Methods of treating alzheimer's disease

Abstract

The disclosure is related to methods of treating a subject with Alzheimer's Disease (AD) using anti-amyloid beta (Aβ) therapy. The patient may be treated based on the measurement and analysis of biomarker levels, such as plasma Aβ42 / 40 ratio, pTau181 and pTau217. The disclosure includes a systematic approach called CP Convert for converting biomarker measurements across different analytical platforms, enabling comparison of pTau217 data from LC-MS / MS, SIMOA, and chemiluminescent enzyme immunoassay platforms. Methods demonstrate that pTau217 has superior diagnostic accuracy compared to Aβ42 / 40 ratio and pTau181 for detecting brain amyloid positivity. Implementation of pTau217 prescreening can significantly reduce Aβ-PET testing, decreasing patient burden and clinical trial costs. The CP Convert methodology can be validated using independent cohorts, demonstrating robust cross-platform conversion for research applications.

Description

METHODS OF TREATING ALZHEIMER’S DISEASEFIELD OF THE INVENTION

[0001] The disclosure is related to methods of treating a subject with Alzheimer’s Disease (AD) using anti-amyloid beta (A0) therapy. The patient may be treated based on the measurement and analysis of biomarker levels.BACKGROUND

[0002] Alzheimer’s Disease (AD) is the most common cause of dementia which affects more than 6.9 million Americans aged 65 and older and 55 million people worldwide.Development of effective therapies for AD have been hampered by expensive clinical trials that take longer to complete than trials in most other therapeutic categories. Effective and accessible diagnosis and screening remain a major barrier to the recruitment in clinical trials and to early detection for initiation of approved therapies for AD.

[0003] The application of amyloid beta (AP) Positron Emission Tomography (PET), often referred to as amyloid-PET or AP-PET, is considered the “gold-standard” parameter for screening of prospective participants in AD clinical trials. Additionally, AP-PET is required to confirm AD diagnosis prior to initiation of anti-Ap therapies, such as Leqembi® (Eisai and Biogen) and Kisunla™ (Eli Lilly), the only disease modifying therapies for AD approved by the FDA. AP-PET molecular imaging has been validated against autopsy findings and enables direct visualization and quantification of fibrillary amyloid deposits in vivo.However, the utility of AP-PET is limited due to high cost, accessibility, and the prevalence of amyloid plaques in cognitively normal individuals, requiring careful clinical assessment alongside the scan.

[0004] Although recently approved biomarkers in CSF such as AP42 / 40 and pTau fragments can substitute for PET imaging, these tests are still relatively invasive due to required lumbar puncture. Recently, the FDA approved the first plasma pTau217 assay, the Lumipulse GpTau217 / beta amyloid 42 plasma ratio by Fujirebio, as an in vitro diagnostics for evaluation of AD making a significant advance toward accessible and non-invasive screening tool. However, this diagnostic for AD requires measuring two biomarkers with the specific assays. Not every lab has access to these assays limiting the accessibly of this diagnostic.Accordingly, there is a need for simple, easy-to-use, accessible, less expensive screening and diagnostic tools using blood biomarkers that can improve screening and enrollment for AD clinical trials and identify patients for treatment with approved therapies.SUMMARY

[0005] The present disclosure relates to methods for treating, selecting, and diagnosing subjects with Alzheimer's Disease (AD) using plasma-based biomarkers, as well as methods for conducting clinical trials and reducing the burden of expensive imaging procedures such as amyloid beta positron emission tomography (AP-PET).

[0006] In one aspect, the invention provides methods of treating subjects with Alzheimer's Disease by administering a therapeutically effective amount of an anti-amyloid beta (AP) therapy to subjects having specific biomarker profiles. The key biomarkers include plasma AP42 / 40 ratio, plasma pTaul81, and plasma pTau217, with specific threshold values that indicate suitability for treatment: plasma AP42 / 40 ratio less than 0.0975 or less than 0.1020, plasma pTaul81 greater than 9.4 pg / mL, and plasma pTau217 greater than 1.2 pg / mL.

[0007] The invention also encompasses methods for selecting subjects for Alzheimer's Disease treatment by measuring these same biomarkers and applying the same threshold criteria to identify appropriate candidates for anti-Ap therapy.

[0008] In another aspect, the disclosure provides methods to addresses the challenge of biomarker measurement across different analytical platforms. The methods include determining biomarker levels using liquid chromatography tandem mass spectrometry (LC-MS / MS), single molecule array technology (Simoa), and chemiluminescent enzyme immunoassay (CLEIA).

[0009] For pTau217 measurements, the disclosure provides specific conversion methodologies between platforms. Simoa-measured pTau217 levels can be converted to LC- MS / MS equivalent values using the relationship: log(Simoa) = -1.41 + 1.11 x log(LC- MS / MS). Similarly, CLEIA-measured pTau217 levels can be converted using the relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

[0010] In another aspect, the disclosure provides methods for conducting clinical trials for anti-Ap therapies while reducing reliance on expensive Ap-PET procedures. These methods include identifying subjects for clinical trials using the plasma biomarker criteria described herein, allowing for enrollment of subjects who have not undergone AP-PET procedures or using biomarker screening to pre-select subjects most likely to benefit from subsequent AP- PET evaluation.

[0011] In certain embodiments, the methods achieve at least a 60% reduction in the number of subjects requiring AP-PET scanning compared to traditional screening approaches by implementing pTau217 prescreening using a cutoff corresponding to 90% sensitivity.

[0012] In another aspect, the disclosure provides diagnostic methods for Alzheimer's Disease based on the same biomarker thresholds, as well as methods for predicting a subject's response to AD treatment. These methods demonstrate that pTau217 achieves superior performance with an area under the curve (AUC) of at least 0.94 in ROC analysis for predicting AP-PET positivity, compared to AP42 / 40 ratio (AUC < 0.85) and pTaul81 (AUC < 0.82).

[0013] In some embodiments, the anti-Ap therapies employed in the methods include both active and passive therapeutic approaches. Passive therapies comprise anti-Ap antibodies,including agents such as PRX012, lecanemab, donanemab, aducanumab, and gantenerumab. Active therapies include vaccine-based approaches.

[0014] In embodiments, methods of the disclosure provide flexibility in incorporating AP- PET procedures within the biomarker-based approach. Methods include scenarios where subjects have not undergone Ap-PET procedures prior to treatment, as well as approaches where AP-PET is performed after biomarker measurement but prior to therapy administration. When AP-PET is utilized, subjects with an Ap brain load of at least 30 centiloids are selected for treatment.

[0015] The methods of the disclosure provide a comprehensive approach to Alzheimer's Disease management that leverages accessible blood-based biomarkers to improve patient selection, reduce healthcare costs, and enhance the efficiency of clinical trials while maintaining therapeutic efficacy through precise biomarker-guided treatment decisions.BRIEF DESCRIPTION OF THE FIGURES

[0016] Fig. 1 A shows an inverse correlation between AP42 / 40 ratio and brain amyloid as measured by AP-PET centiloid (CL) (n=555). A higher AP42 / 40 ratio is associated with lower amyloid burden. Vertical dashed lines represent amyloid CL threshold 30. Fig. IB shows the ROC curve for AP42 / 40 ratio as a predictor of amyloid positivity (AP-PET >30 CL), as well as AUC, and a AP42 / 40 ratio cutoff for a sensitivity of 0.90. AUC, area under the ROC curve; NPV, negative predictive value; PPV, positive predictive value; Se, selectivity; Sp, specificity.

[0017] Fig. 2A shows a direct correlation between pTaul81 level and brain amyloid as measured by AP-PET centiloid (CL) (n=196). A higher pTaul81 level is associated with higher amyloid burden. Fig. 2B shows the ROC curve for pTaul81 as a predictor of amyloid positivity (Ap-PET >30 CL), as well as AUC, and a pTaul81 level cutoff for a sensitivity of0.90.

[0018] Fig. 3A shows a direct correlation between pTau217 level and brain amyloid as measured by AP-PET centiloid (CL) (n=183). A higher pTau217 level is associated with higher amyloid burden. Fig. 3B shows the ROC curve for pTau217 as a predictor of amyloid positivity (AP-PET >30 CL), as well as AUC, and a pTau217 level cutoff for a sensitivity of 0.90.

[0019] Fig. 4 shows a comparison of ROC curves assessing the performance of AP42 / 40 ratio, pTaul81 levels, and pTau217 levels to identify amyloid positivity (AP-PET >30 CL) (n=174). AUCs for AP42 / 40 ratio, pTaul81, and pTau217 were 0.84, 0.81, and 0.95, respectively.

[0020] Fig. 5 A to Fig. 5C show evaluations of the correlations between pTau217 levels from different assay platforms (n=l 16). Correlations were assessed with linear data using simple linear regression. Additionally, conversion equations for pTau217 levels between assay platforms were developed. Fig. 5 A shows significant correlation of pTau217 levels between the Simoa and liquid chromatography-tandem mass spectrometry (LC-MS / MS) assay platforms. Fig. 5B shows significant correlation of pTau217 levels between the Lumipulse and LC-MS / MS assay platforms. Fig. 5C shows significant correlation of pTau217 levels between the Simoa and Lumipulse assay platforms.

