MTBR-tau243, MTBR-tau3R, or a combination thereof in a blood or CSF sample obtained from the subject; or (aii) measuring the phosphorylation occupancy of tau at residue T205, measuring the phosphorylation occupancy of tau at residue T217, and measuring MTBR-tau212 in a blood or CSF sample obtained from the subject, and (b) calculating the time to onset of dementia using the measurements of (ai) or (aii), where the time to onset of dementia is the number of years until clinical dementia assessment that is greater than zero.

[0011] In another aspect, the disclosure encompasses a method of measuring time to onset of dementia in a subject without cognitive or behavioral symptoms of Alzheimer's disease, the method generally comprising: (a) processing a blood or CSF sample from the subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species, wherein the first population of tau species is enriched in N-terminal tau and / or mid domain tau, and the second population of enriched tau species is enriched in MTBR-tau; (bi) measuring tau phosphorylation occupancy at residue T205 in the first population of tau species, and measuring the MTBR-tau299 / MTBR-tau354 ratio in the second population of tau species, and optionally measuring MTBR-tau212 in the second population of tau species; or (bii) measuring the phosphorylation occupancy of tau at residue T205 in a first population of tau species, and measuring the phosphorylation occupancy of tau at residue T217 in the first population of tau species, and measuring MTBR-tau212 in a second population of tau species; and (c) calculating the time to onset of dementia using the measures in (bi) or (bii), where the time to onset of dementia is the number of years to a clinical dementia assessment that is greater than zero. Includes.

[0012] In some embodiments, processing a blood sample or CSF sample from the subject to obtain a first population of enriched tau species and a depleted sample comprises contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope within the N-terminus of tau, or contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope within the mid domain of tau, or contacting the blood sample or CSF sample with a first epitope binding agent that specifically binds to an epitope within the N-terminus of tau and an epitope binding agent that specifically binds to an epitope within the mid domain of tau. and a second epitope binding agent that specifically binds to an epitope within the N-terminus of tau, wherein the first and second epitope binding agents are used sequentially or simultaneously; the epitope binding agent that specifically binds to an epitope within the N-terminus of tau can be HJ8.5 or another epitope binding agent that specifically binds to the same epitope as HJ8.5, and the epitope binding agent that specifically binds to an epitope within the mid domain of tau can be tau1 or another epitope binding agent that specifically binds to the same epitope as tau1.

[0013] In some embodiments, treating the depleted sample to obtain a second population of enriched tau species comprises performing a chemical extraction step to enrich for MTBR-tau species, which may include precipitating proteins of the depleted sample by mixing with an acid, which may be perchloric acid, and wherein the MTBR-tau species are present in the supernatant after removal of the precipitated proteins; or contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope within the MTBR of tau, wherein the epitope binding agent is selected from the group consisting of 77G7, RD3, RD4, UCB1017, or PT76 as described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, or PT76 as described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76, or 7G6, or another epitope-binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76, or 7G6.

[0014] In some embodiments, processing a blood sample or CSF sample from the subject to obtain a first population of enriched tau species and a depleted sample comprises contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope within the N-terminus of tau, or contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope within the mid domain of tau, or contacting the blood sample or CSF sample with a first epitope binding agent that specifically binds to an epitope within the N-terminus of tau and a second epitope binding agent that specifically binds to an epitope within the mid domain of tau, wherein the first and second epitope binding agents are used sequentially or simultaneously, and the epitope binding agent that specifically binds to an epitope within the N-terminus of tau is HJ8.5, or the same epitope as HJ8.5. and treating the depleted sample to obtain a second population of enriched tau species comprises performing a chemical extraction step to enrich for MTBR-tau species, which may include precipitating proteins of the depleted sample by mixing with an acid, which may be perchloric acid, and wherein the MTBR-tau species are present in the supernatant after removal of the precipitated proteins; or contacting the depleted sample with an epitope binding agent that specifically binds at least one epitope within the MTBR of tau, which epitope binding agent may be Vandermeeren's tau 1 epitope binding agent. et al., J Alzheimers Dis, 2018, 65:265-281, or 77G7, RD3, RD4, UCB1017, or PT76 as described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76, or 7G6, or another epitope binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76, or 7G6.

[0015] In each of the above embodiments, the subject may have zero CDRs.

[0016] In some embodiments, the calculated time to onset of dementia measurement can be used to stage the progression of a subject's disease, to stage lesions in the subject's brain, or to select a therapeutic or diagnostic agent for the subject.

[0017] In yet another aspect, the disclosure provides a method for treating a subject without cognitive or behavioral symptoms of Alzheimer's disease by administering to the subject a therapeutic or diagnostic agent, wherein the therapeutic agent reduces Aβ production, prevents or antagonizes Aβ aggregation, or increases brain Aβ clearance, and the therapeutic agent may be a gamma-secretase inhibitor, a beta-secretase inhibitor, passive immunotherapy (including but not limited to, anti-Aβ antibodies, anti-tau antibodies, or anti-ApoE antibodies), or active immunotherapy, or the therapeutic agent prevents or antagonizes tau aggregation, increases neurofibrillary tangle clearance, alters the phosphorylation pattern of tau, and the therapeutic agent may be a tau protein aggregation inhibitor, a kinase inhibitor, a phosphatase activator, passive immunotherapy (including but not limited to, anti-tau antibodies), or active immunotherapy.

[0018] In another aspect, the disclosure provides a method for measuring time since onset of dementia in a subject with cognitive or behavioral symptoms of Alzheimer's disease by: (ai) measuring the phosphorylation occupancy of tau at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio, and optionally measuring MTBR-tau212, phosphorylation occupancy at residue T217 MTBR-tau243, MTBR-tau3R, or a combination thereof in a blood or CSF sample obtained from the subject; or (aii) measuring the phosphorylation occupancy of tau at residue T205, measuring the phosphorylation occupancy of tau at residue T217, and measuring MTBR-tau212 in a blood or CSF sample obtained from the subject, and (b) calculating the time since onset of dementia using the measurements of (ai) or (aii), where the time since onset of dementia is the number of years since clinical dementia assessment that is greater than zero.

[0019] In yet another aspect, the disclosure provides a method for determining whether or not a subject has a tau phosphorylation occupancy at residue T205 in the first population of tau species, and whether or not the subject has a depleted sample, the method comprising: (a) processing a blood sample or a CSF sample from a subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species, the first population of tau species being enriched in N-terminal tau and / or mid domain tau, and the second population of enriched tau species being enriched in MTBR-tau; (bi) measuring the phosphorylation occupancy of tau at residue T205 in the first population of tau species, and measuring the MTBR-tau299 / MTBR-tau354 ratio in the second population of tau species, and optionally measuring the MTBR-tau phosphorylation occupancy in the second population of tau species. and (c) calculating time from onset of dementia using the measurements of (bi) or (bii), wherein time from onset of dementia is the number of years since clinical dementia assessment that is greater than zero.

[0020] In some embodiments, processing a blood sample or CSF sample from the subject to obtain a first population of enriched tau species and a depleted sample comprises contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope within the N-terminus of tau, or contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope within the mid domain of tau, or contacting the blood sample or CSF sample with a first epitope binding agent that specifically binds to an epitope within the N-terminus of tau and an epitope binding agent that specifically binds to an epitope within the mid domain of tau. and a second epitope binding agent that specifically binds to an epitope within the N-terminus of tau, wherein the first and second epitope binding agents are used sequentially or simultaneously; the epitope binding agent that specifically binds to an epitope within the N-terminus of tau can be HJ8.5 or another epitope binding agent that specifically binds to the same epitope as HJ8.5, and the epitope binding agent that specifically binds to an epitope within the mid domain of tau can be tau1 or another epitope binding agent that specifically binds to the same epitope as tau1.

[0021] In some embodiments, processing the depleted sample to obtain a second population of enriched tau species comprises: carrying out a chemical extraction step to enrich for MTBR-tau species, carrying out a chemical extraction step which may include precipitating proteins of the depleted sample by mixing with an acid, which may be perchloric acid, and in which the MTBR-tau species are present in the supernatant after removal of the precipitated proteins; or contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope within the MTBR of tau; The epitope binding agent may be 77G7, RD3, RD4, UCB1017, or PT76 as described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, or 7G6 as described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76, or 7G6, or other epitope binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76, or 7G6. Includes.

[0022] processing a blood sample or a CSF sample from the subject to obtain a first population of enriched tau species and a depleted sample; contacting the blood or CSF sample with an epitope binding agent that specifically binds to an epitope within the N-terminus of tau; contacting the blood or CSF sample with an epitope binding agent that specifically binds to an epitope within the mid domain of tau; contacting the blood or CSF sample with a first epitope-binding agent that specifically binds to an epitope within the N-terminus of tau and a second epitope-binding agent that specifically binds to an epitope within the mid domain of tau, wherein the first and second epitope-binding agents are used sequentially or simultaneously. Includes The epitope binding agent that specifically binds to an epitope within the N-terminus of tau may be HJ8.5 or another epitope binding agent that specifically binds to the same epitope as HJ8.5, and the epitope binding agent that specifically binds to an epitope within the mid domain of tau may be tau1 or another epitope binding agent that specifically binds to the same epitope as tau1; and treating the depleted sample to obtain a second population of enriched tau species comprises performing a chemical extraction step to enrich for MTBR-tau species, which may include precipitating proteins of the depleted sample by mixing with an acid, which may be perchloric acid, and wherein the MTBR-tau species are present in the supernatant after removal of the precipitated proteins; or contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope within the MTBR of tau, which epitope binding agent is a tau binding agent that specifically binds to an ... 2018, 65:265-281, or 7G6 as described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76 or 7G6, or other epitope binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76 or 7G6.

[0023] In some embodiments, the calculated time to onset of dementia measurement can be used to stage the progression of a subject's disease, to stage lesions in the subject's brain, or to select a therapeutic or diagnostic agent for the subject.

[0024] Thus, the present disclosure provides a method for treating a subject without cognitive or behavioral symptoms of Alzheimer's disease, comprising administering to the subject a therapeutic or diagnostic agent.

[0025] In another aspect, the disclosure provides a method for measuring change in cognition in a subject, comprising: (a) measuring phosphorylation occupancy of tau at one or more residues selected from T111, T153, T181, T217, and T231 in a blood or CSF sample obtained from the subject, and measuring at least one of MTBR-tau275, MTBR-tau299, and MTBR-3R in a blood or CSF sample obtained from the subject, and optionally measuring total tau in a blood or CSF sample obtained from the subject; and (b) using the measures of (a) to calculate a change in cognition, which change in cognition can be measured using measures such as delayed recall score from the International Shopping List Test, delayed logical recall score from the Wechsler Memory Scale-Revised, Digit Symbol Coding Score from the Wechsler Adult Intelligence Scale-Revised, or a combination of measures of cognitive impairment. The method includes calculating a change in cognition that is equivalent to the change in cognition as measured by a cognitive composite score consisting of the MMSE total score, the MMSE test total score, and the MMSE total score.

[0026] In some embodiments, measurements of changes in cognition are used to assess the effectiveness of a therapeutic agent.

[0027] Other aspects and iterations of the disclosure are described more fully below.

[0028] The application file contains at least one photograph executed in color. Copies of this patent application publication with color photograph will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0029] [Figure 1]Figure 1 is a schematic diagram of the longest human tau isoform (2N4R). The N-terminus (N-term), mid domain, MTBR and C-terminus (C-term) have been identified for this isoform and will vary in a predictable manner for other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R and 0N3R).

[0030] [Figure 2-1] FIG. 2A is a schematic diagram showing some methods of the present disclosure. One method is the method detailed in the blue box (right - tau chemical extraction). Another method is a combination of the red box (left - IP of N-terminal tau and mid domain tau) and the blue box (right - tau chemical extraction). FIG. 2B is a schematic diagram showing some methods of the present disclosure. One method is the method detailed in the blue box (right - tau chemical extraction). Another method is a combination of the red box (left - IP of N-terminal tau and mid domain tau) and the blue box (right - tau chemical extraction). [Figure 2-2] Same as above.

[0031] [Diagram 3] FIG. 3 is a schematic diagram illustrating the method of the present disclosure.

[0032] [Figure 4] FIG. 4 is a schematic diagram illustrating the method of the present disclosure.

[0033] [Diagram 5]FIG. 5 is an illustration showing that tau pathology evolves through distinct phases in Alzheimer's disease. Measuring four different soluble tau species and insoluble tau in a group of participants with definitive Alzheimer's disease mutations, we show that over a 35-year period (x-axis), tau-related changes unfold (y-axis) and differ based on the stage of the disease and other measurable biomarkers. It starts with the development of phosphorylation of fibrillar amyloid lesions at positions 217 (purple) and 181 (blue) and begins to increase. With increasing neuronal dysfunction (based on metabolic changes), phosphorylation at position 205 (green) begins to increase along with soluble tau (orange). Finally, with the onset of neurodegeneration (based on brain atrophy and cognitive decline), tau PET tangles (red) begin to develop, while phosphorylation at 217 and 181 begins to decrease. Taken together, this highlights the dynamic and divergent patterns of soluble and aggregated tau during disease and their close relationship with amyloid pathology.

[0034] [Figure 6] Figures 6A and 6B show that CSF MTBR-tau299, MTBR-tau306, and MTBR-tau354 exhibit distinct features across the Alzheimer's disease continuum, reflecting tangled status. Figure 6A is a diagram of 4R and 3R tau showing the regions of E2814 binding. Figure 6B shows the concentrations of MTBR-tau299, MTBR-tau306, and MTBR-tau-354 in relation to estimated years to symptom onset. Red dots: symptomatic mutation carriers (MC); orange dots: asymptomatic mutation carriers; blue dots: non-carriers (NC); red curves" LOESS curves for MC; blue curves: LOESS curves for NC.

[0035] [Figure 7]Figure 1 illustrates the secretion of MTBR-tau species into the CSF from brain tau aggregates across clinical stages of Alzheimer's disease. In the preclinical stages of AD, brain tau aggregates are immature, so that the three MTBR-tau species (MTBR-tau-243, MTBR-tau-299, and MTBR-tau-354) are secreted into the CSF at equal concentrations. However, as tau aggregates mature with disease progression and form a rigid core of MTBR-tau-354 (R4 domain), the MTBR-tau-354 species in the CSF is stabilized. The MTBR-tau-299 (R2-R3 domain) species binds to the rigid core structure at later symptom stages, whereas the MTBR-tau-243 species (upstream of the R1 domain) remains exposed, which allows for protease digestion and release into the CSF. Ultimately, an imbalance of these three species in the CSF is observed as a reflection of brain tau aggregate formation.

[0036] [Figure 8] Figure 8A, Figure 8B, and Figure 8C show the ROC and AUC of p-tau species in classifying amyloid PET status. Figure 8A shows pT111, pT153, pS208, and pT231 in mutation carriers. Figure 8B shows pT111, pT153, pS208, and pT231 in both mutation carriers and non-carriers. Figure 8C shows the area under the curve and 95% confidence intervals for the groups.

[0037] [Figure 9] Figure 9A, Figure 9B, and Figure 9C show the ROC and AUC of MTBR-tau species in classifying amyloid PET status. Figure 9A shows MTBR-tau212, MTBR-tau243, MTBR-tau260, and MTBR-tau275 in mutation carriers. Figure 9B shows MTBR-tau212, MTBR-tau243, MTBR-tau260, and MTBR-tau275 in both mutation carriers and non-carriers. Figure 9C shows the area under the curve and 95% confidence intervals for the groups.

[0038] [Figure 10]Figure 10A, Figure 10B, and Figure 10C show the ROC and AUC of MTBR-tau species in classifying amyloid PET status. Figure 10A shows MTBR-tau282, MTBR-tau299, MTBR-tau306, and MTBR-tau354 in mutation carriers. Figure 10B shows MTBR-tau282, MTBR-tau299, MTBR-tau306, and MTBR-tau354 in both mutation carriers and non-carriers. Figure 10C shows the area under the curve and 95% confidence intervals of the groups.

[0039] [Figure 11] Figure 11A, Figure 11B, and Figure 11C show the ROC and AUC of MTBR-tau species in classifying amyloid PET status. Figure 11A shows the MTBR-tau386, MTBR-tau396, MTBR-tau299 / MTBR-tau354 ratio, and MTBR-tau299 / MTBR-tau282 ratio of mutation carriers. Figure 11B shows the MTBR-tau386, MTBR-tau396, MTBR-tau299 / MTBR-tau354 ratio, and MTBR-tau299 / MTBR-tau282 ratio of both mutation carriers and non-carriers. Figure 11C shows the area under the curve and 95% confidence intervals of the groups.

[0040] [Figure 12-1]Figure 12A, Figure 12B, Figure 12C, Figure 12D, Figure 12E, and Figure 12F show box plots of pT111, pT153, and pS208 in mutation carriers. Figure 12A shows that the original value of the phosphorylation ratio pT111 / T111 increases with each PiB quartile (n=47 for Q1, 46 for Q2, 46 for Q3, and 47 for Q4). Figure 12B shows that the original value of the phosphorylation ratio pT153 / T153 increases with each PiB quartile (n=47 for Q1, 46 for Q2, 46 for Q3, and 47 for Q4). Figure 12C shows that the original value of the phosphorylation ratio pS208 / S208 increases with each PiB quartile (n=47 for Q1, 46 for Q2, 46 for Q3, and 47 for Q4). FIG. 12D shows that the standard values ​​of the phosphorylation ratio pT111 / T111 increase with each PiB quartile (n=47 for Q1, 46 for Q2, 46 for Q3, and 47 for Q4). FIG. 12E shows that the standard values ​​of the phosphorylation ratio pT153 / T153 increase with each PiB quartile (n=47 for Q1, 46 for Q2, 46 for Q3, and 47 for Q4). FIG. 12F shows that the standard values ​​of the phosphorylation ratio pS208 / S208 increase with each PiB quartile (n=47 for Q1, 46 for Q2, 46 for Q3, and 47 for Q4). P values ​​are from Wilcoxon rank sum test. [Figure 12-2] Same as above.