[0021] Fig. 6A to Fig. 6C show evaluations of the correlations between pTau217 levels from different assay platforms (n=l 16). Correlations were assessed with log (base 10) transformed data using Deming regression. Additionally, conversion equations for pTau217 levels between assay platforms were developed. Red dots represent amyloid positive subjects (Ap- PET >30 CL) and blue dots represent amyloid negative subjects (AP-PET <30 CL). Fig. 6A shows significant correlation of pTau217 levels between the Simoa and liquid chromatography-tandem mass spectrometry (LC-MS / MS) assay platforms. Fig. 6B show significant correlation of pTau217 levels between the Lumipulse and LC-MS / MS assayplatforms. Fig. 6C shows significant correlation of pTau217 levels between the Simoa and Lumipulse assay platforms.

[0022] Fig. 7 shows a comparison of ROC curves assessing the performance of pTau217 levels, as measured by LC-MS / MS, Simoa and Lumipulse assay platforms, to identify amyloid positivity (AP-PET >30 CL) (n=l 16). AUCs for LC-MS / MS, Simoa and Lumipulse assays were 0.935, 0.929, and 0.912, respectively.

[0023] Fig. 8 shows clinical trial enrollment approaches, using hypothetical enrollment numbers depicting use of AP-PET imaging as the first biomarker assessment for eligibility determination (traditional; left) versus use of pTau217 as a prescreening biomarker test prior to AP-PET imaging (improved; right). The number of subjects and percentage that fail defined eligibility criteria or progress from one step to the next are depicted. Implementing pTau217 prescreening results in reduced number of AP-PET scans which are expensive, time consuming, and burdensome on subjects.DESCRIPTION

[0024] AD is characterized by extracellular inclusions of beta amyloid (AP) peptide- containing plaques and intracellular aggregates of tau (neurofibrillary tangles; NFTs) in the brain. Ap is generated through the sequential cleavage of enzymes to produce the two major forms of Ap - Ap42 and Ap40, with Ap42 being more prone to aggregation. As more Ap42 gets sequestered into plaques in the AD brain, there is a reduction of AP42 / 40 peptide ratio in the blood plasma.

[0025] The tau protein has more than 80 putative phosphorylation sites with each having a different effect on aspects of the biology including aggregation. Phosphorylation of tau on residue 181 (pTaul81) can differentiate AD from healthy controls. In addition, phosphorylation of Tau on residue 217 (pTau217) in plasma has been shown to have better performance in predicting amyloid load than pTau!81.

[0026] In one aspect, the disclosure is directed to determining the level of a blood based biomarker with an assay and, optionally, converting the biomarker level into another level for a different assay.

[0027] In one embodiment, the blood based biomarker is an Ap species. In one embodiment, an Ap species comprises one or more of Ap, a fragment thereof, a cleavage product thereof, posttranslational modifications of any of the foregoing; and monomers, oligomers, fibrils, and plaques that include of any of the foregoing. In a further embodiment, the Ap species comprises one or more of Ap37, Ap38, Ap39, Ap40, Ap42, N-truncated Ap, pyroglutamate Ap (e.g., pGlu-Ap3-42), APP cleavage products, and Ap mid-domain fragments. In another embodiment, the Ap species is a relationship between or calculation based on different Ap species comprising one or more of AP42 / 40 ratio.

[0028] In one embodiment, the blood based biomarker is a tau species. In one embodiment, a tau species comprises one or more of tau, a fragment thereof, a cleavage product thereof, posttranslational modifications of any of the foregoing; and monomers, oligomers, fibrils, tangles, aggregates, and plaques that include of any of the foregoing. In a further embodiment, the tau species comprises one or more of total tau, phosphorylated tau, N- terminal tau, microtubule binding region (MTBR)-tau (e.g., MTBR-tau243), brain derived (BD)-tau. In another embodiment, phosphorylated tau species comprise one or more of pTaul81, pTaul99, pTau205, pTau217, pTau231. In another embodiment, the tau species is a relationship between or calculation based on different tau species, comprising one or more of phosphorylated tau to total tau (pTau / Ttau ratio) and phosphorylated tau to nonphosphorylated tau (pTau / non-pTau ratio).

[0029] In another embodiment, the blood based biomarker is s a relationship between or calculation based on different biomarker species. In one embodiment, the blood based biomarker comprises one or more of pTau / Ap42 ratio and p-tau / Ap40 ratio.

[0030] The terms “level,” “amount,” “quantity,” “value,” and “score” are used interchangeably to refer to a quantification of a biomarker present in a sample or a subject. The level of a biomarker can be the level determined by an assay for the biomarker. Additionally, the level of a biomarker can be a normalized or converted level of the biomarker (e.g., using a conversion equation of the present invention).Assays

[0031] Amyloid beta (AP) Positron Emission Tomography (PET), often referred to as amyloid-PET or AP-PET is frequently used, with or without neuropathological determination, for evaluating the presence of Ap plaques in the brains of individuals with AD or suspected of having AD. AP-PET is correlated with AD and is a biomarker for AD. C- Pittsburgh Compound B (nC-PiB),18F-florbetaben (FBB),18F-florbetapir,18F-flutemetamol and18F-NAV4694 (NAV) are PET tracers that have been shown to bind Ap in AD from the preclinical stage onward through disease progression. Given the number of tracers currently in use, the Centiloid unit (CL) was developed to standardize the results of an AP-PET scan. Based on this scale, a value of around 12CL marks the transition from the absence of Ap pathology to subtle pathology and a value of around 30CL indicates the presence of established pathology. Recently, alternate CL values have been proposed or used in clinical trials as thresholds for amyloid positivity, including 27CL, 24CL, and 11CL.

[0032] Various types of assays can be used with the present invention to determine the level or value of a blood based biomarker in a sample from a subject. In one embodiment, the assay type comprises on or more of chromatography, gas chromatography, liquid chromatography, spectroscopy, colorimetric assays, fluorometric assays, mass spectrometry, immunoassays, chemiluminescent, voltammetry, amperometry, and potentiometry.

[0033] In one another embodiment, the assay is liquid chromatography-tandem mass spectrometry (LC-MS / MS) assay (e.g., Precivity™, C2N Diagnostics), a single moleculearray (Simoa) (e.g., Simoa®, Quanterix), or and a chemiluminescent enzyme immunoassay (CLEIA) (e.g., Lumipulse®, Fujirebio).

[0034] In one embodiment, the assay is analytically validated. Analytical validation or analytically validate, comprises, a process to establish that the performance characteristics of a test, tool or instrument are acceptable in terms of its sensitivity, specificity, accuracy, precision, and other relevant performance characteristics using a specified technical protocol (which may include specimen collection, handling and storage procedures). This is validation of the test’s, tool’s, or instrument’s technical performance, but is not validation of the item’s usefulness. (See, FDA-NIH Biomarker Working Group, BEST (Biomarkers, Endpoints, and other Tools) Resource, 2016-, (ncbi.nlm.nih.gov / books / NBK326791 / pdf / Bookshelf_NBK326791.pdf). Analytical validation supports the biomarker measurement and includes all factors related to the assay system. (See, Piccoli and Sauer, 2019. Points to Consider Document: Scientific and Regulatory Considerations for the Analytical Validation of Assays Used in the Qualification of Biomarkers in Biological Matrices).

[0035] In one embodiment, an assay is clinically validated. Clinical validation or clinically validated comprises, a process to establish that the test, tool or instrument acceptably identifies, measures, or predicts the concept of interest, (see, See, FDA-NIH Working Group, BEST (Biomarkers, Endpoints, and other Tools) Resource, 2016). Clinical validation supports interpretation of the biomarker measurement and is dependent on the clinical performance in predicting the outcome claimed. (See, Piccoli and Sauer, 2019).

[0036] In other embodiments, an assay is cleared, within the meaning of the US FDA or equivalent approval from other regulatory agencies (e.g., an in vitro diagnostic, imaging), approved, within the meaning of the US FDA or equivalent approval from other regulatoryagencies (e.g., a companion diagnostic), a laboratory developed test (LDT), or a Clinical Laboratory Improvement Amendments (CLIA) test.Alzheimer’s Disease Therapies

[0037] A number of therapies are approved or are in development for the treatment of AD. There are currently three FDA approved monoclonal antibodies targeting Ap - lecanemab (Leqembi®, Eisai and Biogen), donanemab (Kisunla™, ,Eli Lilly), and aducanumab (Aduhelm ®, Eisai and Biogen). Several other anti-Ap antibodies have been tested in clinical trials including remtemetug (Eli Lilly), trontinemab (Genentech and Roche), ACU193 (Acumen), and PRX012 (Prothena). A novel humanized IgGl monoclonal antibody that can administered subcutaneously and targets the N-terminus of Ap and binds with high affinity and avidity to aggregated forms of Ap, including protofibrils and plaques is described in U.S. Patent Application Publication No. 2022-0049009, which is incorporated herein in its entirety.

[0038] Other AD therapies include vaccines against AP and other plaque-forming proteins (e.g, tau, alpha-synuclein). In one aspect the disclosure is directed to treatment with a vaccine, for example as described in U.S. Patent Application Publication No. 2023-0183304, which is incorporated herein in its entirety.