[0041] [Figure 13-1]Figures 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H, 13I, 13J, 13K, 13L, 13M, 13N, 13O, 13P, 13Q, 13R and 13S show the concentrations of various tau species in relation to the estimated years to onset of symptoms. FIG. 13A shows pT111 / T111; FIG. 13B shows pT153 / T153; FIG. 13C shows pS208 / S208; FIG. 13D shows pT231 / T231; FIG. 13E shows pT153; FIG. 13F shows pS208; FIG. 13G shows pT231; FIG. 13H shows pT231 / T231; FIG. 13I shows MTBR-tau212; FIG. 13J shows MTBR-tau243; FIG. 13K shows MTBR-tau260; Figure 13L shows MTBR-tau275; Figure 13M shows MTBR-tau282; Figure 13N shows MTBR-tau299; Figure 13O shows MTBR-tau3R; Figure 13P shows MTBR-tau354; Figure 13Q shows MTBR-tau386; Figure 13R shows MTBR-tau396; Figure 13S shows MTBR-tau299 / MTBR-tau282; Figure 13T shows the MTBR-tau299 / MTBR-tau354 ratio. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 13-5] Same as above. [Figure 13-6] Same as above. [Figure 13-7] Same as above. [Figure 13-8] Same as above. [Figure 13-9] Same as above. [Figure 13-10] Same as above. [Figure 13-11] Same as above. [Figure 13-12] Same as above.

[0042] [Figure 14-1]Figure 14A,B,C,D,E,F,G,H,I,J,K,L,M,N,O,P show the annual changes in p-tau and MTBR by DIAN EYO (annual changes were estimated from linear mixed-effects models). EYO cutoffs for cross-sectional area data: -19 for pT111 / T111, -22 for pT153 / T153, -22 for pS208 / S208; EYO cutoffs for percent change: -25 for pT111 / T111 and pT153 / T153, no cutoff for pS208 / S208. All longitudinal analyses were based on log-transformed pT111 / T111 and pS208 / S208, and square-root transformed pT153 / T153. Figure 14A shows pT111 / T111; Figure 14B shows pT153 / T153; Figure 14C shows pS208 / S208; Figure 14D shows pT231 / T231; Figure 14E shows MTBR-tau212; Figure 14F shows MTBR-tau243; Figure 14G shows MTBR-tau260; Figure 14H shows MTBR-tau275; Figure 14I shows MTBR-tau280; Figure 14J shows MTBR-tau290; Figure 14K shows MTBR-tau291; Figure 14K shows MTBR-tau292; Figure 14K shows MTBR-tau293; Figure 14K shows MTBR-tau294; Figure 14K shows MTBR-tau295; Figure 14K shows MTBR-tau296; Figure 14K shows MTBR-tau297; Figure 14K shows MTBR-tau298; Figure 14K shows MTBR-tau299; Figure 14K shows MTBR-tau299. Figure 14J shows MTBR-tau299; Figure 14K shows MTBR-tau3R; Figure 14L shows MTBR-tau299 / MTBR-tau282; Figure 14M shows MTBR-tau299 / MTBR-tau354; Figure 14N shows MTBR-tau354; Figure 14O shows MTBR-tau386; Figure 13P shows MTBR-tau396. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Figure 14-5] Same as above. [Figure 14-6] Same as above. [Figure 14-7] Same as above. [Figure 14-8] Same as above. [Figure 14-9] Same as above.

[0043] [Figure 15]FIG. 15 shows the association between baseline biomarkers and longitudinal rate of change of cognitive composites. [Figure 16-1] Figures 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H, 16I, 16J, 16K, 16L, 16M, 16N, 16O, and 16P show the associations in annual changes between various tau species and overall cognition. Figure 16A shows pT111 / T111; Figure 16B shows pT153 / T153; Figure 16C shows pS208 / S208; Figure 16D shows pT231 / T231; Figure 16E shows MTBR-tau212; Figure 16F shows MTBR-tau243; Figure 16G shows MTBR-tau260; Figure 16H shows MTBR-tau275; Figure 16I shows MTBR-tau280; Figure 16J shows MTBR-tau290; Figure 16J shows MTBR-tau291; Figure 16J shows MTBR-tau292; Figure 16J shows MTBR-tau293; Figure 16J shows MTBR-tau294; Figure 16J shows MTBR-tau295; Figure 16J shows MTBR-tau296; Figure 16J shows MTBR-tau297; Figure 16J shows MTBR-tau298; Figure 16J shows MTBR-tau29 ... pT153 / T153 and MTBR3R have a higher correlation in annual change than other tau species. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above. [Figure 16-5] Same as above. [Figure 16-6] Same as above.

[0044] [Figure 17-1] Figure 7A and Figure 17B show the correlation of phosphorylation occupancy at various sites of tau phosphorylation in mutation carriers (asymptomatic and symptomatic) at baseline and annual change. Figure 17A shows the correlation of pT111 / T111, pT153 / T153 and pS208 / 208 with pT217 / 217. Figure 17B shows the correlation of pT111 / T111, pT153 / T153 and pS208 / 208 with pT205 / 205. [Figure 17-2]Same as above.

[0045] [Figure 18-1] 18A, 18B, 18C, 18D, 18E, 18F, 18G, 18H, 18I, 18J, and 18K show heatmaps of correlations at baseline and annual rate of change for mutation carriers (symptomatic and asymptomatic) and non-carriers. FIG. 18A shows a heatmap of correlations at baseline for MC. All MTBR species and total tau are clustered together (several small clusters). Ratios from the MTBR dataset cluster with other tau species but not with MTBR tau species. pS202 / S202 is not associated with other tau species, i.e., TPPSS (specifying the region of total tau). FIG. 18B shows a heatmap of correlations at baseline using all MC. FIG. 18C shows a heatmap of correlations at baseline using asymptomatic MC. FIG. 18D shows a heatmap of correlations at baseline using symptomatic MC. FIG. 18E shows a heatmap of correlations at baseline using non-carriers. FIG. 18F shows a heatmap that represents the correlation between the annual rates of change of two markers for mutation carriers by color. FIG. 18G shows a heatmap that represents the correlation between the annual rates of change of two markers for asymptomatic mutation carriers by color. FIG. 18H shows a heatmap that represents the correlation between the annual rates of change of two markers for symptomatic mutation carriers by color. FIG. 18I shows a heatmap that represents the absolute value of the correlation between the annual rates of change of two markers for mutation carriers by color. FIG. 18J shows a heatmap that represents the absolute value of the correlation between the annual rates of change of two markers for asymptomatic mutation carriers by color. FIG. 18K shows a heatmap that represents the absolute value of the correlation between the annual rates of change of two markers for symptomatic mutation carriers by color. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above. [Figure 18-6] Same as above. [Figure 18-7] Same as above. [Figure 18-8] Same as above. [Figure 18-9] Same as above. [Figure 18-10] Same as above. [Figure 18-11] Same as above.

[0046] [Figure 19] Figure 19A, Figure 19B, and Figure 19C show the prediction of DIAN EYO in all mutation carriers, asymptomatic mutation carriers, and symptomatic mutation carriers. Semipartial R2 indicates that when VAR1 is included in the model, the R2 of the model is added by x. The square of the semipartial correlation is a measure of the incremental value of R2, and therefore indicates variable importance. The semipartial R2 does not sum to R2 because the total variability of the dependent variable also constitutes a part attributable to the correlation between the independent variables. Figure 19A shows the prediction of DIAN EYO for pT205 / T205, MTBR-tau212, and MTBR-tau299 / MTBR-tau354 in all mutation carriers. Figure 19B shows the prediction of DIAN EYO for pT205 / T205, pT217 / T217, and MTBR-tau212 in asymptomatic mutation carriers. FIG. 19C shows DIAN EYO prediction of pT205 / T205 in symptomatic mutation carriers.

[0047] [Figure 20] Figure 20A and Figure 20B show the tau species abnormality rate for each 5EYO interval.The 95th percentile of each biomarker of NC is used as the threshold for defining each biomarker of MC as normal and abnormal.Figure 20A shows the tau species abnormality rate for each 5EYO interval.Figure 20B is a line graph showing the tau species abnormality rate for each 5EYO interval.

[0048] [Figure 21-1] Figure 21A shows a comparison of effect sizes for annualized percent change (mean to standard deviation ratio-MSR). Figure 21B shows a comparison of effect sizes for annualized percent change (mean to standard deviation ratio-MSR). aMC and baseline EYO >=-25. [Figure 21-2] Same as above.

[0049] [Figure 22] Figures 22A and 22B show that changes in MTBR-tau299 and MTBR-tau354 are affected at the onset of AD (CDR=1). Figure 22A shows MTBR-tau299. Figure 22B shows MTBR-tau534.

[0050] [Diagram 23] FIG. 23 shows that the MTBR-tau299 / MTBR-tau354 ratio increases the discriminatory power of AD staging.

[0051] [Figure 24] Figures 24A and 24B show the performance of CSFMTBR-299 / 354 ratio to predict cognitive scores across the AD continuum. Figure 24A shows MTBR-299 / 354 and CDR-SB. Figure 24B shows MTBR-299 / 354 and MMSE.

[0052] [Diagram 25] FIG. 25 shows a summary table of MTBR-Tau versus cognitive scores.

[0053] [Figure 26] Figure 26A shows the correlation between the MTBR-tau 299 / 354 ratio and pT217% occupancy. Figure 26B shows that pT217% correlates well with E2814-associated MTBR-tau 299 / 354, which summarizes "early stage tau pathology" → pT217% can be used as a surrogate efficacy marker for E2814 clinical trials.

[0054] [Figure 27-1]Figure 27A is a schematic diagram of the tau-derived tryptic peptides (gray bars) quantified and discussed in Figures 27B and 27C. Figures 27B and 27C are graphs showing that brain MTBR tau species, including MTBR tau-243, 299 and 354, are enriched in aggregated Alzheimer's disease brain insoluble extracts compared to control brain extracts, confirming that MTBR tau is specifically deposited in Alzheimer's disease brains. Graphs show (Figure 18B) enrichment profiles of tau peptides from control and Alzheimer's disease brains (n=2, 6-8 brain region samples / group in the discovery cohort) and (Figure 18C) control (amyloid negative, n=8), very mild-moderate Alzheimer's disease (AD) (amyloid positive, CDR=0.5-2, n=5), and severe AD brains (amyloid positive, CDR=3, n=7) (total n=20 in the validation cohort). For internal normalization, the relative abundance of tau peptides was quantified relative to the mid domain (residues 181-190) peptide. Species containing the upstream region of the microtubule-binding region (MTBR) domain (residues 243–254, MTBR-tau-243) and repeat regions 2 (R2)–R3 and R4 (residues 299–317, MTBR-tau-299 and 354–369, MTBR-tau-354, respectively) were highly enriched in the insoluble fraction of Alzheimer's disease brains compared to controls and were specifically enriched by clinical stage of disease progression as measured by CDR. MTBR-tau-299 and MTBR-tau-354 are located inside the filament core, whereas MTBR-tau-243 is located outside the core of Alzheimer's disease aggregates (Fitzpatrick et al., 2017). Of note, residues 195–209 were decreased in Alzheimer's disease brains, which may be due to their hyperphosphorylation. Data are presented as box plots with Tukey's method describing median, interquartiles, minimum, maximum and individual outlier points. Statistical tests of significance: ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. [Figure 27-2] Same as above.

[0055] [Figure 28-1]Figure 28A is a schematic diagram of tau-derived tryptic peptides (gray bars) quantified in Example 3 and further discussed in Figures 19B and 19C, and the general binding sites of antibodies HJ8.5 and tau1. Figure 28B is a graph showing tau profiles in control human CSF. Tau peptides in control human CSF from a cross-sectional cohort of amyloid-negative and CDR=0 patients (n=30) were quantified by tau1 / HJ8.5 immunoprecipitation focusing on the N-terminal to mid domain of tau. To quantify species containing the microtubule-binding region (MTBR) and C-terminal regions, post-immunoprecipitation CSF samples were chemically extracted and analyzed sequentially. Using the tau1 / HJ8.5 immunoprecipitation method (blue circles), peptide recovery dropped dramatically after residue 222; therefore, this method quantified only N-terminal to mid-domain tau (residues 6-23 to 243-254) peptides (Sato et al., 2018). In contrast, chemical extraction of CSF after immunoprecipitation (red squares) allowed quantification of the entire region of tau, including the MTBR to C-terminal region, at concentrations between 0.4 and 7 ng / mL. Data are expressed as mean values. [Figure 28-2] Same as above.

[0056] [Figure 29-1]Figure 29A, Figure 29B, and Figure 29C show that CSF MTBR-tau-243, 299, and 354 species exhibit distinct characteristics across the Alzheimer's disease continuum, reflecting tangled states. Figure 29A: MTBR-tau-243 concentration, Figure 29B: MTBR-tau-299 concentration, and Figure 29C: MTBR-tau-354 concentration. Amyloid-negative CDR=0 (n=30), amyloid-positive CDR=0 (n=18), amyloid-positive CDR=0.5 (n=28), amyloid-positive CDR≧1 (n=12), and amyloid-negative CDR≧0.5 (n=12). MTBR-tau-243 showed a continuous increase with AD progression through all clinical stages. MTBR-Tau-299 and 354 concentrations similarly increased until very mild AD stages (amyloid positive and CDR=0.5) and then saturated (MTBR-Tau-299) or declined (MTBR-Tau-354) at CDR≧1. ****p<0.001, ***p<0.001, **p<0.01, *p<0.05. NS=not significant. [Figure 29-2] Same as above. [Figure 29-3] Same as above.

[0057] [Figure 30-1]Figure 30A, Figure 30B, Figure 30C, Figure 30D show that CSF MTBR-tau-243 is most highly correlated with tau PET measurements across all tau species, including p-tau 217, suggesting that CSF MTBR-tau-243 is the most promising biomarker for recapitulating tau pathology. Correlation between tau PET (AV-1451) SUVR and Figure 30A MTBR-tau-243, Figure 30B MTBR-tau-299, Figure 30C p-tau 217 concentrations, and Figure 30D p-tau 217 phosphorylation occupancy in CSF (control n=15 and Alzheimer's disease (AD) n=20 from the tau PET cohort). Open circles: control, closed squares: AD. MTBR-tau-243 showed the most significant correlation with tau PET SUVR (Spearman r=0.7588, p<0.0001), whereas MTBR-tau-299, p-tau217 concentration and p-tau217 phosphorylation occupancy showed moderate correlations (Spearman r=0.4584, 0.5478, and 0.5555, respectively), with saturation observed in high tau PET samples. [Figure 30-2] Same as above.

[0058] [Diagram 31] FIG. 31 shows MTBR-tau243 versus MTBR-tau212 by chemical extraction "after tau1 / HJ8.1-IP," where CX but not E2814-IP results in a stage-wise increase for MTBR-tau212.

[0059] [Diagram 32] FIG. 32 shows MTBR-tau243 versus MTBR-tau212 by chemical extraction "after tau1 / HJ8.1-IP," where for MTBR-tau212, CX but not E2814-IP results in a stage-wise increase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Tau protein aggregation into neurofibrillary tangles in the central nervous system contributes to the pathogenesis of certain neurodegenerative disorders, including Alzheimer's disease (AD). Although the mechanism of tau destabilization is still not fully understood, tau protein has been found to be hyperphosphorylated in tau aggregates. Furthermore, it has been suggested that aggregates of the microtubule-binding region (MTBR) of tau are enriched in AD brains. However, little is known about the pathophysiology of the corresponding extracellular p-tau and MTBR-containing tau species throughout the progression of AD. Although multiple tau peptides are present in blood and CSF, the very low abundance of these polypeptides has hindered the detection and quantification of tau species in these biological samples.

[0061] Applicants have discovered that certain methods of quantifying tau (e.g., phosphorylation at specific amino acid residues and / or MTBR tau) can be used to track the course of AD from preclinical asymptomatic stages to symptomatic stages. Given the vast variation in tau isoforms, post-translational modifications, abundance and solubility, it has been difficult to use tau species to stage controls and guide treatment decisions before the onset of symptoms associated with Alzheimer's disease. However, Applicants have identified methods to quantify specific combinations of tau species that are particularly useful for identifying the number of years between the onset of AD dementia and the onset of certain pathophysiological changes.

[0062] The methods disclosed herein utilize a unique combination of processing steps that convert biological samples into samples suitable for quantifying various tau species. For example, in some methods disclosed herein, the processing steps enrich for multiple tau proteins while depleting a specific protein. In other methods disclosed herein, the processing steps enrich for multiple MTBR tau proteins while depleting a specific protein. Certain methods disclosed herein are particularly suitable for quantifying mid domain-independent MTBR tau species. Also described herein is the use of mid domain-independent MTBR tau species and tau phosphorylation at certain amino acid residues to measure clinical signs and symptoms of tauopathy, diagnose tauopathy, and directly treat tauopathy. These and other aspects and iterations of the invention are described more fully below.

[0063] I. Definition In order to make the present invention easier to understand, certain terms are defined first.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates.Many methods and materials similar to, modified or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, and preferred materials and methods are described herein.In describing and claiming the present invention, the following terms are used according to the definitions set forth below.

[0064] The term "about" as used herein refers to the variation of quantity for any quantifiable variable, including but not limited to mass, volume, time, distance and amount, which may occur, for example, through typical measuring techniques and instruments. Furthermore, given the solid and liquid manipulation procedures actually used, there are a certain number of inadvertent errors and variations that may occur through differences in the manufacture, source or purity of the components used to make the composition or carry out the method, etc. The term "about" also encompasses these variations, which may be up to ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether or not modified by the term "about", the claims include the equivalents for the quantities.

[0065] As used herein, antibody refers to a complete antibody, i.e., composed of two heavy chains and two light chains, as understood in the art, or any antibody-like molecule having an antigen-binding region, including, but not limited to, fragments such as Fab', Fab, F(ab')2, single domain antibodies, Fv and single chain antibodies Fv. The term antibody also refers to polyclonal antibodies, monoclonal antibodies, chimeric antibodies and humanized antibodies. Techniques for preparing and using various antibody-based constructs and fragments are known in the art. Means for preparing and characterizing antibodies are also known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, which is incorporated herein by reference in its entirety).

[0066] As used herein, the term "aptamer" refers to a polynucleotide, generally RNA or DNA, that has useful biological activity in terms of biochemical activity, molecular recognition or binding properties. Usually, aptamers have molecular activity, such as binding to a target molecule at a specific epitope (region). It is generally accepted that aptamers are specific in binding to polypeptides and can be synthesized and / or identified by in vitro evolution methods. Means for preparing and characterizing aptamers are known in the art, including in vitro evolution methods. See, for example, US 7,939,313, which is incorporated herein by reference in its entirety.

[0067] The term "Aβ" refers to a peptide derived from a region in the carboxy terminus of a larger protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many different forms of Aβ that can be toxic, and although Aβ peptides are typically 37-43 amino acid sequences long, they can have truncations and modifications that change their overall size. They can be found inside or outside the cell, in monomeric, oligomeric and aggregate forms, in soluble and insoluble compartments, and can be complexed with other proteins or molecules. The deleterious or toxic effects of Aβ can result from any or all of the above forms, as well as others not specifically mentioned. For example, two such Aβ isoforms include Aβ40 and Aβ42, with the Aβ42 isoform being particularly fibrillogenic or insoluble and associated with disease states. The term "Aβ" typically refers to multiple Aβ species without distinction between the individual Aβ species. Specific Aβ species are identified by the size of the peptide, e.g., Aβ42, Aβ40, Aβ38, etc.

[0068] As used herein, the term "Aβ42 / Aβ40 value" refers to the ratio of the amount of Aβ42 in a sample obtained from a subject compared to the amount of Aβ40 in the same sample.

[0069] "Aβ amyloidosis" is defined as clinically abnormal Aβ deposits in the brain. Subjects who are determined to have Aβ amyloidosis are referred to herein as "amyloid positive", while subjects who are determined not to have Aβ amyloidosis are referred to herein as "amyloid negative". Accepted indicators of Aβ amyloidosis exist in the art. At the time of this disclosure, Aβ amyloidosis is typically measured directly by amyloid imaging (e.g., PiB PET, fluorbetapir, or other imaging methods known in the art) or indirectly by reducing cerebrospinal fluid (CSF) Aβ42 or reducing CSF Aβ42 / 40 ratio. [11C]PIB-PET images with a mean cortical binding potential score >0.18 are indicative of Aβ amyloidosis, as is a cerebrospinal fluid (CSF) Aβ42 concentration of approximately 1 ng / ml by immunoprecipitation and mass spectrometry (IP / MS). Alternatively, a cutoff ratio of CSF Aβ42 / 40 that maximizes the predictive accuracy of amyloid positivity as determined by PIB-PET can be used. Values ​​such as these, or others known in the art and / or used in the examples, can 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 incorporated herein by reference in its entirety. Subjects with Aβ amyloidosis may or may not be symptomatic, and symptomatic subjects may or may not meet clinical criteria for a disease associated with Aβ amyloidosis.Non-limiting examples of symptoms associated with Aβ amyloidosis can include cognitive dysfunction, behavioral changes, language dysfunction, emotional dysregulation, seizures, dementia, and nervous system structure or function disorders.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 have an increased risk of developing diseases associated with Aβ amyloidosis.

[0070] "Clinical signs of Aβ amyloidosis" refers to the measure of Aβ deposition known in the art.Clinical signs of Aβ amyloidosis can include, but are not limited to, Aβ deposition identified by amyloid imaging (e.g., PiB PET, fluorbetapir, or other imaging methods known in the art) or by reduction in cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio.See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, and Fagan AM et al. Ann Neurol 59(3) 2006, each of which is incorporated herein by reference in its entirety. As described in U.S. Patent Nos. 14 / 366,831, 14 / 523,148 and 14 / 747,453, each of which is incorporated by reference in its entirety, clinical indicia of Aβ amyloidosis can also include measurements of Aβ metabolism, particularly Aβ42 metabolism, either alone or compared to measurements of the metabolism of other Aβ mutants (e.g., Aβ37, Aβ38, Aβ39, Aβ40 and / or total Aβ). Further methods are described in Albert et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 170-179; McKhann et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 263-269; and Sperling et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 280-292, each of which is incorporated herein by reference in its entirety.Importantly, subjects with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition.Subjects with clinical signs of Aβ amyloidosis also have an increased risk of developing diseases associated with Aβ amyloidosis.

[0071] "Candidate for amyloid imaging" refers to a subject identified by a clinician as an individual for whom amyloid imaging is clinically warranted. 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 symptoms associated with Aβ plaques, one or more symptoms associated with CAA, or a combination thereof. A clinician may recommend amyloid imaging for such a subject to direct their clinical care. As another non-limiting example, a candidate for amyloid imaging may be a potential participant (either a control subject or a test subject) in a clinical trial for a disease associated with Aβ amyloidosis.

[0072] "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 aggregates of regularly arranged fibers called amyloid fibrils. Representative Aβ plaque-associated symptoms can include, but are not limited to, neuronal degeneration, cognitive impairment, memory impairment, behavioral changes, emotional dysregulation, seizures, impaired nervous system structure or function, and increased risk of developing or worsening Alzheimer's disease or CAA. Neuronal degeneration can include changes in neuronal structure (including molecular changes, e.g., intracellular accumulation of toxic proteins, protein aggregation, etc., and macro-level changes, e.g., changes in axon or dendrite shape or length, changes in myelin sheath composition, loss of myelin sheath, etc.), changes in neuronal function, loss of neuronal function, neuronal death, or any combination thereof. Cognitive impairment can include, but is not limited to, difficulties with memory, attention, concentration, language, reasoning, creativity, executive function, planning, and organization. Behavioral changes may include, but are not limited to, physical or verbal aggression, impulsivity, reduced inhibitions, apathy, reduced spontaneity, personality changes, alcohol, tobacco or drug abuse, and other addiction-related behaviors. Emotional dysregulation may include, but is not limited to, depression, anxiety, mania, irritability and affective incontinence. Seizures may include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic psychogenic seizures. Disorders of nervous system structure or function may include, but are not limited to, hydrocephalus, parkinsonism, sleep disorders, psychosis, disorders of posture and coordination. This may include movement disorders such as monoparesis, hemiparesis, quadriparesis, ataxia, ballismus and tremor. This may also include sensory loss or dysfunction, including smell, touch, taste, vision, and hearing. Additionally, this can include autonomic nervous system disorders such as bowel and bladder dysfunction, sexual dysfunction, dysregulation of blood pressure and temperature.Finally, this can include hormonal disorders resulting from dysfunction of the hypothalamus and pituitary gland, such as deficiencies and dysregulation of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin and countless other hormones and regulators.

[0073] 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 waiting for medical care or treatment, may be undergoing medical care or treatment, or may have undergone medical care or treatment.

[0074] As used herein, the terms "control population," "normal population," or samples from "healthy" subjects refer to a subject or group of subjects who have been clinically determined to not have a tauopathy or Aβ amyloidosis, or a clinical disease associated with Aβ amyloidosis (including but not limited to Alzheimer's disease) based on qualitative or quantitative test results. "Normal" subjects are usually approximately the same age as the individual being evaluated, including but not limited to subjects of the same age and within a 5-10 year range.

[0075] As used herein, the term "blood sample" refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. The blood sample can be whole blood, plasma or serum, although plasma is typically preferred.

[0076] The term "isoform" as used herein refers to any of several different forms of the same protein variant that arise due to alternative splicing of the mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein, genetic mutation and somatic recombination. The terms "isoform" and "variant" are used interchangeably.

[0077] The term "tau" refers to multiple isoforms encoded by the gene MAPT (or its homologues), as well as species that are C-terminally truncated in vivo, N-terminally truncated in vivo, post-translationally modified in vivo, or any combination thereof.As used herein, the terms "tau", "tau protein" and "tau species" can be used interchangeably.In many animals, including but not limited to humans, non-human primates, rodents, fish, cows, frogs, goats and chickens, tau is encoded by the gene MAPT.In animals where the gene is not identified as MAPT, homologues can be identified by methods well known in the art.

[0078] In humans, there are six isoforms of tau, generated by alternative splicing of exons 2, 3 and 10 of MAPT. These isoforms range from 352 to 441 amino acids in length. Exons 2 and 3 each encode a 29 amino acid insert at the N-terminus (referred to as N), and full-length human tau isoforms may have both inserts (2N), one insert (1N), or no inserts (0N). All full-length human tau isoforms also have three repeats of the microtubule-binding domain (referred to as R). Inclusion of exon 10 at the C-terminus results in the inclusion of a fourth microtubule-binding domain encoded by exon 10. Thus, full-length human tau isoforms may consist of four repeats of the microtubule-binding domain (including exon 10: R1, R2, R3 and R4) or three repeats of the microtubule-binding domain (excluding exon 10: R1, R3 and R4). Human tau may or may not be post-translationally modified. For example, it is known in the art that tau can be phosphorylated, ubiquitinated, glycosylated and glycated.Human tau can also be proteolytically processed or not in vivo at C-terminus, N-terminus, or C-terminus and N-terminus.Thus, the term "human tau" includes 2N3R, 2N4R, 1N3R, 1N4R, 0N3R, and 0N4R isoforms, as well as species that are truncated at C-terminus in vivo, species that are truncated at N-terminus in vivo, species that are post-translationally modified in vivo, or any combination thereof.Alternative splicing of the gene encoding tau occurs in other animals as well.

[0079] 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 as SEQ ID NO: 1. The N-terminus (N-terminus), mid domain, MTBR, and C-terminus (C-terminus) of this isoform are identified in FIG. 1. These regions vary in predictable ways in other tau isoforms (e.g., 2N3R, 1NR4, 1N3R, 0N4R, and 0N3R). Thus, once an amino acid position is identified relative to tau-441, one skilled in the art will be able to determine the corresponding amino acid position of other isoforms. Unless otherwise indicated, the numbering of amino acid residues used in this disclosure is based on tau-441 (e.g., T217 is the threonine residue at position 217 of tau-441).

[0080] As used herein, the term "N-terminal tau" refers to a tau protein, or multiple tau proteins, that includes two or more amino acids at the N-terminus of tau (such as, for example, amino acids 1-103 of tau-441).

[0081] As used herein, the term "mid domain tau" refers to a tau protein, or multiple tau proteins, that includes two or more amino acids of the mid domain of tau (such as, for example, amino acids 104-243 of tau-441).

[0082] As used herein, the term "MTBR tau" refers to a tau protein, or multiple tau proteins, that contain two or more amino acids of the microtubule-binding region (MTBR) of tau (e.g., amino acids 244-368 of tau-441).

[0083] As used herein, the term "C-terminal tau" refers to a tau protein, or multiple tau proteins, that includes two or more amino acids at the C-terminus of tau (such as, for example, amino acids 369 to 441 of tau-441).

[0084] "Proteolytic peptides of tau" refer to peptide fragments of tau protein produced by in vitro proteolytic cleavage. "Tau tryptic peptides" refer to peptide fragments of tau protein produced by in vitro cleavage with trypsin. Tryptic peptides of tau may be referred to herein by their first four amino acids. For example, "LQTA" (first four amino acids of SEQ ID NO:3) refers to the tryptic peptide LQTAPVPMPDLK (SEQ ID NO:3). Non-limiting examples of other tryptic peptides identified by their first four amino acids include IGST (SEQ ID NO:2), VQII (SEQ ID NO:4), LDLS (SEQ ID NO:5), HVPG (SEQ ID NO:6), IGSL (SEQ ID NO:7), VQIV (SEQ ID NO:9), and TPPS (SEQ ID NO:10).

[0085] Diseases associated with tau deposition in the brain are referred to herein as "tauopathies." The term "tau deposition" includes all forms of pathological tau deposition, including, but not limited to, tau aggregates in neurofibrillary tangles, neuropil threads, and dystrophic neurites. Tauopathies known in the art include, but are not limited to, progressive supranuclear palsy, dementia pugilistica, chronic traumatic encephalopathy, frontotemporal dementia and parkinsonism linked to chromosome 17, Ritiko-Bodig disease, Parkinson's dementia of Guam, neurofibrillary tangle senile dementia, ganglioneuroma and neurocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Haravorden-Spatz disease, lipofuscinosis, Pick's disease, corticobasal degeneration (CBD), argyrophilic grain disease (AGD), frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), and frontotemporal dementia (FTD).

[0086] Tauopathies are classified according to the predominance of tau isoforms found in pathological tau deposits. Tauopathies with tau deposits predominantly composed of tau with three MTBRs are referred to as "3R tauopathies". Pick's disease is a non-limiting example of a 3R tauopathy. For clarity, the pathological tau deposits of some 3R tauopathies may be a mixture of 3R and 4R tau isoforms, with the 3R isoforms predominating. It is believed that intracellular neurofibrillary tangles (i.e., tau deposits) in the brains of Alzheimer's disease patients generally contain approximately equal amounts of 3R and 4R isoforms. Tauopathies with tau deposits predominantly composed of tau with four MTBRs are referred to as "4R tauopathies". PSP, CBD, and AGD are non-limiting examples of 4R tauopathies, as are some forms of FTLD. Notably, pathological tau deposits in the brain of some subjects with genetically confirmed FTLD cases, such as some V334M and R406W mutation carriers, display a mixture of 3R and 4R isoforms.

[0087] The clinical manifestation of tauopathy may be tau aggregates in the brain, including but not limited to neurofibrillary tangles.Methods for detecting and quantifying tau aggregates in the brain are known in the art (e.g., tau PET using tau-specific ligands, such as THK5317, THK5351, AV1451, PBB3, MK-6240, RO-948, PI-2620, GTP1, PM-PBB3 and JNJ64349311, JNJ-067, etc.).

[0088] The terms "treat", "treating" or "treatment" as used herein refer to the provision of medical care by a trained and licensed professional to a subject in need thereof. Medical care may be diagnostic testing, therapeutic treatment, and / or prophylactic or preventative measures. The purpose of therapeutic and prophylactic treatment is to prevent or slow (alleviate) undesired physiological changes or diseases / disorders. Beneficial or desired clinical results of therapeutic or prophylactic treatment include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilized (i.e., not worsening) disease state, delayed or slowed disease progression, amelioration or reduction of disease state, and remission (either partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with a disease, condition or disorder, as well as those prone to having the disease, condition or disorder, or those in whom the disease, condition or disorder is to be prevented. Thus, a subject in need of treatment may or may not have symptoms or clinical signs of a disease.

[0089] The phrase "tau therapy" collectively refers to any imaging agent, therapeutic treatment, and / or prophylactic or preventative measures contemplated for or used with subjects at risk of developing a tauopathy or clinically diagnosed as having a tauopathy. Non-limiting examples of imaging agents include functional imaging agents (e.g., fluorodeoxyglucose, etc.) and molecular imaging agents (e.g., Pittsburgh compound B, florbetaben, florbetapir, flutemetamol, radiolabeled tau-specific ligands, radionuclide-labeled antibodies, etc.).Non-limiting examples of therapeutic agents include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-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, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, AF20513, etc.), tau protein aggregation inhibitors (e.g., TRx0237, methylthionimium chloride, etc.), treatments to improve glycemic control (e.g. insulin, exenatide, liraglutide pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma 1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents agents), muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof.

[0090] "Significantly deviating from the mean" refers to a value that is at least 1 standard deviation, preferably at least 1.3 standard deviations, more preferably at least 1.5 standard deviations, or even more preferably at least 2 standard deviations above or below the mean (i.e., the average level of tau species from a normal subject or normal population).

[0091] The phrase "Aβ and tau therapy" collectively refers to any imaging or therapeutic agent contemplated for or for use with a subject at risk of developing Aβ amyloidosis or AD, a subject diagnosed with Aβ amyloidosis, a subject diagnosed with a tauopathy, or a subject diagnosed with AD.

[0092] II. How to Measure Tau The present disclosure provides a method for measuring tau in a biological sample by mass spectrometry. Generally speaking, the disclosed method for measuring tau in a biological sample includes: preparing a biological sample; treating the biological sample by depleting one or more proteins and then purifying tau; cleaving the purified tau with a protease, and then optionally desalting the resulting cleavage product by solid phase extraction to obtain a sample containing tau proteolytic peptides; and performing liquid chromatography-mass spectrometry with the sample containing tau proteolytic peptides to detect and measure the concentration (relative or absolute) of at least one tau proteolytic peptide. Thus, in effect, the disclosed method uses at least one tau proteolytic peptide to detect and measure the amount of tau present in a biological sample.

[0093] In one example, the method of the disclosure includes: (a) providing a biological sample selected from a blood sample or a CSF sample; (b) removing proteins from the biological sample by separating precipitated proteins by protein precipitation to obtain a supernatant; (c) purifying tau from the supernatant by solid phase extraction; (d) cleaving the purified tau with a protease and then, optionally, desalting the resulting cleavage products by solid phase extraction to obtain a sample comprising tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry with the sample comprising tau proteolytic peptides to detect and measure the concentration of at least one tau proteolytic peptide.