[0039] In the various aspects of the disclosure, the subjects that are treated or administered therapeutics as disclosed herein are typically human subjects, often referred to as a “subject,” “individual,” or a “patient,” to the extent they are or will receive treatment, or are or will be evaluated for the possibility of receiving treatment as described herein. In various other aspects of the disclosure, subjects that have Ap-PET values determined, blood based biomarker levels or values determined (e.g., AP40, AP42, AP42 / 40 ratio, pTaul81, pTau217), biomarker levels or values converted, who are screened for Ap-PET scans, or who are selected for therapy based on biomarker level or a converted biomarker level are alsotypically human subjects, often referred to as a “subject,” “individual,” or a “patient,” to the extent they are or will receive treatment, or are or will be evaluated for the possibility of receiving treatment as described herein.Use of blood based biomarkers

[0040] Efficiently and accurately determining the level of a biomarker of interest is important for making medical decisions including diagnosing a subject, determining if a subject should be administered a therapy, administering a subject a treatment, evaluating the effectiveness of a therapy, adjusting, including pausing or stopping, therapy for a subject.

[0041] Characteristics of different biomarker assays can vary. A first biomarker assay may have preferable characteristics (e.g., an established correlation between the measured blood biomarker level and the level of Ap in the brain of a subject as measured by an Ap-PET scan) for the uses described herein, but have other less favorable characteristics (e.g., slow turnaround time to get results). A second assay for the same biomarker may have a rapid turnaround time but does not report the same absolute value for the biomarker level as the first assay and therefore cannot be used in place of the first assay (e.g., determine the subject is positive for brain Ap amyloid).

[0042] Herein, are methods of normalizing (i.e., converting) a biomarker level determined with a second assay to align with the first assay biomarker level. This conversion enables use of the biomarker level determined with second assay as if it were determined with the first assay.Cross-Platform Biomarker Conversion Methodology

[0043] In one aspect, the disclosure is directed to a systematic approach for harmonizing or normalizing biomarker measurements across or between analytical platforms, referred to herein as "CP Convert." CP Convert provides a standardized methodology for convertingbiomarker measurements between different assay platforms, enabling comparison of data from different laboratories, studies, and analytical systems.

[0044] In one aspect, as used in the context of biomarker levels, “convert,” “transform,” and“normalize” are used interchangeably.

[0045] This approach is particularly valuable for pTau217 measurements, which can be performed using various analytical platforms including LC-MS / MS, SIMOA, and chemiluminescent enzyme immunoassays (CLEIA) such as Lumipulse.

[0046] The CP Convert methodology employs statistical methodologies to determine correlation between biomarker levels from different assays and equations to convert biomarker values between the assays. Statistical methods are known by those in the art. In one embodiment, correlation is determined on data in a format comprising one or more of linear data, continuous data, binary data, categorical data, and log transformed data. In another embodiment, regression analysis is conducted on the data comprising one or more of simple linear regression, ordinary least squares (OLS) regression, and Deming regression. In one embodiment, Deming regression analysis of log-transformed data to develop conversion equations that convert or normalize biomarker levels across platforms. CP Convert enables the leveraging of large population studies and established cutoff values across different analytical platforms, reducing the need to repeat expensive validation studies for each platform.

[0047] In one aspect, the disclosure is directed to the use of plasma biomarkers as a minimally invasive tool for clinical trial prescreening. Presented herein are examples from the ASCENT Phase 1 clinical program, which is evaluating PRX012 - a novel humanized IgGl monoclonal antibody that is administered subcutaneously and targets the N-terminus of Ap and binds with high affinity and avidity to aggregated forms of Ap, including protofibrils and plaques.

[0048] In one embodiment, an assay for measuring a biomarker is selected as the reference assay and a different assay for measuring the biomarker is selected as the test assay. The biomarker level determined with the reference assay is the reference biomarker level or reference level. The biomarker level determined with the test assay is a test biomarker level or reference level.

[0049] In one embodiment, the reference assay has preferred analytical or operational characteristics. In one embodiment, the reference assay characteristics comprise high sensitivity, high specificity, high precision, high accuracy, wide dynamic range, low Limit of Detection (LOD), and low Limit of Quantification (LOQ). In another embodiment, the reference assay is analytically validated or clinically validated.

[0050] In one embodiment, a reference biomarker level and a test biomarker level are used to determine the correlation between assays, develop CP Convert equations, and to be converted using a CP Convert equation.

[0051] In one embodiment, a converted biomarker level is test biomarker level that has been converted to a level using a CP Convert equation for the reference assay and the test assy. The converted biomarker level is a mathematical representation of the biomarker level had the biomarker level been determined with the reference assay. As described herein, biomarker levels are transformed from a level determined with a test assay to a normalized level with a conversion equation based of levels for the biomarker determined with the test assay and the reference assay.

[0052] In one embodiment, a CP Convert equation is based on a biomarker levels from assays that have a Pearson correlation coefficient of greater than or equal to 0.8. In some embodiments, the Pearson correlation coefficient is greater than or equal to 0.85, 0.90, 0.91,0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

[0053] In another embodiment, a CP Convert equation reduces the mean difference in raw absolute reference levels and test levels to less than or equal to ±0.5, ±0.45, ±0.4, ±0.35, ±0.3,±0.25, ±0.2, ±0.19, ±0.18, ±0.17, ±0.16, ±0.15, ±0.14, ±0.13, ±0.12, ±0.11, ±0.1, ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02, or ±0.01. In another embodiment, after application of a CP Convert equation to a set of test biomarker levels, the mean difference in raw absolute reference levels and converted levels is less than or equal to ±0.5, ±0.45, ±0.4, ±0.35, ±0.3, ±0.25, ±0.2, ±0.19, ±0.18, ±0.17, ±0.16, ±0.15, ±0.14, ±0.13, ±0.12, ±0.11, ±0.1, ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02, or ±0.01.

[0054] In another embodiment, a CP Convert equation reduces the difference between a reference level and test level to less than or equal to ±0.5, ±0.45, ±0.4, ±0.35, ±0.3, ±0.25, ±0.2, ±0.19, ±0.18, ±0.17, ±0.16, ±0.15, ±0.14, ±0.13, ±0.12, ±0.11, ±0.1, ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02, or ±0.01. In another embodiment, after application of a CP Convert equation to a test biomarker level, the mean difference between the reference level and the converted level is less than or equal to ±0.5, ±0.45, ±0.4, ±0.35, ±0.3, ±0.25, ±0.2, ±0.19, ±0.18, ±0.17, ±0.16, ±0.15, ±0.14, ±0.13, ±0.12, ±0.11, ±0.1, ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02, or ±0.01.

[0055] In one embodiment, a CP Convert equation to convert a biomarker level determined with a test assay is determined by:(a) determining the correlation of (i) biomarker levels determined with a reference assay in a set of samples (the correlation cohort) and (ii) biomarker levels determined with a test assay in the correlation cohort,(b) determining a conversion equation (CP Convert equation) between the two sets of biomarker levels from the correlation cohort, wherein correlation between the reference assay biomarker levels and test assay biomarker levels has Pearson correlation coefficient of greater than or equal to 0.8. In someembodiments, the Pearson correlation coefficient is greater than or equal to 0.85, 0.90, 0.91,0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

[0056] In a further embodiment, the CP Convert equation is determined using linear regression analysis of linear data or Deming regression analysis of log transformed data.

[0057] In another embodiment, a CP Convert equation is validated by applying the CP Convert equation to reference biomarker levels and test biomarker levels in set of samples that are not from the correlation cohort (an evaluation cohort).

[0058] In one embodiment, a CP Convert equation reduces the mean difference in raw absolute values between reference levels and test levels in an evaluation cohort to less than or equal to ±0.5, ±0.45, ±0.4, ±0.35, ±0.3, ±0.25, ±0.2, ±0.19, ±0.18, ±0.17, ±0.16, ±0.15, ±0.14, ±0.13, ±0.12, ±0.11, ±0.1, ±0.09, ±0.08, ±0.07, ±0.06, ±0.05, ±0.04, ±0.03, ±0.02, or ±0.01.

[0059] In another aspect, the disclosure is directed to a method of converting or harmonizing plasma pTau217 measurements across analytical platforms including: (a) measuring plasma pTau217 levels in a first set of samples using a first analytical platform selected from LC- MS / MS, SIMOA, and CLEIA; (b) measuring plasma pTau217 levels in the same set of samples using a second analytical platform different from the first analytical platform; (c) performing Deming regression analysis of log-transformed data from both platforms to generate a conversion equation; (d) validating the conversion equation using an independent cohort; and (e) applying the conversion equation to normalize pTau217 measurements across platforms.

[0060] In some embodiments, the method further comprises determining that the conversion equation reduces median differences in cross-platform results to less than 0.2 pg / mL when applied to independent validation cohorts.

[0061] In another aspect, the disclosure is directed to a method of validating cross-platform biomarker conversion equations including: (a) developing conversion equations using a firstcohort with measurements on multiple analytical platforms; (b) applying the conversion equations to measurements from at least one independent cohorts using the CP Convert methodology; (c) comparing the median differences between converted values and intracohort derived values; and (d) confirming that the conversion equations normalize measurements across platforms within acceptable limits. In embodiments, acceptable limits can include comprise median differences of less than, e.g., 0.20 pg / mL, less than 0.15 pg / mL, less than 0.10 pg / mL, or less than 0.05 pg / mL, between CP Convert-converted values and intra-cohort derived values.Treatment Methods

[0062] In one aspect, the disclosure is directed to a method of treating a subject with Alzheimer’s Disease (AD). The method includes administering to a subject a therapeutically effective amount of an anti-amyloid beta (AP) therapy, wherein the subject has one or more of the following biomarkers: a. plasma AP42 / 40 ratio < 0.0975 or plasma ratio AP42 / 40 < 0.1020, b. plasma pTaul81 > 9.4 pg / mL, and c. plasma pTau217 > 1.2 pg / mL.