[0094] In another example, the method of the disclosure includes: (a) reducing N-terminal tau, mid domain tau, or N-terminal tau and mid domain tau in a biological sample by affinity depletion, where the biological sample is a blood sample or a CSF sample; (b) enriching tau remaining after affinity depletion, which may be referred to as N-terminal-independent tau and / or mid domain-independent tau, by a method comprising: (i) removing additional proteins from the biological sample by protein precipitation and separation of the precipitated proteins to obtain a supernatant, and then purifying tau from the supernatant by solid phase extraction, or (ii) affinity purifying MTBR tau, thereby producing (i) or (ii) enriched tau; (c) cleaving the enriched tau with a protease, and then optionally desalting the resulting cleavage products by solid phase extraction to obtain a sample comprising tau proteolytic peptides; and (d) performing liquid chromatography-mass spectrometry (LC / MS) with the sample comprising tau proteolytic peptides to detect and measure the concentration of at least one tau proteolytic peptide.

[0095] In another example, the method of the disclosure includes: (a) reducing N-terminal tau, mid domain tau, or N-terminal tau and mid domain tau in a biological sample by affinity depletion, where the biological sample is a blood sample or a CSF sample; (b) removing additional proteins from the affinity-depleted sample by separating precipitated proteins by protein precipitation to obtain a supernatant; (c) purifying tau from the supernatant by solid phase extraction; (d) cleaving the purified tau with a protease and then, optionally, desalting the resulting cleavage products by solid phase extraction to obtain a sample comprising tau proteolytic peptides; and (e) performing liquid chromatography-mass spectrometry with the sample comprising tau proteolytic peptides to detect and measure the concentration of at least one tau proteolytic peptide.

[0096] In another example, the method of the present disclosure includes: (a) reducing N-terminal tau, mid domain tau, or N-terminal tau and mid domain tau in a biological sample by affinity depletion, where the biological sample is a blood sample or a CSF sample; (b) affinity purifying MTBR tau from the affinity-depleted sample; (c) cleaving the purified MTBR tau with a protease and then, optionally, desalting the resulting cleavage products by solid phase extraction to obtain a sample comprising proteolytic peptides of MTBR tau; and (d) performing liquid chromatography-mass spectrometry on the sample comprising proteolytic peptides of MTBR tau to detect and measure the concentration of at least one proteolytic peptide of MTBR tau.

[0097] In another example, the method of the present disclosure includes: (a) affinity purifying MTBR tau from a biological sample, where the biological sample is a blood sample or a CSF sample; (b) cleaving the purified MTBR tau with a protease, and then, optionally, desalting the resulting cleavage products by solid phase extraction to obtain a sample containing MTBR tau proteolytic peptides; and (c) performing liquid chromatography-mass spectrometry on the sample containing MTBR tau proteolytic peptides to detect and measure the concentration of at least one MTBR tau proteolytic peptide.

[0098] The present disclosure further contemplates measuring the phosphorylation occupancy of tau at one or more residues in each of the above methods. Phosphorylation occupancy, also referred to as phosphorylation stoichiometry, is measured by quantifying the phosphorylation of tau at one more residue. Using residue T217 for illustration, phosphorylation occupancy is typically expressed as pT217 / T217, where the numerator "pT217" is the amount (relative or absolute) of phosphorylated residue T217, and the denominator "T217" is the amount (relative or absolute) of residue T217. When measuring phosphorylation at more than one residue of tau, the method may further include calculating the ratio or another mathematical relationship between the values. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, S113, T181, S199, S202, S208, T153, T175, T205, S214, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, T181, S208, T153, T175, T205, S214, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, T153, T181, T205, S208, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, T153, T181, T205, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, T153, T181, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T111, T153, T181, T205, S208, and T217. In some embodiments, the methods herein include measuring tau phosphorylation at one or more residues selected from T181, T205, and T217. In some embodiments, the methods herein include measuring tau phosphorylation at T205.In some embodiments, the methods herein include measuring tau phosphorylation at one or more additional residues selected from T205, and optionally T111, T181, S208, T153, T175, S214, T217, and T231. In some embodiments, the methods herein include measuring tau phosphorylation at one or more additional residues selected from T205, and optionally T111, T153, T181, S208, and T217. In some embodiments, the methods herein include measuring tau phosphorylation at one or more additional residues selected from T205, and optionally T181, and T217.

[0099] Generally speaking, phosphorylation occupancy of tau at one or more residues can be measured using any sample containing tau. However, the amino acid residue may affect which sample should be used. For example, if it is desired to measure tau phosphorylation at T111, mid domain tau should be affinity-depleted from blood or CSF samples using antibody tau1 or other affinity purification reagent that binds C-terminus to T111, and affinity-purified tau bound to the affinity purification reagent should be used to measure pT111 / T111. In certain embodiments, samples enriched for mid domain tau (or enriched for N-terminus and mid domain tau) can be used to measure phosphorylation occupancy.

[0100] The present disclosure is not limited to any one particular method for quantitatively assessing site-specific phosphorylation of tau. A suitable method should distinguish between tau isoforms that differ only in the phosphorylation state of a single amino acid, distinguish between p-tau isoforms phosphorylated at different amino acids, and quantify the change in phosphorylation occurring at a specific site independent of the overall change in total tau. The change in phosphorylation stoichiometry occurring at a specific site independent of the overall change in total tau can be quantified by one of three approaches: 1) relative comparison between phosphopeptide isomers, which can be used to estimate the relative abundance of each phosphopeptide that shares the same sequence; 2) standardization of phosphopeptides using any peptide from tau protein as a reference; and 3) absolute quantification for each phosphorylated and non-phosphorylated peptide using labeled synthetic internal standards, where the absolute quantification value for each phosphorylated peptide is standardized with any absolute quantification value obtained for any peptide from tau protein. All three approaches use internal standardization for the comparison of the relative phosphorylation change for each site. Other methods known in the art may also be used. When using an internal synthetic labeled standard for absolute quantification, it is preferable to spike the labeled standard into the sample before treating the sample to enrich for soluble tau.

[0101] In an exemplary embodiment, the site-specific phosphorylation of tau is measured by high-resolution mass spectrometry. Suitable types of mass spectrometry are known in the art. These include, but are not limited to, quadrupole, time-of-flight, ion trap and Orbitrap, as well as hybrid mass spectrometers that combine different types of mass spectrometers in one configuration (e.g., Orbitrap Fusion™ Tribrid™ mass spectrometers from ThermoFisher Scientific). Tau is typically proteolytically digested prior to MS analysis. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C and Arg-N. If affinity purification / depletion is used to generate tau samples, digestion may occur after elution of tau from the immobilized ligand or while tau is bound to the immobilized ligand. Affinity purification / depletion is described in detail in Section II(c). Following one or more cleanup steps, the digested tau peptides may be separated by a liquid chromatography system compatible with a high-resolution mass spectrometer. The chromatography system may be optimized by routine experimentation to generate the desired LC-MS pattern. A variety of LC-MS techniques may be used to quantitatively analyze site-specific tau phosphorylation. Non-limiting examples include selected reaction monitoring, parallel reaction monitoring, selected ion monitoring, and data-independent acquisition. As mentioned above, any quantitative analysis of site-specific tau phosphorylation should consider the overall change in total tau. In an exemplary embodiment, the mass spectrometry protocol outlined in the examples is used.

[0102] The present disclosure further contemplates measuring total tau in each of the above methods. Tau can be found in soluble and insoluble compartments, in monomeric and aggregated forms, in ordered or irregular structures, inside or outside cells, and can be complexed with other proteins or molecules. Thus, the source of the biological sample (e.g., brain tissue, CSF, blood, etc.) and any downstream processing of the biological sample will affect the overall amount of tau isoforms in a given sample. Total tau measurement can be performed by mass spectrometry. Alternatively, total tau can be measured by immunoassay or other methods to quantify tau concentration. In certain embodiments, total tau can be measured by mass spectrometry by quantifying TPSL (first four amino acids of SEQ ID NO: 18) tryptic peptide (i.e., TPSLPTPPTR (SEQ ID NO: 18)) or TPPS (first four amino acids of SEQ ID NO: 10) tryptic peptide (i.e., TPPSSGEPPK (SEQ ID NO: 10)). In certain embodiments, a sample enriched in mid domain tau (or enriched in N-terminal and mid domain tau) can be used to measure total tau.

[0103] In still further embodiments, the present disclosure contemplates determining the presence / absence of one or more proteins in the biological sample and / or measuring the concentration of one or more additional proteins in the biological sample in each of the above methods. In some embodiments, the one or more proteins may be proteins depleted from the biological sample prior to purification of tau. For example, in certain embodiments, N-terminal tau species and / or mid-domain tau species can be identified and / or quantified separately from the tau species (e.g., MTBR tau, C-terminal tau) quantified by the methods disclosed herein. Alternatively, or in addition, Aβ, ApoE, or other proteins of interest can be identified and / or quantified by processing a portion of the biological sample in parallel, by depleting the protein of interest from the biological sample prior to utilization in the methods disclosed herein, or by depleting the protein of interest from the biological sample during the sample processing steps disclosed herein.

[0104] The biological sample, suitable internal standards, and the steps of depleting one or more proteins, purifying tau, cleaving purified tau with a protease, and mass spectrometry are described in more detail below.

[0105] (a) Biological samples Suitable biological samples include blood samples or cerebrospinal fluid (CSF) samples obtained from a subject. In some embodiments, the subject is a human. The human subject may be awaiting medical treatment or treatment, or may be undergoing medical treatment or treatment, or may have undergone medical treatment or treatment. In various embodiments, the human subject may be a healthy subject, a subject at risk of developing a neurodegenerative disease, a subject with signs and / or symptoms of a neurodegenerative disease, or a subject diagnosed with a neurodegenerative disease. In further embodiments, the neurodegenerative disease may be a tauopathy. In certain examples, the tauopathy may be Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), or frontotemporal lobar degeneration (FTLD). In other embodiments, the subject is an experimental animal. In further embodiments, the subject is an experimental animal genetically engineered to express human tau and optionally one or more additional human proteins (e.g., human Aβ, human ApoE, etc.).

[0106] CSF may be obtained by lumbar puncture, with or without an indwelling CSF catheter. Multiple blood or CSF samples collected simultaneously from a subject may be pooled. Blood may be collected by venipuncture, with or without an intravenous catheter, or by finger stick (or equivalent). Once collected, blood or CSF samples may be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cellular debris; use of additives designed to stabilize and preserve specimens prior to analytical testing, etc.). Blood or CSF samples may be used immediately or may be frozen and stored indefinitely. Prior to use in the methods disclosed herein, biological samples may be modified, if necessary or desired, to include protease inhibitors, isotope-labeled internal standards, detergents and chaotropic agents, and / or to deplete other analytes (e.g., proteins, peptides, metabolites, etc.).

[0107] The size of the sample used can or will vary depending on the type of sample, the health status of the subject from whom the sample was obtained, and the analytes (in addition to tau) to be analyzed. The volume of the CSF sample may be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In certain examples, the size of the sample may be about 0.05 mL to about 1 mL of CSF. The amount of the plasma sample may be about 0.01 mL to about 20 mL.

[0108] (b) Isotopically labeled tau internal standard Isotopically labeled tau can be used as an internal standard to account for variation across sample processing and calculate absolute concentrations as appropriate. Generally, isotopically labeled tau internal standards are added prior to any significant sample processing, but can be added multiple times if necessary. See, for example, the methods shown in Figures 2-4.

[0109] Several isotopically labeled tau internal standards are described herein. All incorporate a heavy isotope label into at least one amino acid residue. One or more full-length isoforms may be used. Alternatively, or in addition, tau isoforms and / or peptide fragments of tau with post-translational modifications may be used, as known in the art. Generally speaking, the incorporated labeled amino acid residue should increase the mass of the peptide without affecting its chemical properties, and the mass shift caused by the presence of the isotope label should be sufficient to allow mass spectrometry to distinguish between the internal standard (IS) and the endogenous tau analyte signal. As shown herein, suitable heavy isotope labels include, but are not limited to: 2 H, 13 C, and 15 N. Typically, about 1-10 ng of an internal standard is usually sufficient.

[0110] (c) depleting one or more proteins The method of the present disclosure comprises depleting one or more proteins from a sample.The term "deplete" means to reduce in amount or number.Therefore, a protein-depleted sample may have any amount of protein that is measurably less than the amount in the original sample, including the absence of the amount of protein.

[0111] Proteins can be depleted from samples by methods that specifically target one or more proteins, such as affinity depletion, solid-phase extraction, or other methods known in the art. Targeted depletion of a protein or proteins may be used in situations where downstream analysis of the protein is desired (e.g., identification, quantification, analysis of post-translational modifications, etc.). For example, Aβ peptides can be identified and quantified by methods known in the art after affinity depletion of Aβ with a suitable epitope binding agent. As another non-limiting example, apolipoprotein E (ApoE) status can be determined by methods known in the art following affinity depletion of ApoE and identification of ApoE isoforms. Targeted depletion can also be used to isolate other proteins for subsequent analysis, including but not limited to apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, 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 remove proteins that cofound mass spectrometry. For example, in certain embodiments of the present disclosure, N-terminal tau proteins and / or mid domain tau proteins are depleted from samples prior to further sample processing for analysis by mass spectrometry. Downstream analysis of depleted tau proteins may or may not be performed, but both options are contemplated by the methods of the present disclosure.

[0112] In some embodiments, target depletion may occur by affinity depletion. Affinity depletion refers to a method of depleting a protein of interest from a sample by utilizing its specific binding properties for a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support, such as a bead, resin, tissue culture plate, etc. The immobilization of the ligand to the solid support may occur after the interaction of the ligand with the protein has occurred. Suitable ligands include antibodies, aptamers, and other epitope binding agents. The molecule may be a polymer or other material that selectively absorbs the protein of interest. As a non-limiting example, polyhydroxymethylene substituted with fatty oxoethylated alcohols (e.g., PHM-L LIPOSORB, Sigma Aldrich) can be used to selectively absorb lipoproteins (including ApoE) from serum. Two or more affinity depletion agents can be combined to sequentially or simultaneously deplete multiple proteins.

[0113] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using at least one epitope binding agent that specifically binds to an epitope within amino acids 1-243, inclusive, (or within a similarly defined region for the 0N or 1N isoforms) of Tau-441. In various embodiments, one, two, three or more epitope binding agents can be used. When two or more epitope binding agents are used, they can be used sequentially or simultaneously.

[0114] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within the N-terminus of tau (e.g., amino acids 1-103 of tau-441, inclusive) and an epitope binding agent that specifically binds to an epitope within the mid domain of tau (e.g., amino acids 104-243 of tau-441, inclusive). The epitope binding agents can be used sequentially or simultaneously.

[0115] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1-35, inclusive, of Tau-441, and an epitope binding agent that specifically binds to an epitope within amino acids 104-243, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms). The epitope binding agents can be used sequentially or simultaneously.

[0116] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1-103, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); an epitope binding agent that specifically binds to an epitope within amino acids 104-243, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents can be used sequentially or simultaneously.

[0117] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1-35, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); an epitope binding agent that specifically binds to an epitope within amino acids 104-243, inclusive, of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms); and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents can be used sequentially or simultaneously.

[0118] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1-103 of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms), inclusive; and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents can be used sequentially or simultaneously.

[0119] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 1-35 of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms), inclusive; and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents can be used sequentially or simultaneously.

[0120] In some embodiments, the methods of the disclosure include affinity depleting one or more proteins from a sample using an epitope binding agent that specifically binds to an epitope within amino acids 104-243 of Tau-441 (or within a similarly defined region for the 0N or 1N isoforms), inclusive; and an epitope binding agent that specifically binds to an epitope of amyloid beta. The epitope binding agents can be used sequentially or simultaneously.

[0121] In each of the above embodiments, the epitope binding agent may comprise an antibody or an aptamer. In some embodiments, the epitope binding agent that specifically binds to amyloid beta is HJ5.1 or is 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 1-103 of Tau-441, inclusive, is HJ8.5 or is an epitope binding agent that binds to the same epitope as HJ8.5 and / or competitively inhibits HJ8.5. In some embodiments, the epitope binding agent that specifically binds to an epitope within amino acids 104-221 of Tau-441, inclusive, is Tau1 or is an epitope binding agent that binds to the same epitope as Tau1 and / or competitively inhibits Tau1. Methods for identifying the epitope to which an antibody specifically binds and assays to assess competitive inhibition between two antibodies are known in the art.

[0122] Alternatively, samples can be depleted of proteins by more conventional methods, such as ultrafiltration or protein precipitation with acids, organic solvents or salts. Generally, these methods are used to ensure the reduction of high abundance and high molecular weight proteins, resulting in the enrichment of low molecular weight and / or low abundance proteins and peptides (e.g., tau, Aβ, etc.).

[0123] In some embodiments, proteins can be depleted from a sample by precipitation. Briefly, precipitation involves adding a precipitating agent to the sample and mixing thoroughly, incubating the sample with the precipitating agent to precipitate proteins, and separating the precipitated proteins by centrifugation or filtration. The resulting supernatant can then be used in downstream applications. The amount of reagent required can be determined experimentally by methods known in the art. Suitable precipitating agents include perchloric acid, trichloroacetic acid, acetonitrile, methanol, and the like. In an exemplary embodiment, proteins are depleted from a sample by acid precipitation. In a further embodiment, proteins are depleted from a sample by acid precipitation using perchloric acid.