[0063] In another aspect, the disclosure is directed to a method for selecting a subject for treatment for Alzheimer’s Disease (AD). The method includes measuring one or more subject’s fluid biomarkers selected from plasma AP42 / 40 ratio, plasma pTaul81 and plasma pTau217, and selecting the subject for treatment when the level of one or more of the biomarkers are as follows: a. plasma AP42 / 40 ratio < 0.0975 or plasma AP42 / 40 < 0.1020, b. plasma pTaul81 > 9.4 pg / mL, and c. plasma pTau217 > 1.2 pg / mL.In this aspect, the treatment can be associated with participation in a clinical trial. During the trial, the subject may be administered either a test anti-Ap therapy or a control.

[0064] In this aspect, the treatment can be associated with participation in a clinical trial. During the trial, the subject may be administered either a test anti-Ap therapy or a control. In various aspects of the disclosure, the subject that is treated or considered for a clinical trial, has not undergone an AP-Positron Emission Tomography (PET) procedure, or has not undergone an Ap PET procedure that did not produce a result that is indicative of a need for the therapy. In other aspects, prior to a first administration of the anti-Ap therapy, the subject has not undergone an AP-Positron Emission Tomography (PET) procedure, or n AP PET procedure that did not produce a result that is indicative of a need for the therapy. An Ap- PET procedure indicative of a need for the therapy includes, for example, a measured Ap brain load of > 30 centiloids. In addition other parameters may be determined that are indicative of whether a subject needs therapy. In one aspect, the subject undergoes the Ap- PET procedure after the fluid biomarker level(s) are measured but prior to a first administration of the anti-Ap therapy.Clinical Trial Methods

[0065] In a further aspect, the disclosure is directed to a method for conducting a clinical trial for an anti-Ap therapy on subjects that have not undergone an AP-Positron Emission Tomography (PET) procedure or on subjects that have not undergone an AP-Positron Emission Tomography (PET) procedure that produced a result indicative of a need for therapy. The method includes identifying a subject for the clinical trial by measuring one or more of the subject’s fluid biomarkers selected from plasma AP42 / 40 ratio, pTaul81 and pTau217, and selecting the subject for the trial when the level of one or more of the biomarkers are as follows:a. plasma Ap 42 / 40 ratio < 0.0975 or plasma Ap 42 / 40 < 0.1020, b. plasma pTaul81 > 9.4 pg / mL, and c. plasma pTau217 > 1.2 pg / mL.

[0066] In another aspect, the disclosure is directed to a method of reducing the number of subjects selected for an AP-PET procedure prior to enrollment in a clinical trial for an anti- Ap therapy. The method includes selecting a subject for an AP-PET procedure when the patient has one or more biomarker levels selected from: a. plasma Ap 42 / 40 ratio < 0.0975 or plasma Ap 42 / 40 < 0.1020, b. plasma pTaul81 > 9.4 pg / mL, and c. plasma pTau217 > 1.2 pg / mL.According to this aspect, the subject may undergo an AP-PET procedure prior to enrollment in a clinical trial. In addition, the subject may have an Ap brain load of > 30 centiloid, as measured by the AP-PET procedure.

[0067] Still further, the disclosure is directed to a method of reducing the number of subjects selected for an AP-PET procedure prior to enrollment in a clinical trial for an anti-Ap therapy. The method includes selecting a subject for an AP-PET procedure when the patient has one or more biomarker levels selected from: a. plasma Ap 42 / 40 ratio < 0.0975 or plasma Ap 42 / 40 < 0.1020, b. plasma pTaul81 > 9.4 pg / mL, and c. plasma pTau217 > 1.2 pg / mL.Diagnostic Methods

[0068] In another aspect, the disclosure is directed to a method of diagnosing Alzheimer’sDisease in a subject, including measuring levels of one or more biomarkers in the subject, which biomarkers are selected from plasma A042 / 4O ratio, plasma pTaul81, and plasma pTau217. The subject is diagnosed with Alzheimer’s Disease when: a. the plasma A042 / 4O ratio is < 0.0975 or plasma ratio A042 / 4O is < 0.1020, or b. the plasma pTaul81 > 9.4 pg / mL, or c. the plasma pTau217 > 1.2 pg / mL,

[0069] In another aspect, the disclosure is directed to a method of predicting a subject’s response to a treatment for Alzheimer’s Disease, including measuring levels of one or more biomarkers in the subject, which biomarkers are again selected from plasma A042 / 4O ratio, plasma pTaul81, and plasma pTau217. The subject is predicted to be responsive to a therapeutically effective amount of an anti-amyloid beta ( A|3) therapy when: a. the plasma A042 / 4O ratio is < 0.0975 or plasma ratio A042 / 4O is < 0.1020, or b. the plasma pTaul81 > 9.4 pg / mL, or c. the plasma pTau217 > 1.2 pg / mL,

[0070] In various aspects, the selected subject undergoes an A0-PET procedure prior to a first administration of the anti-A0 therapy. In some aspects, the subject has an A|3 brain load of > 30 centiloid, as measured by the A0-PET procedure.Cross-Platform Conversion Methods

[0071] As described elsewhere herein, rigorous A0-PET studies were conducted with equally rigorous liquid chromatography-tandem mass spectrometry (LC-MS / MS)-based measurement of biomarkers in large AD or AD-vulnerable populations. Receiver operator characteristic (ROC) and area under the curve (AUC) analyses were performed to determine robust cutoffs for AP42 / 40 ratios, pTaul81, and pTau217 that are indicative of early disease(predicting AP-PET Alzheimer’s positivity). Bridging biomarker analyses pertaining to pTau217 were conducted with other assays using different detection technologies including a single molecule array (Simoa) (e.g., Simoa®, Quanterix) and a chemiluminescent enzyme immunoassay (CLEIA) (e.g., Lumipulse®, Fujirebio), which showed that strong positive correlations existed among the assays. Such correlations allow the leveraging of the large population PET and LC-MS / MS-based studies (and resulting ROC, AUC analyses, biomarker cutoff values, etc.) for use with assays for measuring pTau217 that are, e.g., faster, easier, less expensive than LC-MS / MS. That is, Simoa and CLEIA assays may be used to measure plasma pTau217 in a subject and convert the measured values to LC-MS / MS equivalent (i.e., converted) values, which can then be used with the cutoff values determined using LC-MS / MS.

[0072] However, one of ordinary skill in the art can also utilize an alternate assay, one other than LC-MS / MS, Simoa, and CLEIA, to detect pTau217 and utilize the LC-MS / MS cutoff described herein. One of ordinary skill can measure pTau217 in the alternate assay in parallel with measurement in the same sample(s) with LC-MS / MS, Simoa, and / or CLEIA. A routine correlation study of pTau217 measurements from the alternate assay type(s) the against LC-MS / MS measurements in the same samples allows the use of the plasma pTau217 cutoff of > 1.2 pg / mL directly. If the alternate assay is measured against CLEIA and / or Simoa assays on the same samples, a routine correlation study will allow conversion of alternate assay results into corresponding CLEIA and / or Simoa assay results. These corresponding CLEIA and / or Simoa assay results can then be converted into converted LC- MS / MS results based on the correlations described herein, e.g., using the CP Convert methodology and equations.

[0073] For example, alternate assay pTau217 results can be converted to Simoa-based results based on a routine correlation study between the alternate assay and Simoa assay, whichSimoa-based results can be converted to LC-MS / MS-pTau217 levels utilizing the relationship: log(Simoa) = -1.41 + 1.11 x log(LC-MS / MS). For an alternate assay measuring pTau217, results can be converted to CLEIA-based results based on a routine correlation study between the alternate assay and CLEIA assay, which CLEIA-based results can be converted to LC-MS / MS-pTau217 levels utilizing the relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS). The (LC-MS / MS-derived) plasma pTau217 cutoff of > 1.2 pg / mL may be used on the converted pTau217 results, which leverages the rigorous PET and LC- MS / MS studies on large AD or AD-vulnerable populations, along with related ROC and AUC analyses, without having to repeat them.Simoa Platform Methods

[0074] In another aspect, the disclosure is directed to a method of treating a subject with Alzheimer’s Disease including administering to the subject a therapeutically effective amount of an anti-Ap therapy, in which one or more plasma pTau217 levels are determined for the subject by a single molecule array technology, such as Quanterix Simoa, yielding one or more Simoa-based plasma pTau217 levels (“Simoa-pTau217”, or as evident from context). The Simoa-pTau217 levels can then be converted into corresponding LC-MS / MS-based plasma pTau217 levels (“LC-MS / MS-pTau217”, or generally, “plasma pTau217”) using the CP Convert methodology or equations. A therapeutically effective amount of an anti-Ap therapy can be administered to the subject when the subject has a (converted) plasma pTau217 > 1.2 pg / mL. In some embodiments, the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + 1.11 x log(LC-MS / MS).

[0075] In another aspect, the disclosure is directed to a method for selecting a subject for treatment for Alzheimer’s Disease including determining one or more subject plasmapTau217 levels by single molecule array technology, such as Quanterix Simoa, to make one or more Simoa-pTau217 levels. The one or more Simoa-pTau217 levels can be converted to one or more LC-MS / MS-based plasma pTau217 levels using CP Convert, whereupon the subject is selected for treatment when the level of (converted) LC-MS / MS-pTau217 > 1.2 pg / mL. In some embodiments, the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + 1.11 x log(LC-MS / MS).