[0124] As a non-limiting example, samples can be depleted of protein by acid precipitation using perchloric acid. As used herein, "perchloric acid" refers to 70% perchloric acid unless otherwise indicated. In some embodiments, perchloric acid is added at a final concentration of about 1% v / v to about 15% v / v. In other embodiments, perchloric acid is added at a final concentration of about 1% v / v to about 10% v / v. In other embodiments, perchloric acid is added at a final concentration of about 1% v / v to about 5% v / v. In other embodiments, perchloric acid is added at a final concentration of about 3% v / v to about 15% v / v. In other embodiments, perchloric acid is added at a final concentration of about 3% v / v to about 10% v / v. In other embodiments, perchloric acid is added at a final concentration of about 3% v / v to about 5% v / v. In other embodiments, perchloric acid is added at 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 at a final concentration of about 3.5% v / v. Following addition of perchloric acid, the sample is thoroughly mixed (e.g., by vortex mixing) and typically held for about 10 minutes or longer at a low temperature to promote precipitation. For example, the sample may be held for about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes. In other examples, the sample may be held for about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes. In other examples, the sample may be held for about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes. In other examples, the sample is held for about 30 minutes. The sample is then centrifuged at a low temperature to pellet precipitated proteins, and the supernatant containing soluble tau (i.e., the acid-soluble fraction) is transferred to a new container. As used in the above context, "low temperature" refers to a temperature of 10°C or lower. For example, low temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C. In some embodiments, a narrower temperature range may be preferred, for example, about 3°C ​​to about 5°C, or even about 4°C. In certain embodiments, low temperature may be achieved by placing the sample on ice.

[0125] Two or more methods from either or both of the above approaches can be combined to sequentially or simultaneously deplete multiple proteins. For example, one or more proteins can be selectively depleted (targeted depletion), followed by depletion of high abundance / molecular weight proteins. Alternatively, high abundance / molecular weight proteins can be depleted first, followed by targeted depletion of one or more proteins. In yet another alternative, high abundance / molecular weight proteins can be depleted first, followed by a first round of targeted depletion of one or more proteins, followed by a second round of targeted depletion of one or more different proteins that are different from the first round target. Other iterations will be readily apparent to those skilled in the art.

[0126] (d) Purifying tau Another step of the method disclosed herein includes purifying tau, particularly MTBR tau.In some examples, MTBR tau is N-terminal independent and / or mid domain independent MTBR tau.Purified tau can be partially or completely purified.

[0127] In some embodiments, the method of the present disclosure includes 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 that adsorbs tau, one or more washing steps, and eluting tau from the adsorbent. Suitable adsorbents include reversed-phase adsorbents. Suitable reversed-phase adsorbents are known in the art and include, but are not limited to, alkyl-bonded silica, aryl-bonded silica, styrene / divinylbenzene material, N-vinylpyrrolidone / divinylbenzene material. In an exemplary embodiment, the reversed-phase material is a polymer containing N-vinylpyrrolidone and divinylbenzene, or a polymer containing styrene and divinylbenzene. In an exemplary embodiment, the adsorbent is Oasis HLB (Waters). Before contacting with the supernatant containing tau, the adsorbent is typically preconditioned (e.g., with a buffer containing a water-miscible organic solvent and then a mobile phase) according to the manufacturer's instructions or as known in the art. In addition, the supernatant may be acidified, since some reversed-phase materials retain ionized analytes more strongly than others. The use of volatile components in the mobile phase and for elution is preferred, since it facilitates drying of the sample. In exemplary embodiments, 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 an equivalent thereof. In some examples, the washing may use 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 use 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 using a liquid phase comprising about 20% v / v to about 50% v / v acetonitrile (ACN), or an equivalent thereof. In some examples, tau may be eluted using 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 may be eluted using a liquid phase comprising about 30% v / v to about 50% v / v ACN or about 30% v / v to about 40% v / v ACN.The eluate may be dried by methods known in the art, such as vacuum drying (eg, speed-vac), lyophilization, evaporation under a stream of nitrogen, etc.

[0128] In some embodiments, the method of the present disclosure includes purifying MTBR tau by affinity purification. Affinity purification refers to a method of concentrating a protein of interest by utilizing its specific binding properties to a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support, such as a bead, a resin, a tissue culture plate, etc. The immobilization of the ligand to the solid support may occur after the interaction of the ligand with the protein has occurred. Suitable ligands include antibodies, aptamers, and other epitope binding agents. Purification of MTBR tau by affinity purification includes contacting a sample containing tau with a suitable immobilized ligand, one or more washing steps, and elution of MTBR tau from the immobilized ligand.

[0129] In some embodiments, the methods of the disclosure include purifying MTBR tau by affinity purification using at least one epitope binding agent that specifically binds to an epitope within amino acids 235-368, inclusive, of tau-441, or within amino acids 244-368, inclusive, of tau-441 (or within a similarly defined region of other full-length isoforms). In various embodiments, one, two, three or more epitope binding agents can be used. When two or more epitope binding agents are used, they can be used sequentially or simultaneously. Non-limiting examples of suitable epitope binding agents include 77G7, RD3, RD4, UCB1017, and PT76 described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, and antibodies E2814 and 7G6 described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, as well as other epitope binding agents that specifically bind to the same epitope as those antibodies. In further embodiments, the methods of the disclosure include purifying MTBR tau by affinity purification using an epitope binding agent that specifically binds to an epitope within R1 of MTBR tau, an epitope binding agent that specifically binds to an epitope within R2 of MTBR tau, an epitope binding agent that specifically binds to an epitope within R3 of MTBR tau, an epitope binding agent that specifically binds to an epitope within R4 of MTBR tau, an epitope binding agent that specifically binds to an epitope unique to 3R tau, an epitope binding agent that specifically binds to an epitope unique to 4R tau, an epitope binding agent that specifically binds to an epitope spanning R1 and R2 of MTBR tau, an epitope binding agent that specifically binds to an epitope spanning R2 and R3 of MTBR tau, an epitope binding agent that specifically binds to an epitope spanning R3 and R4 of MTBR tau, or any combination thereof. In certain examples, the methods of the disclosure include purifying MTBR tau by affinity purification using an epitope binding agent that specifically binds to an epitope including amino acids 316-355 of tau-441 (or the same region of other full-length isoforms).In various embodiments, one, two, three or more epitope binding agents may be used. When more than one epitope binding agent is used, they may be used sequentially or simultaneously.

[0130] In each of the above embodiments, the epitope binding agent may comprise an antibody or an aptamer. In some embodiments, the epitope binding agent that specifically binds to an epitope within R3 and R4 of MTBR tau is 77G7, or an epitope binding agent that binds to the same epitope as 77G7 and / or competitively inhibits 77G7 (BioLegend). In some embodiments, the epitope binding agent that specifically binds to an epitope unique to 3R tau is RD3 (de Silva et al., Neuropathology and Applied Neurobiology, 2003, 29: 288-302), or an epitope binding agent that binds to the same epitope as RD3 and / or competitively inhibits RD3. In some embodiments, the epitope binding agent that specifically binds to an epitope unique to 4R tau is RD4 (de Silva et al., Neuropathology and Applied Neurobiology, 2003, 29: 288-302) or is an epitope binding agent that binds to the same epitope as RD4 and / or competitively inhibits RD4.

[0131] (e) Cleavage of purified tau with a protease Another step of the method disclosed herein includes cleaving 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 elution of tau from the immobilized ligand or while tau is bound. Suitable proteases include, but are not limited to, trypsin, Lys-N, Lys-C and Arg-N. In a preferred embodiment, the protease is trypsin. The resulting cleavage product is a composition comprising proteolytic peptides of tau. When the protease is trypsin, the resulting cleavage product comprises tryptic peptides of tau. Following proteolytic cleavage, the resulting cleavage product is desalted, typically by solid phase extraction.

[0132] (f) LC-MS Another step of the method disclosed herein includes performing liquid chromatography-mass spectrometry (LC-MS) with a sample containing proteolytic peptides of tau to detect and measure the concentration of at least one proteolytic peptide of tau. Thus, in effect, the disclosed method uses one or more proteolytic peptides of tau to detect and measure the amount of tau protein present in a biological sample.

[0133] In embodiments where trypsin is the protease, the proteolytic peptides of tau that indicate the presence of MTBR tau include, but are not limited to, those listed in Table A. When alternative enzymes are used for digestion, the resulting proteolytic peptides may differ slightly, but can be easily determined by one of skill in the art. Without wishing to be bound by theory, it is believed that the variation in the amount of tryptic peptides between two biological samples of the same type reflects the differences in the MTBR tau species that make up those biological samples. As disclosed herein, the amount of certain proteolytic peptides of MTBR tau, as well as the ratio of certain proteolytic peptides of MTBR tau, may provide clinically meaningful information to guide treatment decisions. Thus, methods that allow for the detection and quantification of tryptic peptides of MTBR tau are useful in the diagnosis and treatment of many neurodegenerative diseases. [Table A]

[0134] Tau proteolytic peptides can be separated by a liquid chromatography system compatible with a high resolution mass spectrometer. A suitable LC-MS system is equipped with an ID <1.0 mm column and can use a flow rate of less than about 100 μl / min. In a preferred embodiment, a nanoflow LC-MS system is used (e.g., a column with an ID of about 50-100 μm and a flow rate of <1 μL / min, preferably about 100-800 nL / min, more preferably about 200-600 nL / min). In an exemplary embodiment, the LC-MS system is equipped with an ID 0.05 mm column and can use a flow rate of about 400 nL / min.

[0135] As known in the art, tandem mass spectrometry can be used to improve resolution, or technology can be improved to achieve the resolution of tandem mass spectrometry with a single mass analyzer.Suitable types of mass analyzers are known in the art.These include, but are not limited to, quadrupole, time-of-flight, ion trap and Orbitrap, and hybrid mass analyzers that combine different types of mass analyzers into one configuration (e.g., Orbitrap Fusion™ Tribrid™ mass analyzer, Orbitrap Fusion™ Lumos™ mass analyzer, Orbitrap Tribrid™ Eclipse™ mass analyzer, Q Exactive mass analyzer, each from ThermoFisher Scientific). In an exemplary embodiment, the LC-MS system can be equipped with a mass spectrometer selected from Orbitrap Fusion™ Tribrid™ mass spectrometer, Orbitrap Fusion™ Lumos™ mass spectrometer, Orbitrap Tribrid™ Eclipse™ mass spectrometer, or mass spectrometers with similar or improved focusing and ion transmission in quadrupole format. A suitable mass analysis protocol can be developed by optimizing the number of ions collected before analysis (e.g., AGC settings with orbitrap) and / or injection time. In an exemplary embodiment, the mass analysis protocol outlined in the examples is used.

[0136] III. Staging the subject In one aspect, the present disclosure provides a method for measuring time to onset of dementia in a subject without cognitive or behavioral symptoms of Alzheimer's disease, comprising: i ) measuring the phosphorylation occupancy of tau at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio and, where appropriate, measuring MTBR-tau212, the phosphorylation occupancy at residue T217 MTBR-tau243, and / or MTBR-tau3R in a blood sample or CSF sample obtained from the subject; orii ) measuring the phosphorylation occupancy of tau at residue T205, measuring the phosphorylation occupancy of tau at residue T217, and measuring MTBR-tau 212 in a blood sample or CSF sample obtained from the subject; and (b) i ) or (a ii ), where time to onset of dementia is the number of years to a Clinical Dementia Rating (CDR) greater than zero. In an exemplary embodiment, subjects with no cognitive or behavioral symptoms of Alzheimer's disease have a CDR of zero.

[0137] In another aspect, the disclosure provides a method of measuring time to onset of dementia in a subject without cognitive or behavioral symptoms of Alzheimer's disease, comprising: (a) processing a blood or CSF sample from the subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species, wherein the first population of tau species is enriched in N-terminal tau and / or mid domain tau, and the second population of enriched tau species is enriched in MTBR-tau; (b) obtaining a blood or CSF sample from the subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species, wherein the first population of tau species is enriched in N-terminal tau and / or mid domain tau, and the second population of enriched tau species is enriched in MTBR-tau; i ) measuring the phosphorylation occupancy of tau at residue T205 in a first population of tau species and measuring the MTBR-tau299 / MTBR-tau354 ratio in a second population of tau species and, optionally, measuring MTBR-tau212 in the second population of tau species; or ii ) measuring the phosphorylation occupancy of tau at residue T205 in a first population of tau species, and measuring the phosphorylation occupancy of tau at residue T217 in the first population of tau species, and measuring MTBR-tau212 in a second population of tau species; and (c) (b i ) or (b ii) to calculate time to onset of dementia, where time to onset of dementia is the number of years to clinical dementia assessment greater than zero. In exemplary embodiments, subjects with cognitive or behavioral symptoms of Alzheimer's disease have a CDR greater than zero, greater than or equal to 0.5, or greater than or equal to 1. As non-limiting examples, subjects with cognitive or behavioral symptoms of Alzheimer's disease may have a CDR of 0.5, 1, 1.5, or 2.

[0138] In another aspect, the disclosure provides a method for measuring time from onset of dementia in a subject having cognitive or behavioral symptoms of Alzheimer's disease, comprising: i ) measuring the phosphorylation occupancy of tau at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio and, where appropriate, measuring MTBR-tau212, the phosphorylation occupancy at residue T217 MTBR-tau243, and / or MTBR-tau3R in a blood sample or CSF sample obtained from the subject; or ii ) measuring the phosphorylation occupancy of tau at residue T205, measuring the phosphorylation occupancy of tau at residue T217, and measuring MTBR-tau 212 in a blood sample or CSF sample obtained from the subject; and (b) i ) or (a ii ), where time since dementia onset is the number of years since clinical dementia assessment that is greater than zero.

[0139] In another aspect, the disclosure provides a method for measuring time from onset of dementia in a subject having cognitive or behavioral symptoms of Alzheimer's disease, comprising: (a) processing a blood or CSF sample from the subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species, wherein the first population of tau species is enriched in N-terminal tau and / or mid domain tau, and the second population of enriched tau species is enriched in MTBR-tau; (b) obtaining a blood or CSF sample from the subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species, wherein the first population of tau species is enriched in N-terminal tau and / or mid domain tau, and the second population of enriched tau species is enriched in MTBR-tau;i ) measuring the phosphorylation occupancy of tau at residue T205 in a first population of tau species and measuring the MTBR-tau299 / MTBR-tau354 ratio in a second population of tau species and, optionally, measuring MTBR-tau212 in the second population of tau species; or ii ) measuring the phosphorylation occupancy of tau at residue T205 in a first population of tau species, and measuring the phosphorylation occupancy of tau at residue T217 in the first population of tau species, and measuring MTBR-tau212 in a second population of tau species; and (c)(b i ) or (b ii ), where time since onset of dementia is the number of years since clinical dementia assessment that is greater than zero.

[0140] In another aspect, the disclosure provides a method for measuring time from onset of dementia in a subject having cognitive or behavioral symptoms of Alzheimer's disease, comprising: i (b) measuring the phosphorylation occupancy of tau at residue T205, measuring the rate of change of MTBR-tau (e.g., MTBR-tau 299), and, where appropriate, measuring MTBR-tau 212 in a blood sample or CSF sample obtained from the subject; and (b) (a i ) or (a ii ), where time since dementia onset is the number of years since clinical dementia assessment that is greater than zero.

[0141] In another aspect, the disclosure provides a method for measuring changes in cognition in a subject, the method comprising: (a) measuring phosphorylation occupancy of tau at one or more residues selected from T111, T153, T181, T217 and T231 in a blood or CSF sample obtained from the subject, and measuring at least one of MTBR-tau275, MTBR-tau299, and MTBR-tau3R (MTBR-tau306) in a blood or CSF sample obtained from the subject, and optionally measuring total tau in a blood or CSF sample obtained from the subject; and (b) calculating the change in cognition using the measurements of (a).

[0142] In some of the above embodiments, calculating the time to onset of dementia comprises determining the significant deviation of the tau species level measured in the control population without brain amyloid plaques as measured by PET imaging and / or Aβ42 / 40 measurement in CSF from the mean. "Significantly deviate from the mean" refers to a value that is at least 1 standard deviation, preferably at least 1.3 standard deviation, more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ, respectively, where σ is the standard deviation defined by normal distribution measured in the control population without brain amyloid plaques as measured by PET imaging and / or Aβ42 / 40 measurement in CSF). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean, or the rate of change over time, can be used to calculate the time to onset of dementia in a subject.

[0143] Biological samples can be obtained from subjects that may or may not be asymptomatic. "Asymptomatic subjects" refer to subjects that do not show any signs or symptoms of AD. However, subjects may exhibit signs or symptoms of AD (e.g., memory loss, forgetting things, mood or behavior changes, etc.), but do not show sufficient cognitive or functional impairment for a clinical diagnosis of mild cognitive impairment or dementia. In a further embodiment, the subject may have one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. In an alternative embodiment, the subject may not have a genetic mutation known to cause dominantly inherited Alzheimer's disease. Alzheimer's disease that does not have a specific familial association is referred to as sporadic Alzheimer's disease.

[0144] Another aspect of the present disclosure includes a method for diagnosing the stage of Alzheimer's disease of a subject.In various embodiments, "stage of AD" may be defined as the amount of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, etc.) that has passed since the onset of dementia caused by AD.Although there are criteria for clinical diagnosis of AD, in clinical practice, the time of symptom onset for a given subject is often unclear, or the diagnosis of either dementia or AD is doubtful.Therefore, there is a need in the art for a test that objectively diagnoses the stage of AD of a subject.