[0076] CLEIA Platform Methods

[0077] In another aspect, the disclosure is directed to a method of treating a subject with Alzheimer’s Disease including administering to the subject a therapeutically effective amount of an anti-Ap therapy, in which one or more plasma pTau217 levels are determined for the subject by a chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels. The CLEIA-pTau217 levels can then be converted into corresponding LC-MS / MS-based plasma pTau217 levels. A therapeutically effective amount of an anti-Ap therapy can be administered to the subject when the subject has a (converted) plasma pTau217 > 1.2 pg / mL. In some embodiments, the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

[0078] In another aspect, the disclosure is directed to a method for selecting a subject for treatment for Alzheimer’s Disease including determining one or more subject plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels. The one or more CLEIA-pTau217 levels can be converted to one or more LC-MS / MS-based plasma pTau217 levels using CP Convert, whereupon the subject is selected for treatment when the level of (converted) LC-MS / MS-pTau217 > 1.2 pg / mL. In some embodiments, the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).Clinical Trial Methods

[0079] In another aspect, the disclosure is directed to a method for conducting a clinical trial for an anti-Ap therapy on subjects that have not undergone an AP-Positron Emission Tomography (PET) procedure, the method including identifying a subject for the clinical trial by measuring one or more subject’s plasma pTau217 levels by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa-pTau217 levels. The Simoa-pTau217 levels can be converted to one or more LC-MS / MS-pTau217 levels using CP Convert, and the subject is selected for the trial when the (converted) LC-MS / MS-pTau217 level is >1.2 pg / mL In some embodiments, the one or more Simoa-pTau217 levels are converted to one or more LC- MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + 1.11 x log(LC-MS / MS).

[0080] In another aspect, the disclosure is directed to a method for conducting a clinical trial for an anti-Ap therapy on subjects that have not undergone an AP-Positron Emission Tomography (PET) procedure, the method including identifying a subject for the clinical trial by measuring one or more subject’s plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels. The CLEIA-pTau217 levels can be converted to one or more LC-MS / MS-pTau217 levels using CP Convert, and the subject is selected for the trial when the (converted) LC- MS / MS-pTau217 level is >1.2 pg / mL In some embodiments, the one or more CLEIA- pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

[0081] In embodiments, the CP Convert methodology (equations) for conversion between Lumipulse and SIMOA assays can be validated using data from one or more independent cohorts. In such independent cohort(s), pTau217 concentration can be measured for each sample, and, preferably, compared to assess correlation for intra-laboratory comparability. Such intra-lab oratory cross-platform pTau217 results should demonstrate strong correlation, though the mean difference in raw absolute values between the two assay platforms may be elevated (e.g., 0.3 pg / mL, 0.4 pg / mL, 0.5 pg / mL). The robustness of the CP Convert equations can be demonstrated by their application to independent cohort(s) that are different from the initial cohort (ASCENT) used to generate them.Methods of Reducing A0-PET utilization

[0082] In another aspect, the disclosure is directed to a method of reducing the number of subjects selected for an AP-PET procedure prior to enrollment in a clinical trial for an anti- Ap therapy, the method including determining one or more plasma pTau217 levels from a subject by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa- pTau217 levels. The one or more Simoa-pTau217 levels can be converted to one or more LC- MS / MS-based plasma pTau217 levels using CP Convert, whereupon the subject is selected for an AP-PET procedure when the level of (converted) LC-MS / MS-pTau217 > 1.2 pg / mL. In some embodiments, the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + 1.11 x log(LC-MS / MS).

[0083] In another aspect, the disclosure is directed to a method of reducing the number of subjects selected for an AP-PET procedure prior to enrollment in a clinical trial for an anti- Ap therapy, the method including determining one or more plasma pTau217 levels from a subject by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) tomake one or more CLEIA-pTau217 levels. The one or more CLEIA-pTau217 levels can be converted to one or more LC-MS / MS-based plasma pTau217 levels using CP Convert, whereupon the subject is selected for the A0-PET procedure when the level of (converted) LC-MS / MS-pTau217 > 1.2 pg / mL. In some embodiments, the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

[0084] In an embodiment, a method of reducing AP-PET testing burden in clinical trials is provided, including: (a) implementing pTau217 prescreening using a cutoff corresponding to 90% sensitivity; (b) selecting subjects for Ap-PET procedures based on pTau217 levels above the cutoff; and (c) achieving at least a 60% reduction in the number of subjects requiring AP-PET scanning compared to traditional screening approaches.

[0085] In embodiments of the method of reducing AP-PET testing burden in clinical trials, the pTau217 cutoff is 1.2 pg / mL as measured by LC-MS / MS or equivalent values determined using CP Convert methodology for other analytical platforms.

[0086] In an embodiment, a method of comparing pTau217 performance with other bloodbased biomarkers is provided, including: (a) measuring plasma AP42 / 40 ratio, pTaul81, and pTau217 in the same population of subjects; (b) performing head-to-head ROC analysis to determine AUC values for predicting AP-PET positivity; and (c) demonstrating that pTau217 achieves superior performance with an AUC > 0.94, compared to AP42 / 40 ratio (AUC < 0.85) and pTaul81 (AUC < 0.82).

[0087] In each of the forgoing aspects, the anti-Ap therapy may include active or passive therapies, for example the antibodies and vaccines are described herein.EXAMPLES

[0088] Data from subjects who underwent screening for enrollment in anti-amyloid immunotherapy phase 1 clinical trials, ASCENT trials (Othair Prothena Limited, Dublin,Ireland), was evaluated. All subjects consented to study participation using IRB / IEC approved informed consent forms prior to any study procedures. Subjects (aged 55 - 85) with mild cognitive impairment or mild dementia (stage 2, 3 or 4) according to the National Institute on Aging and Alzheimer’s Association (NIA-AA) criteria (Jack 2018) and an MMSE score >18 were evaluated by amyloid PET (AP-PET) using one of the following three 18F-labeled tracers, florbetapir (AV45), flutemetamol (GE067), or florbetaben (BAY 94- 9172). An AP-PET between 30 centilod (CL) and 130 CL was a requirement for enrollment.

[0089] At the time of AP-PET imaging, whole blood samples were collected in K2EDTA tubes, processed for plasma following centrifugation at 500-700xg for 15 minutes at 4°C. Plasma was aliquoted into polypropylene tubes and subsequently stored on dry ice until frozen at -80°C until analysis. Samples were analyzed for Abeta 42 / 40 (n=555), pTau217 (n=199) and pTaul81 (n=199) using liquid chromatography-tandem mass spectrometry LC- MS / MS (C2N Diagnostics, Madison, WI).

[0090] Amyloid positive, Ap positive, or AP-PET positive was defined as an AP-PET value of > 30 centiloids (CL). Receiver operated characteristic (ROC) analyses evaluated biomarker ability to identify amyloid positive subjects.

[0091] Example 1: A]J42 / 40 Ratio Performance in Detecting Amyloid Positivity

[0092] The correlation of AP42 / 40 ratio with AP-PET centiloid level in subjects was determined. FIG. 1 A shows the AP42 / 40 ratios on the y-axis and the AP-PET CL values on the x-axis. Lower plasma AP42 / 40 ratios are correlated with greater brain amyloid pathology. As seen in FIG. 1 A, numerous subjects who underwent a costly and time consuming AP-PET scan had values below 30CL vertical dashed line) and were not selected for enrollment in the study.

[0093] Receiver operating characteristic (ROC) analysis was conducted to evaluate the ability of AP42 / 40 ratio to detect amyloid positivity. ROC curves for AP42 / 40 ratio as a positive predictor of AP-PET positivity is shown in FIG. IB. For AP42 / 40 ratio, the area under the curve (AUC; which gives a measure of classification ability) is 0.77 (n=555).

[0094] Previously, an AP42 / 40 ratio of 0.0975 was found to be a cutoff that maximized sensitivity and specificity for Ap-PET positivity in a subject (West et al., Mol Neurodegener. 2021 ; 16(l):30). In West 2021, the overall percent accuracy between the plasma AP42 / 40 ratio and amyloid positivity was 75% at the 0.0975 AP42 / 40 ratio cutoff. Here, that cutoff of AP42 / 40 ratio 0.0975 had a sensitivity of 0.79 (true positive rate), meaning that cutoff correctly identified subjects who are Ap-PET positive 79% of the time. To improve use of AP42 / 40 ratio, an optimized cutoff (AP42 / 40 ratio 0.102) was determined by setting the sensitivity to 0.90 (a true positive rate of 90%). AP42 / 40 ratio 0.102 is depicted as the horizontal dashed line in FIG 1 A. See Tables 1 A and IB below comparing performance of the AP42 / 40 ratio cutoffs. NPV, negative predictive value; PPV, positive predictive value; Se, selectivity; Sp, specificity.Table 1A. AP42 / 40 ratio <0.0975 cutoff (West 2021)Subject ResultsPerformancePrevalence Amyloid Pos: 167 / 555 = 30%Se = 132 / 167 = 79%Sp = 230 / 388 = 59%PPV = 132 / 290 = 46%NPV = 230 / 265 = 87%Table IB. AP42 / 40 ratio <0.102 optimized cutoff (high sensitivity)Subject ResultsPerformancePrevalence Amyloid Pos: 167 / 555 = 30%Se = 150 / 167 = 90%Sp = 172 / 388 = 44%PPV = 150 / 366 = 41%NPV = 172 / 189 = 91%

[0095] As seen in FIG. 1 A, numerous subjects who underwent a costly and time consuming AP-PET scan had values below 30CL (vertical dashed line). However, A majority of amyloid negative subjects had AP42 / 40 ratio less than 0.102 and a majority of amyloid positive subjects had a AP42 / 40 ratio greater than 0.102. Thus, AP42 / 40 ratio can be used to identify Ap positivity.

[0096] Example 2: pTaul81 Performance in Detecting Amyloid Positivity

[0097] The correlation of pTaul81 levels with AP-PET centiloid level in subjects was determined. FIG. 2A shows the plasma pTaul81 level in pg / mL on the y-axis and the AP- PET centiloids on the x-axis. Higher plasma pTaul81 levels are correlated with greater brain amyloid pathology. To achieve a 90% sensitivity, a reference value of 9.4pg / mL for plasma pTaul81 levels was selected (horizontal dashed line).

[0098] Receiver operating characteristic (ROC) analysis was conducted to evaluate the ability of pTaul81 levels to detect amyloid positivity. ROC curves for pTaul81 levels as a positive predictor of AP-PET positivity is shown in FIG. 2B. For pTaul81, the AUC is 0.82 (n=196). Performance of the pTaul81 level cutoff of 9.4 pg / mL is included in Table 2 below. Table 2. pTau!81 9.4 pg / mL cutoffSubject ResultsPerformancePrevalence PET Pos: 60 / 194 = 31%Se = 54 / 60 = 90%Sp = 88 / 136 = 65%PPV = 54 / 102 = 53%NPV = 88 / 94 = 94%

[0099] As seen in FIG. 2 A, numerous subjects who underwent a costly and time consuming AP-PET scan had values below 30CL (vertical dashed line). However, A majority of amyloid negative subjects had a pTaul81 level less than 9.4 pg / mL and a majority of amyloid positivesubjects had a pTaul81 level greater than 9.4 pg / mL. Thus, pTaul81 can be used to identify Ap positivity.

[0100] Example 3: pTau217 Performance in Detecting Amyloid Positivity

[0101] The correlation of pTau217 levels with Ap-PET centiloid level in subjects was determined. FIG. 3A shows the plasma pTau217 level in pg / mL on the y-axis and the AP- PET centiloids on the x-axis. Higher plasma pTau217 levels are correlated with greater brain amyloid pathology. To achieve a 90% sensitivity, a reference value of 1.2 pg / mL for plasma pTau217 levels was selected (horizontal dashed line).

[0102] Receiver operating characteristic (ROC) analysis was conducted to evaluate the ability of pTau217 levels to detect amyloid positivity. ROC curves for pTau217 levels as a positive predictor of AP-PET positivity is shown in FIG. 3B. For pTau217, the AUC is 0.95 (n=183). Performance of the pTau217 level cutoff of 1.2 pg / mL is included in Table 3 below. Table 3. pTau217 1.2 pg / mL cutoff.Subject ResultsPerformancePrevalence PET Pos: 60 / 183 = 33%Se = 54 / 60 = 90%Sp = 106 / 123 = 86%PPV = 54 / 71= 76%NPV = 106 / 112 = 95%

[0103] As seen in FIG. 3 A, numerous subjects who underwent a costly and time consuming AP-PET scan had values below 30CL (vertical dashed line). However, A majority of amyloid negative subjects had a pTau217 level less than 1.2 pg / mL and a majority of amyloid positive subjects had a pTau217 level greater than 1.2 pg / mL. Thus, pTau217 can be used to identify Ap positivity.

[0104] Example 4. Comparison of A|J42 / 40, pTaul81, and pTau217 as Predictors of Amyloid Positivity

[0105] As shown in Examples 1-3, AP42 / 40, pTaul81, and pTau217 correlated linearly with AP-PET centiloid values. Additionally, AUC for each was greater than 0.8, demonstrating that each biomarker can distinguish between Ap-PET scores >30 centiloids and <30 centiloids at least 80% of the time.

[0106] To compare performance of each biomarker, the ROC curves were calculated for matched overlaid and the difference in AUCs were calculate. See, Fig. 4 and Table 4.Table 4.

[0107] As shown in Table 4, pTau217 had the highest AUC, and is the best biomarker for identifying amyloid positivity.

[0108] Example 5. pTau217 bridging study: correlation among LC-MS / MS, Simoa, and Lumipulse analytical platforms

[0109] To investigate the impact and interchangeability of the analytical method on pTau217 performance to identify amyloid positive subjects, three pTau217 assay platforms were evaluated.

[0110] As described above, pTau217 levels were determined using liquid chromatographytandem mass spectrometry LC-MS / MS (C2N Diagnostics, Madison, WI). Additionally, a subset of matched plasma samples was analyzed for pTau217 using the Simoa® Janssen Assay (p217+ tau) assay on the HD-X automated immunoassay analyzer (Quanterix, Billerica, MA) and the Lumipulse® G pTau217 plasma assay on the Fujirebio Lumipulse®G1200 fully automated analyzer (Fujirebio, Malvern, PA). All assays were run under regulatory classification as Laboratory developed tests (LDTs) according to manufacturers’ instructions and individual laboratory SOPs.[OHl] In the sub-analysis, 118 subjects had a pTau217 result by LC-MS / MS, and one result from either Lumi pulse or Simoa (n=l 16 for each comparison). Of these 118 subjects, 59 were amyloid positive, and 59 were amyloid negative.

[0112] For statistical analysis of the data, absolute concentrations values (mean ±SD) for Ap- PET positive subjects and Ap-PET negative subjects were compared, correlation coefficients across assay platforms were examined. Finally, equations to convert pTau217 levels between the assay platforms were developed.Absolute pTau217 Concentrations

[0113] Absolute concentrations pTau217 is dependent on analytical platform used for sample analysis. The absolute concentrations of pTau217 measured across platforms demonstrated a clear trend of LC-MS / MS having the highest values with a mean ± SD of 1.859 pg / mL ± 1.634 pg / mL (n=l 18). Lumipulse results were approximately 7-fold lower with a mean ± SD of 0.275 pg / mL ± 0.278 pg / mL (n=l 17). Simoa returned the lowest absolute values, mean ± SD of 0.077 pg / mL ±0 .068 pg / mL (n=l 17), which was 24-fold lower than LC-MS / MS.

[0114] These trends were observed when pTau217 levels from each platform were averaged based on amyloid positivity status. See, Table 5.Table 5. pTau217 concentrations (pg / ml).

[0115] The three tested assays are equally capable of detecting pTau217 and may be used with assay-specific cutoffs, including for the methods described herein. However, as result of these differences in absolute pTau217 levels, direct comparisons of absolute concentrations among analytical methods are not possible.

[0116] Correlation of the assays was evaluated.Linear Correlation

[0117] The correlations among LC-MS / MS, Simoa, and Lumipulse assay platforms for detection of pTau217 were assessed using linear data and simple linear regression using software from Graphpad (Boston, MA).

[0118] Figures 5 A, 5B, and 5C show the correlation between Simoa and LC-MS / MS, Lumipuse and LC-MS / MS, and Simoa and Lumipuse. Correlations were significant with correlation coefficients (Pearson’s r) of 0.93, 0.96, and 0.95 between Simoa and LC-MS / MS, Lumipuse and LC-MS / MS, and Simoa and Lumipuse, respectively.

[0119] Given the high concordance of the assays, based on these linear regression analyses, equations for the conversion of values among the individual assays were developed to enable platform comparison of pTau217 readouts. Conversion equations were develop using matched values for each platform comparison (n=l 16).

[0120] For interconversion of LC-MS / MS data and Simoa data, the following equation can be used: Simoa = 0.006 + 0.038 x LC-MS / MS

[0121] For interconversion of LC-MS / MS data and Lumipulse data, the following equation can be used: CLEIA = - 0.023 + 0.1625 x LC-MS / MS

[0122] For interconversion of Simoa data and Lumipulse data, the following equation can be used: Simoa = 0.012 + 0.230 x LumipulseLog Transformed Correlation

[0123] To improve assessment of the correlations among LC-MS / MS, Simoa, and Lumipulse assay platforms for detection of pTau217, correlations were assessed using log (base 10) transformed data and Deming regression using R software (The R Foundation, Vienna, Austria).

[0124] Figures 6A, 6B, and 6C show the correlation between Simoa and LC-MS / MS, Lumipuse and LC-MS / MS, and Simoa and Lumipuse. Correlations were significant with high correlation coefficients (Pearson’s r) between Simoa and LC-MS / MS, Lumipuse and LC- MS / MS, and Simoa and Lumipuse.

[0125] Given the high concordance of the assays, based on these Deming regression analyses, equations for the conversion of values among the individual assays were developed to enable platform comparison of pTau217 readouts. Conversion equations were develop using matched values for each platform comparison (n=l 16).