[0145] Instead of or in addition to using site-specific tau phosphorylation, measurement of MTBR-tau species, and optionally measurement of total tau, in any of the above embodiments, ratios calculated from measured phosphorylation levels and / or MTBR-tau levels, or ratios calculated from measured phosphorylation levels and / or MTBR-tau levels and total tau can be used. Both approaches are detailed in the examples. Mathematical operations other than ratios may also be used. For example, the examples use site-specific tau phosphorylation values ​​and / or MTBR tau values ​​in various statistical models (e.g., linear regression, LME curves, LOESS curves, etc.) together with other known biomarkers (e.g., APOE ε4 status, age, sex, cognitive test scores, functional test scores, etc.). The selection of measurements and the selection of mathematical operations may be optimized to maximize the specificity of the method. For example, diagnostic accuracy may be assessed by the area under the ROC curve, and in some embodiments, a ROC AUC value of 0.7 or greater (e.g., 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, etc.) is set as the threshold.

[0146] Brain amyloid plaques in humans are routinely measured by amyloid positron emission tomography (PET). For example, 11 C-Pittsburgh Compound B (PiB) PET imaging is commonly used to detect Aβ plaque pathology. The standard uptake value ratio (SUVR) of cortical PiB-PET is used to reliably identify significant cortical Aβ plaques and classify subjects as PIB positive (SUVR≧1.25) or negative (SUVR<1.25). Thus, in the above embodiment, a control population without brain amyloid plaques as measured by PET imaging may refer to a population of subjects with cortical PiB-PET SUVR<1.25. Other values ​​of PiB binding (e.g., mean cortical binding potential) or analysis of regions of interest other than cortical regions may also be used to classify subjects as PIB positive or negative. Other PET imaging agents may also be used.

[0147] A control population that does not have brain amyloid plaques as measured by Aβ42 / 40 measurement in CSF may refer to a population of subjects that have an Aβ42 / 40 measurement of <0.12 as measured by mass spectrometry, as described in Patterson et al, Annals of Neurology, 2015.

[0148] Figures 5 and 6 show the dynamic patterns of tau phosphorylation and MTBR-tau levels in isolated tau samples in relation to the years to AD dementia onset. Figure 20 shows the rates of various tau species abnormalities per 5EYO interval. The phosphorylation levels of T217, which deviate significantly from the mean, first occur approximately 21 years before the onset of AD dementia. The changes in MTBR-tau299 in R2 occur approximately 22 years before onset, close to when changes in p-tau217 occupancy were first detected. The increase in MTBR-tau354 in R4, potentially due to deposition in brain tangles, saturates in the later clinical stages. Notably, the ratio of MTBR-tau299 / 354, which recapitulates tau pathophysiology, is highly correlated with pT217 occupancy (i.e., from approximately 21 years before AD dementia onset). The rate of change in MTBR-tau3R begins to increase approximately 20 years before AD dementia onset and is highly correlated with AD progression. The rate of change in MTBR-tau243 begins to increase approximately 15 years before the onset of AD dementia. Changes in TBR-tau212 levels are highly correlated with AD progression. Significant deviations in T205 phosphorylation levels from the mean first occur approximately 13 years before the onset of AD dementia.

[0149] In one example, a method of the disclosure includes providing a biological sample obtained from a subject, and (ai) measuring tau phosphorylation occupancy at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio, and optionally measuring MTBR-tau212, phosphorylation occupancy at residue T217, MTBR-tau243, and / or MTBR-tau3R in a blood sample or CSF sample obtained from the subject, or (aii) measuring tau phosphorylation occupancy at residue T205, measuring tau phosphorylation occupancy at residue T217, and measuring MTBR-tau212 in a blood sample or CSF sample obtained from the subject; (b) calculating time to onset of dementia using the measurements of (ai) or (aii), where time to onset of dementia is the number of years until clinical dementia assessment that is greater than zero; and (c) calculating tau phosphorylation at T217 and / or MTBR-tau3R in a blood sample or CSF sample obtained from the subject. and determining that the subject is about 10 to about 25 years, or about 10 to about 20 years, from onset of dementia due to AD if the tau phosphorylation at T217 and T205, MTBR-tau299 / MTBR-tau354 ratio is about 1.5σ or more above the mean and tau phosphorylation at T205 is less than about 1.5σ above the mean, where σ is the standard deviation defined by a normal distribution of tau phosphorylation at T217 and T205, MTBR-tau299 / MTBR-tau354 ratio and MTBR-tau212 measured in a control population that does not have brain amyloid plaques as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. In various embodiments, tau phosphorylation and / or the MTBR-tau299 / MTBR-tau354 ratio at T217 may be greater than the mean of a control population by about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2σ, or more than 2σ. In other embodiments, tau phosphorylation and / or the MTBR-tau299 / MTBR-tau354 ratio at T217 may be greater than the mean of a control population by about 1.85σ, about 1.9σ, about 1.95σ, about 2σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or more than 2.5σ.In each of the above embodiments, phosphorylation of tau at T205 may be greater than the average or control population average by about 1.3σ, about 1.35σ, about 1.4σ, about 1.45σ, about 1.5σ, about 1.51σ, about 1.55σ, about 1.6σ, about 1.7σ, about 1.8σ, about 1.9σ, about 2.0σ or less. Alternatively, phosphorylation of tau at T205 may be greater than the average of a control population by about 2.0σ, about 2.05σ, about 2.1σ, about 2.2σ, about 2.3σ, about 2.4σ, about 2.5σ, or less than 2.5σ. In a further example, tau phosphorylation at T217 and / or MTBR-tau299 / MTBR-tau354 ratio may be greater than about 2σ above the mean of the control population, and tau phosphorylation at T205 may be greater than about 2σ or less above the mean of the control population. In addition to using a threshold (e.g., at least one standard deviation above or below the mean), in some embodiments, the degree of absolute change above or below the mean, or the rate of change over time, may be used to classify subjects. In still further embodiments, the measured levels of tau phosphorylation at T205 and / or T217, as well as the MTBR-tau299 / MTBR-tau354 ratio value and / or the MTBR-tau212 value may be used in various mathematical operations to improve predictive power compared to each other. For example, a ratio may be calculated from the measured phosphorylation levels. Mathematical operations other than ratios may also be used.

[0150] In another example, the method of the disclosure includes: (a) providing a first and a second biological sample obtained from a subject, where "first" and "second" refer to the order in which the samples were collected, and measuring tau species as described above; (b) calculating the change in site-specific phosphorylation at each measured residue, and the change in the MTBR-tau299 / MTBR-tau354 ratio value and, where appropriate, the MTBR-tau212 value; and (c) diagnosing the stage of AD in the subject if the phosphorylation level T217 and / or the MTBR-tau299 / MTBR-tau354 ratio decrease or remain the same and the phosphorylation level at T205 increases. The first and second isolated tau samples may be collected at intervals of days, weeks or months. Typically, the tau phosphorylation at the specific sites listed in (a)(i), (a)(ii) or (a)(iii) is also about 1.5σ or more for both samples, where σ is the standard deviation defined by the normal distribution of tau phosphorylation at T217 and T205, T181 and T205 or T181, T205 and T217 measured in a control population that does not have brain amyloid plaques as measured by PET imaging and / or Aβ42 / 40 measurement in CSF. In yet further embodiments, the measured levels of tau phosphorylation at the specific sites and the values ​​of MTBR-tau species described in (a)(i), (a)(ii) may be used in various mathematical operations to improve predictive power compared to each other. For example, a ratio may be calculated from the measured phosphorylation levels. Mathematical operations other than ratios may also be used.

[0151] Methods for measuring tau phosphorylation and MTBR-tau are described in Section II and are incorporated herein by reference. However, those skilled in the art will appreciate that absolute values ​​may vary depending on the protocol and source / standard of internal standard used for absolute amount quantification.

[0152] In some embodiments above, processing a blood sample or CSF sample from a subject to obtain a first population of enriched tau species and a depleted sample may include contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope in the N-terminus of tau, or contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope in the mid domain of tau, or contacting the blood sample or CSF sample with a first epitope binding agent that specifically binds to an epitope in the N-terminus of tau and a second epitope binding agent that specifically binds to an epitope in the mid domain of tau. The first and second epitope binding agents can be used sequentially or in combination. In some examples, the epitope binding agent that specifically binds to an epitope in the N-terminus of tau is HJ8.5, or another epitope binding agent that specifically binds to the same epitope as HJ8.5. In some examples, the epitope binding agent that specifically binds to an epitope within the mid domain of tau is tau1 or another epitope binding agent that specifically binds to the same epitope as tau1.

[0153] In other embodiments of the above, treating the depleted sample to obtain a second population of enriched tau species may include performing a chemical extraction step to enrich for MTBR-tau species. In certain embodiments, the chemical extraction step may include mixing an acid to precipitate proteins of the depleted sample, which may be perchloric acid, and the MTBR-tau species being present in the supernatant after removal of the precipitated proteins. Alternatively, treating the depleted sample to obtain a second population of enriched tau species may include contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope within the MTBR of tau. In certain embodiments, the epitope binding agent may be 77G7, RD3, RD4, UCB1017, or PT76 as described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, or E2814 or 7G6 as described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76, E2814 or 7G6, or other epitope binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76, E2814 or 7G6.

[0154] In yet another embodiment of the above, processing a blood sample or CSF sample from a subject to obtain a first population of enriched tau species and a depleted sample may include contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope in the N-terminus of tau, or contacting the blood sample or CSF sample with an epitope binding agent that specifically binds to an epitope in the mid domain of tau, or contacting the blood sample or CSF sample with a first epitope binding agent that specifically binds to an epitope in the N-terminus of tau and a second epitope binding agent that specifically binds to an epitope in the mid domain of tau; and processing the depleted sample to obtain a second population of enriched tau species includes performing a chemical extraction step to enrich MTBR-tau species, or contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope in the MTBR of tau. The first and second epitope binding agents can be used sequentially or in combination. In certain examples, the epitope binding agent that specifically binds to an epitope within the N-terminus of tau is HJ8.5, or another epitope binding agent that specifically binds to the same epitope as HJ8.5; and the epitope binding agent that specifically binds to an epitope within the mid domain of tau is tau1, or another epitope binding agent that specifically binds to the same epitope as tau1. The chemical extraction step may include precipitating proteins of the depleted sample by mixing an acid, which may be perchloric acid, and wherein the MTBR-tau species are present in the supernatant following removal of the precipitated proteins.An epitope binding agent that specifically binds to at least one epitope within the MTBR of tau can be 77G7, RD3, RD4, UCB1017, or PT76 as described in Vandermeeren et al., J Alzheimers Dis, 2018, 65:265-281, or E2814 or 7G6 as described in Roberts et al., Acta Neuropathol Commun, 2020, 8: 13, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76, E2814 or 7G6, or other epitope binding agents that specifically bind the same epitope as 77G7, RD3, RD4, UCB1017, PT76, E2814 or 7G6.

[0155] IV. Treatment Method The present disclosure also encompasses the use of the measurements of time to onset of dementia described herein for staging the disease progression of a subject; for staging the lesions in the brain of a subject; for selecting a diagnostic agent for a subject; and for selecting a therapeutic agent or class of therapeutic agents for a subject according to the disease stage and underlying disease lesions of the subject. Thus, another aspect of the present disclosure is a method for treating a subject, comprising administering to the subject a therapeutic or diagnostic agent selected for the subject in consideration of the measurement of time to onset of dementia of the subject.

[0156] The terms "treat", "treating" or "treatment" as used herein refer to the provision of medical care by a trained and licensed professional to a subject in need thereof. Medical care can be diagnostic testing, therapeutic treatment, and / or prophylactic or preventative measures. The purpose of therapeutic and prophylactic treatment is to prevent or slow (alleviate) undesired physiological changes or diseases / disorders. Beneficial or desired clinical results of therapeutic or prophylactic treatment include, but are not limited to, alleviation of symptoms, attenuation of the extent of disease, stabilized (i.e., not worsening) disease state, delayed or slowed disease progression, amelioration or reduction of disease state, and remission (either partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with a disease, condition or disorder, as well as those prone to having the disease, condition or disorder, or those in whom the disease, condition or disorder is to be prevented. In some embodiments, the subject undergoing treatment is asymptomatic. "Asymptomatic subject" as used herein refers to a subject that does not show any signs or symptoms of AD. In another embodiment, the subject may exhibit signs or symptoms of AD (e.g., memory loss, misplacing things, changes in mood or behavior, etc.), but does not show sufficient cognitive or functional impairment for a clinical diagnosis of dementia due to Alzheimer's disease. Although symptomatic or asymptomatic subjects may have Aβ amyloidosis, prior knowledge of Aβ amyloidosis is not a prerequisite for treatment. In still further embodiments, the subject may be diagnosed with AD. In any of the above-mentioned embodiments, the subject may have one of the genetic mutations known to cause dominantly inherited Alzheimer's disease. In an alternative embodiment, the subject may not have a genetic mutation known to cause dominantly inherited Alzheimer's disease.

[0157] In one embodiment, a method for treating a subject as described above may comprise providing a biological sample obtained from the subject, and (ai) measuring the phosphorylation occupancy of tau at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio, and optionally measuring MTBR-tau212, the phosphorylation occupancy at residue T217 MTBR-tau243, and / or MTBR-tau3R in a blood or CSF sample obtained from the subject, or (aii) measuring the phosphorylation occupancy of tau at residue T205, measuring the phosphorylation occupancy of tau at residue T217, and measuring MTBR-tau212; and (b) administering a pharmaceutical composition to the subject if the measured levels deviate significantly from the mean value in a control population without brain amyloid plaques, as measured by PET imaging and / or Aβ42 / 40 measurement in CSF.

[0158] In another embodiment, the method for treating a subject as described above comprises: (a) providing a first and a second biological sample obtained from the subject and measuring in each sample; (ai) measuring the phosphorylation occupancy of tau at residue T205 in a blood sample or CSF sample obtained from the subject, measuring the MTBR-tau299 / MTBR-tau354 ratio and, optionally, measuring the phosphorylation occupancy at residue T217 of MTBR-tau212. The method may include measuring MTBR-Tau243, and / or MTBR-Tau3R, or (aii) measuring phosphorylation occupancy of tau at residue T205, measuring phosphorylation occupancy of tau at residue T217, and measuring MTBR-Tau212; (b) calculating time to onset of dementia using the measurements or changes in measurements of (ai) or (aii), where time to onset of dementia is the number of years to clinical dementia assessment that is greater than zero; and (C) administering the pharmaceutical composition to the subject if the calculated measurements or changes deviate significantly from the mean value in a control population without brain amyloid plaques, as measured by PET imaging and / or Aβ42 / 40 measurements in CSF. "Significantly deviate from the mean" refers to a value that is at least 1 standard deviation, preferably at least 1.3 standard deviation, more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (i.e., 1σ, 1.3σ, 1.5σ or 1.5σ, respectively, where σ is the standard deviation defined by the normal distribution measured in a control population that does not have brain amyloid plaques as measured by PET imaging and / or Aβ42 / 40 measurement in CSF).In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean can be used as a criterion for treating a subject.

[0159] Instead of or in addition to using the measurement of site-specific tau phosphorylation, in any of the above embodiments, the ratio calculated from the measured level of MTBR-tau species can be used.Mathematical operations other than ratios can also be used.For example, in the examples, site-specific tau phosphorylation values ​​are used in various statistical models (e.g., linear regression, LME curve, LOESS curve, etc.) together with other known biomarkers (e.g., APOE ε4 status, age, sex, cognitive test score, functional test score, etc.).

[0160] Numerous imaging and therapeutic agents contemplated for or used with subjects at risk of developing Aβ amyloidosis or AD, subjects diagnosed with Aβ amyloidosis, subjects diagnosed with tauopathy, or subjects diagnosed with AD target specific pathophysiological changes. For example, Aβ targeted therapies are generally designed to reduce Aβ production, antagonize Aβ aggregation, or increase brain Aβ clearance, tau targeted therapies are generally designed to change tau phosphorylation patterns, antagonize tau aggregation, or increase NFT clearance, various therapies are designed to reduce CNS inflammation or brain insulin resistance, etc. The efficacy of these various agents can be improved by administering the agents to subjects staged by the methods disclosed herein.

[0161] In exemplary embodiments, the efficacy of imaging and therapeutic agents contemplated for, or used together with, a subject at risk of developing Aβ amyloidosis or AD, a subject diagnosed with Aβ amyloidosis, a subject diagnosed with a tauopathy, or a subject diagnosed with AD (collectively referred to herein as "Aβ and tau therapy") can be improved by administering Aβ or tau therapy to a subject having certain tau phosphorylation levels at T205 and / or T217, and / or MTBR-tau299 / MTBR-tau354 ratios, and, where appropriate, measuring MTBR-tau212 and / or MTBR-tau3R and / or MTBR-243 as measured by the methods disclosed herein and shown in the Figures and Examples.

[0162] For example, in embodiments where the time to onset of dementia is measured to be about 20 years, about 25 years, or about 30 years or more, a suitable treatment may be a primary prevention therapy that prevents pathological amyloid deposition (i.e., amyloid deposition greater than would be expected for the subject's age). Non-limiting examples include therapeutic agents that reduce Aβ production, prevent or antagonize Aβ aggregation, or increase brain Aβ clearance, including but not limited to gamma-secretase inhibitors, beta-secretase inhibitors, passive immunotherapy (including but not limited to anti-Aβ antibodies, anti-tau antibodies, or anti-ApoE antibodies), active immunotherapy.

[0163] In embodiments where the time to dementia onset measurement is about 25 years to about 15 years, or about 20 years to about 15 years, a suitable treatment may be a secondary prevention therapy that prevents further pathological amyloid beta deposition or reduces the subject's existing amyloid beta plaque burden. Non-limiting examples include therapeutic agents that reduce Aβ production, prevent or antagonize Aβ aggregation, or increase cerebral Aβ clearance, including but not limited to gamma-secretase inhibitors, beta-secretase inhibitors, and passive immunotherapy (including but not limited to anti-Aβ antibodies, anti-tau antibodies, or anti-ApoE antibodies).