[0126] For interconversion of LC-MS / MS data and Simoa data, the following equation can be used: log(Simoa) = -1.41 + 1.11 x log(LC-MS / MS)

[0127] For interconversion of LC-MS / MS data and Lumipulse data, the following equation can be used: log(CLEIA) = -0.88 + 1.16x log(LC-MS / MS)

[0128] For interconversion of Simoa data and Lumipulse data, the following equation can be used: log(CLEIA) = 0.6 + 1.05x log(Simoa)Performance of pTau217 Assays Platforms as Predictors of Amyloid Positivity

[0129] Performance of pTau217 measured by LC-MS, Simoa and Lumipulse assays to detect positive Ap-PET subjects (>30 centiloids) was evaluated using ROC analysis. Figure 7 shows the ROC curves for all three pTau217 assay platforms. AUCs were 0.935, 0.929; and 0.912 for LC-MS / MS, Simoa, and Lumipulse, respectively.Conclusion

[0130] The three tested pTau217 assay platforms performed equally for detecting Ap positivity, with the mass spectrometry assay having slightly higher AUC than the immunoassays. The absolute concentrations reported by individual pTau217 assay platforms differ significantly. Consequently, it is important to use assay specific cutoff values or use converted values.

[0131] However, the wide range of pTau217 values used to create conversion equations across platforms / technologies enables these equations to be more broadly applicable to various independent cohorts. Therefore, the conversion equations described herein can be used to determine converted values.

[0132] The high correlation of pTau217 assays means pTau217 testing for identifying and selecting a subject for inclusion in a clinal trial or for administration of a therapy is not limited to a specific assay. pTau217 testing may be used with any detection technology that can accurately and precisely determine pTau217 concentrations and demonstrate an excellent correlation coefficient with existing methods, such as Simoa, Lumipulse and LC-MS / MS.

[0133] Example 6. Biomarkers reduce the need for A]J-PET scans prior to administration of therapy.

[0134] Blood-based biomarkers offer a minimally invasive, more accessible, and potentially more cost-effective alternative with the potential to facilitate earlier diagnosis, especially in primary care settings, and enable enrichment of desired clinical trial populations by identifying individuals most likely to have AD pathology, thereby reducing screening failures and optimizing trial designs.

[0135] Using the correlation of plasma biomarkers with AP-PET positivity, the utility of plasma biomarkers to identify patients with brain Ap deposits who would be selected for therapy was evaluated.

[0136] Here, the impact of implementing plasma biomarker prescreening on the need for AP- PET screening in selecting subjects for a clinical trial (e.g., for anti-Ap therapies for AD) was assessed. To evaluate the impact that biomarker screening has on identifying amyloid positive subjects, and what savings may be realized, a clinical trial screening process was designed. The impact was calculated using parameters from a real-world example of a clinical study. A Ap-PET positivity prevalence of 25% based on a conservative estimate of the prevalence observed in the real-world ASCENT clinical trial enrollment paradigm was used.

[0137] Modeled screening cascades with and without plasma AP42 / 40 ratio or pTau217 prescreening were assessed for selecting 100 subjects for enrollment in a clinical trial. Biomarker cutoffs were set at 90% sensitivity for the assays. For the LC-MS / MS assays, AP42 / 40 ratio cutoff was set at 0.1020 and the pTau217 cutoff was set at 1.2 pg / mL. See Figure 8 for flowcharts of the screening cascades.Traditional Screening Approach

[0138] Under the traditional screening approach (no blood based biomarker prescreen before the AP-PET scan), to enroll 100 subjects for the clinical trial, 1000 subjects enter screening and 430 fail enrollment criteria prior to receiving an AP-PET scan,. The remaining 570 subjects undergo an AP-PET scan. Of those, -75% are not Ap positive and only 144 subjects continue for further screening. Based on other screening criteria, 100 of those 144 subjects are be enrolled and randomized and administered a therapy (e.g., PRX012) or placebo.

[0139] Optimized Approach - Biomarker Prescreening

[0140] In contrast, if an AP42 / 40 ratio prescreen is implemented before an AP-PET scan, 1002 subjects enter screening. Subjects not failing enrollment criteria have their AP42 / 40 ratio determined. 408 subjects have an AP42 / 40 ratio above the cutoff and then undergo a AP-PET scan. Based on the same remaining enrollment criteria, 99 of the 408 subjects thatunderwent a AP-PET scan are enrolled, randomized, for administration of a drug (e.g., PRX012) or placebo. Using AP42 / 40 ratio in screening results in a reduction of about 28% of subjects ((408-570) / 570) that need to undergo AP-PET scan prior to administration of a therapy (e.g., PRX012).

[0141] Likewise, if a pTau217 prescreen is implemented before an AP-PET scan, 1117 subjects enter screening and based on the same criteria, 480 do not meet enrollment criteria. 637 subjects then have their pTau217 determined. 427 subjects have a pTau217 level below the cutoff and only 210 subjects undergo an AP-PET scan. Because the pTau217 prescreening selected subjects likely to be Ap positive, only about 32% of the subjects (67 subjects) are not Ap positive, by AP-PET scan. The remaining 143 subjects continue for further screening. Based on the remaining screening criteria, 100 of those 143 subjects are enrolled and randomized and administered a therapy (e.g., PRX012) or placebo. Using pTau217 in screening results in a reduction of about 63% of subjects ((210-570)7570) that need to undergo AP-PET scan prior to administration of a therapy (e.g., PRX012).

[0142] Results of the model suggest that implementing a biomarker prescreen before an Ap- PET scan would require slightly more participants to be screened to achieve the same number of subjects enrolled to be treated. Approximately 11% more individuals need to be screened to obtain the same number of enrolled participants (n=100) in the optimized model that integrates pTau217 prescreening to identify individuals who should receive a confirmatory amyloid PET scan. Despite this increase in subjects who undergo early screening, the model suggests there is a significant decrease in the number of subjects that undergo expensive and burdensome AP-PET testing. Approximately 67% (427 out of 637) screened individuals fail eligibility criteria based on having a pTau217 level that does not meet the designated cutoff (90% sensitivity for this context of use). Thus, biomarker screening prior to an AP-PET scan reduces patient burden by eliminating unnecessary PET scans.

[0143] pTau217 screening achieved a meaningful decrease in the need for Ap-PET testing.Calculations indicate that using pTau217 in prescreening to predict Ap-PET status decreases the number of AP-PET scans needed in screening by over 60%.

[0144] Overall, these results support the use of plasma biomarkers (e.g., pTau217) as a minimally invasive and logistically practical tool for identifying and selecting a patient for anti-Ap therapy that reduces the need for burdensome, costly, and time consuming AP-PET scan prior to administration of an anti-amyloid beta therapy.

Claims

What is claimed is:

1. A method of treating a subject with Alzheimer’s Disease (AD) comprising administering to a subject a therapeutically effective amount of an anti -amyloid beta (A0) therapy, wherein the subject has one or more biomarker levels comprising:(a) plasma A042 / 4O ratio < 0.0975 or plasma ratio A042 / 4O < 0.1020,(b) plasma pTaul81 > 9.4 pg / mL, and(c) plasma pTau217 > 1.2 pg / mL.

2. A method for selecting a subject for treatment for Alzheimer’s Disease (AD) comprising measuring one or more subject’s biomarkers selected from plasma A042 / 4O ratio, pTaul81 and pTau217; and selecting the subject for treatment when the level of one or more of the biomarkers comprises:(a) plasma A042 / 4O ratio < 0.0975 or plasma A042 / 4O < 0.1020,(b) plasma pTaul81 > 9.4 pg / mL, and(c) plasma pTau217 > 1.2 pg / mL.

3. The method of claim 1 or claim 2, wherein the one or more biomarker levels is determined by liquid chromatography tandem mass spectrometry (LC-MS / MS, e.g., C2N Diagnostics).

4. The method of any one of claims 1-3, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa-pTau217 levels;(e) converting the one or more Simoa-pTau217 levels to one or more LC-MS / MS- pTau217 levels;(f) administering to the subject a therapeutically effective amount of an antiamyloid beta (A0) therapy, wherein the subject has a LC-MS / MS-pTau217 > 1.2 pg / mL.

5. The method of claim 2, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa-pTau217 levels;(e) converting the one or more Simoa-pTau217 levels to one or more LC-MS / MS- pTau217 levels;(f) selecting the subject for treatment when the level of LC-MS / MS-pTau217 > 1.2 pg / mL.

6. The method of claim 4 or claim 5, wherein the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + l.l l x log(LC-MS / MS).

7. The method of claim 1, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels;(e) converting the one or more CLEIA-pTau217 levels to one or more LC-MS / MS-pTau217 levels;(f) administering to the subject a therapeutically effective amount of an antiamyloid beta (A0) therapy, wherein the subject has a plasma pTau217 > 1.2 pg / mL.

8. The method of claim 2, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels;(e) converting the one or more CLEIA-pTau217 levels to one or more LC- MS / MS-pTau217 levels;(f) selecting the subject for treatment when the level of LC-MS / MS-pTau217 > 1.2 pg / mL.

9. The method of claim 7 or claim 8, wherein the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

10. The method of anyone of claims 2-9, wherein the treatment comprises participation in a clinical trial.

11. The method of claim 10, wherein the subject is administered either a test anti-Ap therapy or a control.

12. The method of any one of claims 1-11, wherein the subject has not undergone an A0-Positron Emission Tomography (PET) procedure.

13. The method of any one of claims 1-11, wherein prior to a first administration of the anti-AP therapy the subject has not undergone an AP-Positron Emission Tomography (PET) procedure.

14. The method of any one of claims 1-11, wherein the subject has not undergone an AP- Positron Emission Tomography (PET) procedure providing a result that is indicative of a need for the therapy.

15. The method of claim 14, wherein an AP-PET result that indicative of a need for the therapy comprises a measured Ap brain load of > 30 centiloids.