[0164] In the embodiment where the time to dementia onset measurement is about 15 years or less, the suitable treatment may be a secondary prevention therapy that prevents or antagonizes tau aggregation or targets neurofibrillary tangles, in addition to a treatment that prevents further pathological amyloid beta deposition or reduces the subject's existing amyloid beta plaque load.Non-limiting examples include therapeutic agents that prevent or antagonize tau aggregation or target neurofibrillary tangles, including tau protein aggregation inhibitors, kinase inhibitors, phosphatase activators, passive immunotherapy (including but not limited to anti-tau antibodies).

[0165] Non-limiting examples of anti-Aβ antibodies include solanezumab (LY2062430; Eli Lilly), aducanumab (BI-IB037; Biogen), crenezumab (MABT102A, RG7412, Genentech and Roche), gantenerumab (RO4909832, RG14502; Roche), bapinezumab (Janssen and Pfizer), BAN2401 (Eisai), LY3002813 (Lilly), RO7126209 (Roche), AAB-003, and GK933776.

[0166] Non-limiting examples of anti-tau antibodies include semolinemab (AC Immune and Genentech), ABBV-8E12 (Abbvie), BIIB092 (Biogen), BIIB076 (Biogen), LY3303560 (Lilly), RO7105705 (Roche / Genentech), JNJ-63733657 (Janssen), Lu AF87908 (Lundbeck).

[0167] Non-limiting examples of anti-ApoE antibodies include HJ6.3, HAE-4 (WUSTL, Denali Therapeutics).

[0168] Non-limiting examples of therapeutic agents include cholinesterase inhibitors, N-methyl D-aspartate (NMDA) antagonists, antidepressants (e.g., selective serotonin reuptake inhibitors, atypical antidepressants, aminoketones, selective serotonin and norepinephrine reuptake inhibitors, tricyclic antidepressants, etc.), gamma-secretase inhibitors, beta-secretase inhibitors, anti-Aβ antibodies (including antigen-binding fragments, variants or derivatives thereof), anti-tau antibodies (including antigen-binding fragments, variants or derivatives thereof), anti-T ... or derivatives), anti-TREM2 antibodies (including antigen-binding fragments, variants or derivatives thereof), TREM2 agonists, 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.), serotonin receptor 6 antagonists, p38 alpha MAPK inhibitors, recombinant granulocyte macrophage colony stimulating factor, passive immunotherapy, active vaccines (e.g., CAD106, A F20513, etc.), tau protein aggregation inhibitors (e.g. TRx0237, methylthionimium chloride, etc.), treatments to improve glycemic control (e.g. insulin, exenatide, liraglutide pioglitazone, etc.), anti-inflammatory agents, phosphodiesterase 9A inhibitors, sigma 1 receptor agonists, kinase inhibitors, phosphatase activators, phosphatase inhibitors, angiotensin receptor blockers, CB1 and / or CB2 endocannabinoid receptor partial agonists, beta-2 adrenergic receptor agonists, nicotinic acetylcholine receptor agonists, 5-HT2A inverse agonists, alpha-2c adrenergic receptor antagonists, 5-HT1A and 1D receptor agonists, glutaminyl-peptide cyclotransferase inhibitors, selective inhibitors of APP production, monoamine oxidase B inhibitors, glutamate receptor antagonists, AMPA receptor agonists, nerve growth factor stimulants, HMG-CoA reductase inhibitors, neurotrophic agents, muscarinic M1 receptor agonists, GABA receptor modulators, PPAR-gamma agonists, microtubule protein modulators, calcium channel blockers, antihypertensives, statins, and any combination thereof.In an exemplary embodiment, the pharmaceutical composition may include a kinase inhibitor. Suitable kinase inhibitors may inhibit thousand-and-one amino acid kinase (TAOK), CDK, GSK-3β, MARK, CDK5 or Fyn. In another exemplary embodiment, the pharmaceutical composition may include a phosphatase activator. As a non-limiting example, the phosphatase activator may increase the activity of protein phosphatase 2A. In some embodiments, the treatment is a pharmaceutical composition that includes a tau targeted therapy, including, but not limited to, an active pharmaceutical ingredient that alters tau phosphorylation patterns, antagonizes tau aggregation, or increases the clearance of pathological tau isoforms and / or aggregates. In some embodiments, the treatment is an anti-Aβ antibody, an anti-tau antibody, an anti-TREM2 antibody, a TREM2 agonist, a gamma-secretase inhibitor, a beta-secretase inhibitor, a kinase inhibitor, a phosphatase activator, a vaccine, or a tau protein aggregation inhibitor.

[0169] Non-limiting and specific examples of classes of therapeutic agents are also provided in the table below. [Table B] [Table C]

[0170] For example, if the tau phosphorylation at T217 and the MTBR-tau299 / MTBR-tau354 ratio values ​​are greater than the control population average by about 1.5σ or more, and the tau phosphorylation at T205 is close to the average or is greater than the control population average by less than about 1.5σ or more, preferred therapeutic agents can include those designed to prevent the subject from becoming amyloid positive (e.g., amyloid-targeted therapies designed to reduce Aβ production, antagonize Aβ aggregation, etc.). As another example, if the tau phosphorylation at T217 and the MTBR-tau299 / MTBR-tau354 ratio values ​​are greater than the control population average by about 1.5σ or more, and the tau phosphorylation at T205 is greater than the control population average by about 1.5σ or more, preferred therapeutic agents can include those designed to prevent the subject's existing plaque burden from increasing or decreasing due to amyloid deposition. As another example, if tau phosphorylation and MTBR-tau299 / MTBR-tau354 ratio values ​​at T217, and T205 are greater than about 1.5σ above the average of the control population, preferred therapeutic agents can include those designed to prevent amyloid deposition from increasing or decreasing the subject's existing plaque burden, to prevent tau aggregation, or to target NFTs. As another example, if tau phosphorylation and MTBR-tau299 / MTBR-tau354 ratio values ​​at T217 and T205 are greater than the average of the control population by about 1.5σ or more, tau phosphorylation and MTBR-tau299 / MTBR-tau354 ratio values ​​at T217 plateau or decline, and total tau and / or tau phosphorylation at T205 increases, preferred therapeutic agents can include those designed to prevent amyloid deposition from increasing or decreasing the subject's existing plaque burden, to prevent tau aggregation, or to target NFTs, as well as those specific to AD patients. The details disclosed herein can be used similarly to administer therapeutic agents designed for other targets (e.g., CNS inflammation, ApoE, etc.), including but not limited to those identified in the preceding paragraphs. EXAMPLES

[0171] The following examples illustrate various iterations of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention. However, those skilled in the art should understand in light of this disclosure that modifications can 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 described or shown in the accompanying drawings shall be interpreted as illustrative and not limiting in meaning.

[0172] [Example 1] Several sample processing methods have been developed - immunoprecipitation (IP) of N-terminal tau and mid-domain tau as described by Sato et al., 2018; chemical extraction (CX); and a process combining IP and CX methods to enrich MTBR tau (PostIP-CX). The CX and PostIP-CX methods were specifically developed to detect and quantify MTBR tau. An overview of these methods is shown in Figure 2.

[0173] Briefly, CSF (approximately 475 μL) was added as an internal standard. 15 The tau-441 (2N4R) homogeneously labeled solution (approximately 10 μL of 100 pg / μL or approximately 5 μL of 200 pg / μL) was mixed with the tau-441 (2N4R) homogeneously labeled solution. N-terminal tau and mid-domain tau species were immunoprecipitated with tau1 and HJ8.5 antibodies, followed by trypsin digestion and processing as previously described (Sato et al., 2018).

[0174] For the CX method, CSF (approximately 475 μL) was used as the internal standard. 15The supernatant was mixed with a solution containing N-tau-441 (2N4R) uniformly labeled (approximately 10 μL of a solution at 100 pg / μL, or approximately 5 μL of a solution at 200 pg / μL). Tau was then chemically extracted. Highly abundant CSF proteins were precipitated using 25 μL of perchloric acid. After mixing and incubating on ice for 15 min, the mixture was centrifuged at 20,000 g for 15 min at 4° C., and the supernatant was further purified using an Oasis HLB 96-well μElution plate (Waters) according to the following steps: The plate was washed once with 300 μL of methanol and equilibrated once with 500 μL of 0.1% FA in water. The supernatant was added to an Oasis HLB 96-well μElution plate and allowed to adsorb onto the solid phase. The solid phase was then washed once with 500 μL of 0.1% FA in water. Elution buffer (100 μL; 35% acetonitrile and 0.1% FA in water) was added and the eluate was dried by Speed-vac. The dried sample was dissolved with 50 μL of trypsin solution (10 ng / μL) in 50 mM TEABC and incubated at 37° C. for 20 h.

[0175] For the PostIP-CX method, the post-immunoprecipitation CSF (i.e., the supernatant remaining after the IP method above) was treated as described for the CX method.

[0176] After trypsin digestion, all samples were purified by solid-phase extraction on a C18 TopTip. The purification process was spiked with 5 fmol each of the AQUA internal standard peptides of residues 354–369 (MTBR tau-354) and 354–368 (tau 368) for differential quantification. Before elution of the samples, 3% hydrogen peroxide and 3% FA in water was added to the beads, followed by overnight incubation at 4 °C to oxidize peptides containing methionine. The eluates were lyophilized and resuspended in 27.5 μL of 2% acetonitrile in water and 0.1% FA before MS analysis on a nanoAcquity UPLC system coupled to an Orbitrap Fusion Lumos Tribrid or Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operated in PRM mode.

[0177] The CX and PostIP-CX methods produced samples containing MTBR tau that was detectable and quantifiable by mass spectrometry. No quantifiable signal for MTBR tau was obtained with the IP method. Although not demonstrated, it is believed that alternative methods for detecting and quantifying MTBR tau with similar sensitivity may also be used.

[0178] [Example 2] CSF tau analysis by MS: CSF (455 μL) was used as an internal standard. 15 The tau species, which are mainly composed of the N-terminal to mid domain region, were immunoprecipitated with tau1 and HJ8.5 antibodies. The immunoprecipitated tau species were treated and digested as previously described (Sato et al., 2018). Then, 20 μL of 10 ... 15 N-tau internal standard (100 pg / μL) was spiked into the post-immunoprecipitated CSF. Tau was then chemically extracted as previously reported (Barthelemy et al., 2016b) with some modifications. Highly abundant CSF proteins were precipitated using 25 μL of perchloric acid. After mixing and incubating on ice for 15 min, the mixture was centrifuged at 20,000 g for 15 min at 4 °C, and the supernatant was further purified using Oasis HLB 96-well μElution plates (Waters) according to the following steps: The plate was washed once with 300 μL of methanol and equilibrated once with 500 μL of 0.1% FA in water. The supernatant was added to the Oasis HLB 96-well μElution plate and allowed to adsorb onto the solid phase. The solid phase was then washed once with 500 μL of 0.1% FA in water. Elution buffer (100 μL; 35% acetonitrile and 0.1% FA in water) was added and the eluate was dried by Speed-vac. The dried sample was dissolved with 50 μL of trypsin solution (10 ng / μL) in 50 mM TEABC and incubated at 37° C. for 20 h.

[0179] After incubation of both immunoprecipitated and chemically extracted samples, each tryptic digest was purified by solid-phase extraction on a C18 TopTip. The purification process was spiked with 5 fmol each of the AQUA internal standard peptides of residues 354-369 (MTBR tau-354) and 354-368 (tau 368) for differential quantification. Before elution of the samples, 3% hydrogen peroxide and 3% FA in water was added to the beads, followed by overnight incubation at 4 °C to oxidize peptides containing methionine. The eluates were lyophilized and resuspended in 27.5 μL of 2% acetonitrile in water and 0.1% FA before MS analysis on a nanoAcquity UPLC system coupled to an Orbitrap Fusion Lumos Tribrid or Orbitrap Tribrid Eclipse mass spectrometer (Thermo Scientific) operated in PRM mode. Nineteen CSF tau peptides were quantified (Table 1). The schematic procedure for CSF tau analysis is described in Figure 2A.

[0180] Statistical analysis: Differences in biomarker values ​​were assessed by one-way ANOVA unless otherwise specified. A two-tailed p<0.05 was considered statistically significant and corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) method (Benjamini and Hochberg, 1995), with the FDR set at 5%. Spearman correlations were used to assess associations between tau biomarkers and cognitive test measures and tau PET SUVR. [Table 1]

[0181] [Example 3] An additional sample processing method, termed "PostIP-IP," was developed and compared to the PostIP-CX method described in Examples 1 and 2. An exemplary workflow of the PostIP-IP method is provided in Figures 3 and 4.

[0182] CSF samples obtained from the LOAD100 cohort described in Example 2 were processed by the PostIP-CX method (Example 1) or the PostIP-IP method (this example) and then analyzed by LC-MS as generally described in Example 2.

[0183] [Example 4] Advances in methods for identifying and quantifying increasing soluble tau species from plasma and cerebrospinal fluid (CSF) provide an opportunity to better understand the role of tau-related pathology in Alzheimer's disease (AD). Recent studies of dominantly inherited and sporadic Alzheimer's disease have demonstrated that hyperphosphorylation at specific locations of tau protein, N-terminal fragments and microtubule binding region (MTBR), are associated with various aspects of the Alzheimer's disease cascade. However, the potential unique information provided by the various soluble tau species needs to be evaluated. The Dominantly Inherited AD Network (DIAN) study provides an opportunity to investigate the association of multiple soluble tau species with disease progression and multiple biomarkers of amyloid and neurodegeneration.

[0184] In this example, more than 12 CSF phosphorylated tau (p-tau) and truncated MTBR species were quantified from 227 mutation carriers (MCs) (152 asymptomatic MCs, 77 symptomatic MCs) and 141 non-carriers enrolled in DIAN. The optimal combination of p-tau and MTBR species to predict expected years to symptom onset (EYO) was evaluated using linear regression with stepwise selection. Correlation heatmaps and hierarchical clustering were used to explore association patterns of baseline levels and longitudinal rates of change between tau / p-tau species and other biomarkers.

[0185] DIAN is an international, multicenter registry of individuals (mutation carriers and non-carriers, asymptomatic and symptomatic) who are the biological adult children of parents with known AD-causing mutations in the APP, PSEN1, or PSEN2 genes; in DIAN, individuals are assessed with uniform measures at enrollment and longitudinally thereafter using standardized instruments. Standard measures include: (1) the Unified Data Set (UDS) clinical and cognitive battery, as well as additional neuropsychological and personality measures; (2) Alzheimer's Disease Neuroimaging Initiative (ADNI) structural imaging (magnetic resonance imaging, i.e., MRI, functional imaging (18-fluorodeoxyglucose positron emission tomography, i.e., FDG PET), and amyloid imaging (Pittsburgh Compound-B, i.e., PIB) PET protocol); (3) collection of body fluids (blood, CSF) for DNA analysis and assay of putative biomarkers of AD, according to the ADNI protocol, and (4) uniform histopathological examination of brain tissue of autopsied individuals. In DIAN, symptomatic individuals are those with a Clinical Dementia Rating greater than zero (CDR>0). The Clinical Dementia Rating is a well-known measure used to quantify the severity of dementia symptoms. Symptom risk in DIAN is defined by EYO. EYO was defined as the parent's age at dementia diagnosis minus the participant's current age. For participants who were symptomatic at baseline, assessed with a CDR > 0, EYO was defined as the reported age at actual symptom onset minus the age at each clinical assessment.

[0186] Detection and quantification of phosphorylated tau: sample processing Human CSF was pooled from a cohort of 80 participants including amyloid-negative and cognitively normal (CDR=0) controls (n=47, age 60+) and amyloid-positive and CDR>0 AD patients (n=33, age 60+). Five and seven pools of 500 μL CSF aliquots were generated from the control and AD groups, respectively. Upon initial collection, CSF was spun down at 1,000×g for 10 min to remove cellular debris and immediately frozen at −80°C. Protease inhibitor cocktails were added during the experiment. Tau was immunoprecipitated and desalted as previously described (Sato et al., 2018) with some modifications. Briefly, CNBr-activated Sepharose beads (GE Healthcare 17-0430-01) were crosslinked separately to antibodies tau1 and HJ8.5 at a concentration of 3 mg antibody per gram of beads. Samples were spiked with AQUA peptides (ThermoFisher Scientific) to provide 10 fmol of phosphorylated tau and 100 fmol of non-phosphorylated tau for each sequence of interest per microliter of sample. Tau and p-tau concentrations are calculated using these internal standards. Soluble tau was immunoprecipitated in detergent (1% NP-40), chaotropic reagent (5 mM guanidine) and protease inhibitor (Roche Complete Protease Inhibitor Cocktail). Anti-tau1 and HJ8.5 antibodies conjugated to Sepharose beads were diluted 10-fold and 5-fold, respectively, in inactivated Sepharose beads, and 30 μL of a 50% slurry of antibody beads was rotated with the solution for 90 minutes at room temperature. Beads were washed three times in 25 mM triethylammonium bicarbonate buffer (TEABC, Fluka 17902). Bound tau was digested on-bead with 400 ng MS-grade trypsin (Promega, V5111) for 16 h at 37° C. The digest was loaded onto a TopTip C18 (Glygen, TT2C18.96), desalted, and eluted according to the manufacturer's instructions.The eluted peptides were dried by vacuum centrifugation (CentriVap Concentrator Labconco) and resuspended in 25 μL of a solution of 2% acetonitrile and 0.1% formic acid in MS grade water.

[0187] Human blood is treated in substantially the same manner as described above, although larger volumes of blood (eg, about 500 μL to about 10 mL) may be used.

[0188] Additional details are described in PCT / US2019 / 030725, the disclosure of which is incorporated herein by reference.

[0189] Detection and quantification of MTBR-tau: sample processing For detection and quantification of MTBR tau species, CSF was processed as shown in FIG. 4. Immunoprecipitation with tau1 and HJ8.5 was as generally described above. E2814 is an anti-tau antibody (Eisai). See, e.g., Roberts et al., Acta Neuropathol Commun, 2020, 8(1): 13. Trypsin digestion and desalting using TopTip C18 was performed as generally described above. Human blood is processed in essentially the same manner, although larger volumes of blood (e.g., about 0.5 mL to about 10 mL) may be used.

[0190] Detection and quantification of phosphorylated tau and MTBR tau: Mass spectrometry A 5 μL aliquot of the peptide resuspension was injected into a nano-Acquity LC for MS analysis. The nano-Acquity LC (Waters Corporation, Milford, MA) was fitted with an HSS T3 75 μm × 100 μm, 1.8 μm column and peptides were separated using a gradient of solutions A and B at a flow rate of 0.5 μL / min. Solution A consisted of 0.1% formic acid in MS grade water and solution B consisted of 0.1% formic acid in acetonitrile. Peptides were eluted from the column with a gradient of 2% to 20% solution B for 28 min, then 20% to 40% solution B for an additional 13 min, followed by an increase to 85% solution B for an additional 3 min to clean the column. The Orbitrap Fusion Lumos was equipped with a Nanospray Flex electrospray ion source (Thermo Fisher Scientific, San Jose, CA). Peptide ions sprayed into the ion source from a 10 μm SilicaTip emitter (New Objective, Woburn, Ma) were targeted to the quadrupole and isolated in the quadrupole. They were fragmented by HCD and the ion fragments were detected in the Orbitrap (resolution 30,000 or 60,000, mass range 150-1200 m / z). Monitoring of hydrophilic peptides (SSRcalc<9, all leucine-free) for peptide profiling was performed on an HSS T3 300 μm × 100 μm, 1.8 mm column at a flow rate of 4 μl / min, elution occurred with a gradient of 2% to 12% solution B, and the spray was run on a 30 mm SilicaTip emitter.

[0191] A list of some MS transitions is shown below. The peptides are listed in the left column. The nomenclature "K.IATPR.G" indicates that the tryptic peptide is IATPR (SEQ ID NO: 16). The "." in "K.IATPR.G" is used by the software program to mark the cleavage site.

[0192] result The best combination of tau biomarkers for predicting EYO was the phosphorylation occupancy at T205 (pT205 / T205), the MTBR-tau299 / MTBR-tau354 ratio, and MTBR-tau212 for all mutation carriers (MCs) (r 2 =0.6). pT205 / T205, pT217 / T217, and MTBR-tau212 were the best combinations for asymptomatic MC (r 2 = 0.47), and pT205 / T205 alone was most predictive for symptomatic MC (r 2 = 0.12). Baseline phosphorylation occupancy (pT217 / T217, pT181 / T181, pT153 / T153, pT111 / T111, pT205 / T205, pS208 / S208) and two MTBR ratios (MTBR-tau299 / MTBR-tau354, MTBR-tau299 / MTBR-tau282)) clustered together with amyloid pathology (PiB PET, CSF Aβ42 / Aβ40 ratio), whereas MTBR-tau was more associated with total tau and neuronal injury / neuroinflammation biomarkers (YKL40, NGRN, VILIP1, SNAP25). Annual changes in pT153 / T153 were most highly correlated with annual changes in PiB PET (-0.43) and CSF Aβ42 / Aβ40 (0.67); whereas annual changes in pT217 / T217, pT181 / T181, total tau, pT153 / T153, MTBR-3R, pT111 / T111, pT231 / T231, MTBR-tau275, and MTBR-tau299 were highly correlated (r>0.5) with annual changes in the cognitive composite.

[0193] This study further highlights the diversity of soluble tau-related changes that occur with AD progression. Importantly, this study suggests that there are distinct stages in the progression of tau-related pathological changes that track disease progression and changes in specific non-tau biomarkers. These findings may aid in understanding the role of tau in AD and the outcome of therapeutic trials. These findings are also utilized in the diagnosis, prognosis, and treatment of AD. As a non-limiting example, these findings can be used to determine whether a subject should undergo additional diagnostic testing and / or select an appropriate test (e.g., amyloid-based PET, tau-based PET, etc.). As another non-limiting example, these findings can be used to select a therapeutic agent or class of therapeutic agents for a subject that is tailored to the subject's disease stage and underlying disease pathology (as measured by the tau biomarker). [Table 2] [Table 3] [Table 4]

[0194] [Example 5] Background: Brain tau aggregation is a pathological hallmark of Alzheimer's disease (AD), and the tau microtubule-binding region (MTBR) is the core of AD tau tangles. Recently, tau residues 243-254 in R1 (MTBR-tau243), 299-317 in R2 (MTBR-tau299), and 354-369 in R4 (MTBR-tau354) were identified as being specifically enriched in sporadic AD brain tangles. The corresponding soluble MTBR species in CSF increased with high correlation with clinical progression and tau pathology measured by tau-PET. Extracellular MTBR-tau was recently discovered in human AD, making MTBR-tau a preferred target for antibodies targeting this region. In this study, we sought to determine when these MTBR-tau alterations occurred and their relationship to clinical, cognitive and biomarker changes in DIAN.

[0195] METHODS:We developed a highly accurate method to quantify more than 12 tau species in CSF. To profile MTBR-tau, we performed immunoprecipitation using anti-MTBR, antibody E2814, which is dual epitope for R2 and R4, and potential tau agents. We analyzed CSF from 227 mutation carriers (MC) (152 asymptomatic, 77 symptomatic) and 141 non-carriers enrolled in the DIAN observational cohort.

[0196] Results: CSFMTBR-tau was specifically increased in MCs and changed at different time points. The change in MTBR-tau299 in R2 occurred at estimated symptom onset year (EYO)-22, close to when the change in p-tau217 occupancy was first detected (EYO-21). The increase in MTBR-tau354 in R4 saturated at later clinical stages, potentially due to deposition in brain tangles. Notably, the ratio of MTBR-tau299 / 354, which recapitulates tau pathophysiology, was highly correlated (r=0.82) with p-tau217 occupancy, an early amyloid marker, especially in asymptomatic MCs, indicating MTBR-tau and p-tau217 as biomarkers for evaluating anti-MTBR therapy in tau-PET-negative populations.

[0197] Conclusion: We identified MTBR species, a soluble measure of tau tangles that are altered years before symptom onset and continue into the symptomatic stage, suggesting that early intervention may be performed with anti-MTBR-tau antibodies such as E2814. Finally, we used these results to design the tau next-generation platform in DIAN trial unit (DIAN-TU) E2814 drug, which targets soluble MTBR-tau in an independent cohort of asymptomatic and symptomatic carriers. In sporadic AD and DIAD, CSF "E2814-associated" MTBR was specifically increased in AD. E2814-associated MTBR-tau profiles across the AD continuum appear to be similar between sAD and DIAD. Increased in early stages (preclinical stages) of sAD and DIAD (-20 EYO). Levels of E2814-associated MTBR decline after AD onset (CDR>1, EYO>0). At MTBR-260 (late R1), 270 (early R2), 282 (mid R2), and 299 (R2-R3), E2814-associated MTBR-tau levels correlate well with CX levels. At MTBR-243 (upstream R1) and 354 (R4), the correlation between E2814-associated MTBR-tau levels and CX levels is low, potentially due to MTBR cleavage and recruitment to tau aggregates in the brain. E2814-IP studies revealed the presence of multiple cleavage sites in CSFMTBR-tau, e.g., 254 (upstream R1)-260 (early R1) and 354 (R4)-386 (C-terminus). Longitudinal assessment revealed a specific trajectory in which MTBR-tau 299 and 354 increase before AD onset, whereas they decrease after AD onset (→reflecting brain tau aggregates). "E2814-associated MTBR-tau" in CSF can predict cognitive measures in AD when considering the MTBR-299 / 354 ratio. "E2814-associated MTBR-tau" in CSF reproduces tau pathology (tau-PET) in "early stage AD" only when considering the MTBR-299 / 354 ratio.

Claims

1. A method for measuring time to onset of dementia in a subject, comprising: (a ｉ ) measuring the phosphorylation occupancy of tau at residue T205, measuring the MTBR-tau299 / MTBR-tau354 ratio, and optionally measuring MTBR-tau212, phosphorylation occupancy at residue T217, MTBR-tau243, MTBR-tau3R, or a combination thereof in a blood sample or cerebrospinal fluid (CSF) sample obtained from said subject; or (a ｉｉ ) measuring the phosphorylation occupancy of tau at residue T205, measuring the phosphorylation occupancy of tau at residue T217, and measuring MTBR-tau212 in a blood sample or CSF sample obtained from said subject; and (b)(a ｉ ) or (a ｉｉ calculating time to onset of dementia using said measure of dementia severity, where time to onset of dementia is the number of years to a Clinical Dementia Rating (CDR) greater than zero. Including, Wherein the object is (i) is not accompanied by cognitive or behavioral symptoms of Alzheimer's disease; or (ii) having one or more cognitive or behavioral symptoms of Alzheimer's disease.

2. A method for measuring time to onset of dementia in a subject, comprising: (a) processing a blood or cerebrospinal fluid (CSF) sample from the subject to obtain a first population of tau species and a depleted sample, and then processing the depleted sample to obtain a second population of tau species; wherein said first population of tau species is enriched in N-terminal tau and / or mid domain tau, and said second population of enriched tau species is enriched in MTBR-tau; (b ｉ ) measuring the phosphorylation occupancy of tau at residue T205 in said first population of tau species, and measuring the MTBR-tau299 / MTBR-tau354 ratio in said second population of tau species, and optionally measuring MTBR-tau212 in said second population of tau species; or (b ｉｉ ) measuring the phosphorylation occupancy of tau at residue T205 in said first population of tau species, and measuring the phosphorylation occupancy of tau at residue T217 in said first population of tau species, and measuring MTBR-tau212 in said second population of tau species; and (c)(b) ｉ ) or (b ｉｉ calculating time to onset of dementia using said measure of 0.5 mg / kg / day, where time to onset of dementia is the number of years to a Clinical Dementia Rating (CDR) greater than zero; Including, Wherein the object is (i) is not accompanied by cognitive or behavioral symptoms of Alzheimer's disease; or (ii) having one or more cognitive or behavioral symptoms of Alzheimer's disease.

3. processing a blood or CSF sample from said subject to obtain a first population of enriched tau species and a depleted sample; contacting the blood sample or the CSF sample with an epitope binding agent that specifically binds to an epitope within the N-terminus of tau; contacting the blood sample or the CSF sample with an epitope binding agent that specifically binds to an epitope within the mid domain of tau; or contacting the blood sample or the CSF sample with a first epitope binding agent that specifically binds to an epitope within the N-terminus of tau and a second epitope binding agent that specifically binds to an epitope within the mid domain of tau; wherein said first and second epitope binding agents are used sequentially or simultaneously; Including; 3. The method of claim 2, wherein the epitope binding agent that specifically binds to an epitope within the N-terminus of tau is HJ8.5 or another epitope binding agent that specifically binds to the same epitope as HJ8.5, and wherein the epitope binding agent that specifically binds to an epitope within the mid domain of tau is tau1 or another epitope binding agent that specifically binds to the same epitope as tau1.

4. processing the depleted sample to obtain a second population of enriched tau species; carrying out a chemical extraction step to enrich for MTBR-tau species; wherein optionally said chemical extraction step comprises mixing an acid to precipitate proteins of said depleted sample, wherein optionally said acid is perchloric acid, and wherein said MTBR-tau species are present in the supernatant after removal of said precipitated proteins; or contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope within the MTBR of tau; wherein optionally the epitope binding agent is 77G7, RD3, RD4, UCB1017, PT76 or 7G6, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76 or 7G6, or another epitope binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76 or 7G6. The method of claim 2 , comprising:

5. processing a blood or CSF sample from said subject to obtain a first population of enriched tau species and a depleted sample; contacting the blood sample or the CSF sample with an epitope binding agent that specifically binds to an epitope within the N-terminus of tau; contacting the blood sample or the CSF sample with an epitope binding agent that specifically binds to an epitope within the mid domain of tau; or contacting the blood sample or the CSF sample with a first epitope binding agent that specifically binds to an epitope within the N-terminus of tau and a second epitope binding agent that specifically binds to an epitope within the mid domain of tau; wherein the first and second epitope binding agents are used sequentially or simultaneously; Including, Optionally, the epitope binding agent that specifically binds to an epitope within the N-terminus of tau is HJ8.5 or another epitope binding agent that specifically binds to the same epitope as HJ8.5, and optionally, the epitope binding agent that specifically binds to an epitope within the mid domain of tau is tau1 or another epitope binding agent that specifically binds to the same epitope as tau1; and processing the depleted sample to obtain a second population of enriched tau species; carrying out a chemical extraction step to enrich for MTBR-tau species; wherein optionally said chemical extraction step comprises mixing an acid to precipitate proteins of the depleted sample, wherein optionally said acid is perchloric acid, and wherein said MTBR-tau species are present in the supernatant after removal of precipitated proteins; or contacting the depleted sample with an epitope binding agent that specifically binds to at least one epitope within the MTBR of tau; wherein optionally the epitope binding agent is 77G7, RD3, RD4, UCB1017, PT76 or 7G6, or an antigen-binding fragment of 77G7, RD3, RD4, UCB1017, PT76 or 7G6, or another epitope binding agent that specifically binds to the same epitope as 77G7, RD3, RD4, UCB1017, PT76 or 7G6. The method of claim 2 , comprising:

6. 6. The method of any one of claims 1 to 5, wherein the subject is free of cognitive or behavioral symptoms of Alzheimer's disease and has a zero CDR.

7. The method of claim 1, wherein the subject has one or more cognitive or behavioral symptoms of Alzheimer's disease and has a CDR of 0.5 or more, a CDR of 1 or more, or a CDR of 2 or more.

8. The measured value of time to onset of dementia is (i) staging the progression of disease in a subject; (ii) staging a lesion in the brain of a subject; or (iii) selecting a therapeutic or diagnostic agent for a subject; 8. The method according to any one of claims 1 to 7, for use in 9. A therapeutic or diagnostic agent for use in the treatment of Alzheimer's disease in a subject in need thereof, said therapeutic or diagnostic agent selected in accordance with claim 8.

10. The diagnostic agent is (a) a tau PET tracer, optionally selected from the group consisting of flortaucipir, MK-6240, PI-2620, RO-948, GPT1, JNJ-067, JNJ-64349311, APN-1607, SNFT-1, PM-PBB3, AV1451, THK5351 and THK5317; or (b) an Aβ PET tracer, optionally selected from the group consisting of FDDNP, AV-45, GE067, BAY 94-9172, PI, GDF, NaF, p5+14, florbetapir, florbetaben, and flutemetamol; The therapeutic or diagnostic agent according to claim 9.

11. The therapeutic agent (a) reducing Aβ production, preventing or antagonizing Aβ aggregation, or increasing brain Aβ clearance; Optionally, the therapeutic agent is a gamma-secretase inhibitor, a beta-secretase inhibitor, passive immunotherapy or active immunotherapy; or (b) preventing or antagonizing tau aggregation, increasing neurofibrillary tangle clearance, and altering the phosphorylation pattern of tau; Optionally, the therapeutic agent is a tau protein aggregation inhibitor, a kinase inhibitor, a phosphatase activator, passive immunotherapy or active immunotherapy. The therapeutic or diagnostic agent according to claim 9.

12. The therapeutic agent is (a) a gamma secretase inhibitor or a beta secretase inhibitor; (b) an anti-Aβ antibody, an anti-tau antibody, or an anti-ApoE antibody; or (c) a cholinesterase inhibitor, an NMDA receptor antagonist, or an anti-inflammatory agent; The therapeutic or diagnostic agent according to claim 9.

13. 1. A method for measuring changes in cognition in a subject, comprising: (a) measuring the phosphorylation occupancy of tau at one or more residues selected from T111, T153, T181, T217 and T231 in a blood sample or cerebrospinal fluid (CSF) sample obtained from the subject; and Measuring at least one of MTBR-tau275, MTBR-tau299, and MTBR-3R in a blood or CSF sample from the subject; and Optionally, measuring total tau in a blood or CSF sample obtained from said subject; and (b) calculating change in cognition using the measures of (a); and optionally, said subject is (i) there are zero CDRs; or (ii) The method, wherein the CDR is 0.5 or greater.

14. 14. The method of claim 13, wherein the change in cognition is equivalent to the change in cognition as measured by a cognitive composite score consisting of the Delayed Recall score from the International Shopping List Test, the Logical Memory Delayed Recall score from the Wechsler Memory Scale-Revised, the Digit Symbol Coding test total score from the Wechsler Adult Intelligence Scale-Revised, and the MMSE total score.

15. Use of a measure of change in cognition as described in claim 13 or 14 to assess the effectiveness of a therapeutic agent in a subject. (a) an epitope binding agent comprising: (i) an epitope binding agent that specifically binds to an epitope within the N-terminus of tau; (ii) an epitope binding agent that specifically binds to an epitope within the mid domain of tau; and (iii) an epitope binding agent that specifically binds to an epitope within the NTBR of tau; one or more epitope binding agents selected from: (b) Instructions for use, comprising: (i) measuring the time to dementia in a subject according to claims 1 to 7, or (ii) measuring cognitive changes in a subject according to claim 13 or 14. Instructions for Kit including:

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