16. The method of any one of claims 1-11, wherein the subject undergoes an AP-PET procedure after measuring the one or more biomarkers.

17. The method of any one of claims 2-11, wherein the subject undergoes an AP-PET procedure after measuring the one or more biomarkers but prior to a first administration of the anti-AP therapy.

18. The method of claim 17, further comprising selecting the subject for treatment when the subject has an Ap brain load of > 30 centiloid, as measured by the AP-PET procedure.

19. A method for conducting a clinical trial for an anti-A0 therapy on subjects that have not undergone an AP-Positron Emission Tomography (PET) procedure, the method comprising identifying a subject for the clinical trial by measuring one or more subject’s biomarkers selected from plasma A042 / 4O ratio, pTaul81 and pTau217; and selecting the subject for the trial when the level of one or more of the biomarkers comprises:(a) plasma A|3 42 / 40 ratio < 0.0975 or plasma A|3 42 / 40 < 0.1020,(b) plasma pTaul81 > 9.4 pg / mL, and(c) plasma pTau217 > 1.2 pg / mL.

20. The method of claim 19, wherein the one or more subject biomarkers is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa-pTau217 levels;(e) converting the one or more Simoa-pTau217 levels to one or more LC-MS / MS- pTau217 levels;(f) selecting the subject for the trial when the LC-MS / MS-pTau217 level is >1.2 pg / mL.

21. The method of claim 20, wherein the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + l. l l x log(LC-MS / MS).

22. The method of claim 19, wherein the one or more subject biomarkers is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels;(e) conv converting the one or more CLEIA-pTau217 levels to one or more LC- MS / MS-pTau217 levels;(f) selecting the subject for the trial when the LC-MS / MS-pTau217 level is >1.2 pg / mL.

23. The method of claim 22, wherein the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

24. A method of reducing the number of subjects selected for an A0-PET procedure prior to enrollment in a clinical trial for an anti-A0 therapy, the method comprising selecting a subject for an A0-PET procedure when the subject has one or more biomarker levels selected from:(a) plasma A0 42 / 40 ratio < 0.0975 or plasma A0 42 / 40 < 0.1020,(b) plasma pTaul81 > 9.4 pg / mL, and(c) plasma pTau217 > 1.2 pg / mL.

25. The method of claim 24, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more plasma pTau217 levels from a subject by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa-pTau217 levels;(e) converting the one or more Simoa-pTau217 levels to one or more LC-MS / MS- pTau217 levels;(f) selecting the subject for an A0-PET procedure when the LC-MS / MS-pTau217 level is >1.2 pg / mL.

26. The method of claim 25, wherein the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + l. l l x log(LC-MS / MS).

27. The method of claim 24, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels;(e) conv converting the one or more CLEIA-pTau217 levels to one or more LC- MS / MS-pTau217 levels;(f) selecting the subject for an A0-PET procedure when the LC-MS / MS-pTau217 level is >1.2 pg / mL.

28. The method of claim 27, wherein the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

29. The method of claim 19 or claim 24, wherein the one or more biomarker levels is determined by liquid chromatography tandem mass spectrometry (LC-MS / MS).

30. The method of any one of claims 24-29, wherein the selected subject undergoes an A0-PET procedure prior to enrollment in a clinical trial.

31. The method of any one of claims 24-28, wherein subject has an A0 brain load of > 30 centiloid, as measured by the A0-PET procedure.

32. A method of reducing the number of subjects selected for an A0-PET procedure prior to administration of an anti-A0 therapy, the method comprising selecting a subject for an A0- PET procedure when the subject has one or more biomarker levels selected from:(a) plasma A0 42 / 40 ratio < 0.0975 or plasma A0 42 / 40 < 0.1020,(b) plasma pTaul81 > 9.4 pg / mL, and(c) plasma pTau217 > 1.2 pg / mL.

33. The method of claim 32, wherein the one or more biomarker levels is determined by liquid chromatography tandem mass spectrometry (LC-MS / MS).

34. The method of claim 32, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by single molecule array (Simoa, e.g., Quanterix) to make one or more Simoa-pTau217 levels;(e) converting the one or more Simoa-pTau217 levels to one or more LC-MS / MS- pTau217 levels;(f) selecting the subject for the A0-PET procedure when the LC-MS / MS- pTau217 level is >1.2 pg / mL.

35. The method of claim 34, wherein the one or more Simoa-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(Simoa) = -1.41 + l. l l x log(LC-MS / MS).

36. The method of claim 32, wherein the one or more biomarker levels is plasma pTau217, the method further comprising(d) determining one or more subject plasma pTau217 levels by chemiluminescent enzyme immunoassay (CLEIA, e.g., Fujirebio Lumipulse) to make one or more CLEIA-pTau217 levels;(e) converting the one or more CLEIA-pTau217 levels to one or more LC- MS / MS-pTau217 levels;(f) selecting the subject for the A0-PET procedure when the LC-MS / MS- pTau217 level is >1.2 pg / mL.

37. The method of claim 36, wherein the one or more CLEIA-pTau217 levels are converted to one or more LC-MS / MS-pTau217 levels utilizing the following relationship: log(CLEIA) = -0.88 + 1.16 x log(LC-MS / MS).

38. The method of any one of claims 32-37, wherein the selected subject undergoes an A0-PET procedure prior to a first administration of the anti-A0 therapy.

39. The method of any one of claims 32-37, wherein subject has an A0 brain load of > 30 centiloid, as measured by the A0-PET procedure.

40. A method of converting plasma pTau217 measurements across analytical platforms comprising:(a) measuring plasma pTau217 levels in a first set of samples using a first analytical platform selected from LC-MS / MS, SIMOA, and CLEIA;(b) measuring plasma pTau217 levels in the same set of samples using a second analytical platform different from the first analytical platform;(c) performing Deming regression analysis of log-transformed data from both platforms to generate a conversion equation;(d) validating the conversion equation using an independent cohort; and(e) applying the conversion equation to normalize pTau217 measurements across platforms using CP Convert methodology.

41. A method of validating cross-platform biomarker conversion equations comprising:(a) developing conversion equations using a first cohort with measurements on multiple analytical platforms;(b) applying the conversion equations to measurements from at least one independent cohort using CP Convert methodology;(c) comparing the median differences between converted values and intra-cohort derived values; and(d) confirming that the conversion equations normalize measurements across platforms within acceptable limits.

42. The method of claim 42, wherein the acceptable limits comprise median differences of less than 0.15 pg / mL between CP Convert-converted values and intra-cohort derived values.

43. A method of reducing AP-PET testing burden in clinical trials comprising:(a) implementing pTau217 prescreening using a cutoff corresponding to 90% sensitivity;(b) selecting subjects for Ap-PET procedures based on pTau217 levels above the cutoff; and(c) achieving at least a 60% reduction in the number of subjects requiring AP-PET scanning compared to traditional screening approaches.

44. The method of claim 43, wherein the pTau217 cutoff is 1.2 pg / mL as measured by LC- MS / MS or equivalent values determined using CP Convert methodology for other analytical platforms.

45. A method of comparing pTau217 performance with other blood-based biomarkers comprising:(a) measuring plasma AP42 / 40 ratio, pTaul81, and pTau217 in the same population of subjects;(b) performing head-to-head ROC analysis to determine AUC values for predicting AP-PET positivity; and(c) demonstrating that pTau217 achieves superior performance with an AUC > 0.94, compared to AP42 / 40 ratio (AUC < 0.85) and pTaul81 (AUC < 0.82).

46. The method of any of claims 1-45, wherein the anti-AP therapy comprises active or passive therapies.

47. The method of claim 46, wherein the passive therapy comprises an anti-AP antibody.

48. The method of claim 47, wherein the anti-A0 antibody is PRX012.

49. The method of claim 47, wherein the anti-A0 antibody is lecanemab.

50. The method of claim 47, wherein the anti-A0 antibody is donanemab.

51. The method of claim 47, wherein the anti-A0 antibody is aducanumab.

52. The method of claim 47, wherein the anti-A0 antibody is gantenerumab.

53. The method of claim 46, wherein the active therapy comprises a vaccine.

54. A method of diagnosing Alzheimer’s Disease in a subject, comprising measuring levels of one or more biomarkers in the subject, the biomarkers selected from the group consisting of plasma A042 / 4O ratio, plasma pTaul81, and plasma pTau217, wherein when(a) the plasma A042 / 4O ratio is < 0.0975 or plasma ratio A042 / 4O is < 0.1020, or(b) the plasma pTaul81 > 9.4 pg / mL, or(c) the plasma pTau217 > 1.2 pg / mL, the subject is diagnosed with Alzheimer’s Disease.

55. A method of predicting a subject’s response to a treatment for Alzheimer’s Disease, comprising measuring levels of one or more biomarkers in the subject, the biomarkers selected from the group consisting of plasma A042 / 4O ratio, plasma pTaul81, and plasma pTau217, wherein when(a) the plasma A042 / 4O ratio is < 0.0975 or plasma ratio A042 / 4O is < 0.1020, or(b) the plasma pTaul81 > 9.4 pg / mL, or(c) the plasma pTau217 > 1.2 pg / mL, the subject is predicted to be responsive to a therapeutically effective amount of an anti-amyloid beta (A0) therapy.

56. The method of claim 54 or 55, wherein pTau217 measurements are obtained using CPConvert methodology to enable cross-platform comparison of results.

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