TANGO was a Phase 2 clinical study designed to assess the safety and efficacy of gosuranemab, an anti-tau monoclonal antibody, in participants with mild cognitive impairment due to Alzheimer’s disease (AD) or with mild AD dementia. Despite robust target engagement of unbound N-terminal tau, the clinical efficacy endpoint was not met. In this exploratory analysis of TANGO participants, we examine plasma p-tau217 levels to assess the feasibility of using this biomarker to identify patients with AD pathology and predict disease progression. Plasma was collected from 554 randomized TANGO participants at baseline and up to Week 78. Plasma p-tau217 was measured using the ALZpath Quanterix Simoa assay. Plasma p-tau181 was measured using the Quanterix Simoa p-tau181 V2 Advantage assay. Tau PET was measured in a sub-study of 357 participants at baseline and Weeks 52 and 78 using 18 F-MK-6240. Amyloid PET was measured at screening using 18 F-florbetapir in n = 322 participants Clinical decline was measured using the CDR-SB, MMSE, ADAS-Cog13 and ADCS-ADL cognitive assessment scales. Statistical analysis was performed using Spearman correlation coefficient. Plasma p-tau217 and plasma p-tau181 were correlated at baseline (Spearman = 0.7617, p-value <0.0001). Baseline plasma p-tau217 levels were correlated with both baseline amyloid PET using SUVR in a composite region of interest (Spearman = 0.4294, p-value <0.0001) and baseline Tau PET using SUVR in composite regions of interest corresponding to Braak stages I-II, III-IV, and V-VI (Spearman range = 0.4075-0.6163, p = value <0.0001). Participants with higher concentrations of plasma ptau217 at baseline showed a greater rate of clinical decline at Week 78. The correlations of plasma p-tau217 with amyloid and tau PET at baseline suggest a relationship with both underlying pathological hallmarks of AD. Similar to results from observational cohorts, correlation of baseline plasma p-tau217 and clinical decline observed during the TANGO trial supports the utility of plasma p-tau217 as a potential prognostic biomarker of disease. Data from additional studies are needed to define appropriate use cases and relevant thresholds for plasma p-tau217 as a biomarker of disease pathology that can be used to prescreen patients in clinical trials.

Plasma biomarkers for Alzheimer's disease (AD) are increasingly being used to assist in making an etiological diagnosis for cognitively impaired (CI) individuals or to identify cognitively unimpaired (CU) individuals with AD pathology who may be eligible for prevention trials. However, a better understanding of the timing of plasma biomarker changes is needed to optimize their use in clinical and research settings. The aim of this study was to evaluate the timing of change of key AD plasma biomarkers (Aβ42/Aβ40, p-tau217, p-tau181, GFAP and NfL) from six different companies, along with established AD biomarkers, using AD progression timelines based on amyloid and tau PET. We used data from the Alzheimer's Disease Neuroimaging Initiative (ADNI), including 784 individuals with longitudinal 18 F-florbetapir amyloid PET and 359 individuals with longitudinal 18 F-flortaucipir tau PET, to estimate age at amyloid and tau positivity, defined as the age at the first positive PET scan. Of these, longitudinal plasma biomarker measures were available from 190 individuals with an estimated age at amyloid positivity and 70 individuals with an estimated age at tau positivity. Age at tau positivity was a stronger predictor of symptom onset than age at amyloid positivity in 17 individuals who progressed from CU to CI during their participation in the ADNI study (Adj R 2 = 0.86 vs. Adj R 2 = 0.38), and therefore was used to estimate symptom onset age for all individuals with an estimated age at tau positivity. Generalized additive mixed models (GAMMs) were used to model biomarker trajectories across years since amyloid positivity, tau positivity, and symptom onset, and to identify the earliest timepoint of biomarker abnormality when compared to a reference group of amyloid- and tau-negative CU individuals, as well as time periods of significant change in biomarkers. All plasma biomarkers except NfL became abnormal prior to amyloid and tau positivity. Plasma Aβ42/Aβ40 was the first biomarker to reach abnormality consistently across timelines and plasma GFAP became abnormal early in the tau timeline. Plasma Aβ42/Aβ40 levels reached a plateau, while plasma p-tau217, p-tau181, GFAP and NfL increased throughout disease progression. Some differences in the timing of change were observed across biomarker assays. The primary utility of plasma Aβ42/Aβ40 may lie in early identification of individuals at high risk of AD. In contrast, p-tau217, p-tau181, GFAP and NfL increase throughout the estimated timelines, supporting their potential as biomarkers for staging and monitoring disease progression.

Introduction: Blood tests have the potential to improve the accuracy of Alzheimer disease (AD) clinical diagnosis, which will enable greater access to AD-specific treatments. This study compared leading commercial blood tests for amyloid pathology and other AD-related outcomes. Methods: Plasma samples from the Alzheimers Disease Neuroimaging Initiative were assayed with AD blood tests from C2N Diagnostics, Fujirebio Diagnostics, ALZPath, Janssen, Roche Diagnostics, and Quanterix. Outcomes measures were amyloid positron emission tomography (PET), tau PET, cortical thickness, and dementia severity. Logistic regression models assessed the classification accuracies of individual or combined plasma biomarkers for binarized outcomes, and Spearman correlations evaluated continuous relationships between individual plasma biomarkers and continuous outcomes. Results: Measures of plasma p-tau217, either individually or in combination with other plasma biomarkers, had the strongest relationships with all AD outcomes. Discussion: This study identified the plasma biomarker analytes and assays that most accurately classified amyloid pathology and other AD-related outcomes.

There is a need for scalable, robust and precise Alzheimer’s Disease (AD) plasma phosphorylated Tau (pTau) biomarker tests to support clinical trials and clinical practice. This study evaluated the performance of novel plasma pTau181 and pTau217 assays developed on the LUMIPULSE G1200 automated immunoassay platform and compared them to Quanterix homebrew assays. Homebrew Quanterix Simoa assays for plasma pTau181 and pTau217 were transferred to the LUMIPULSE G instrument using recombinantly expressed versions of respectively α-pTau181 mAb ADx252 and α-pTau217 mAb RD-084 (capture) both combined with an ALP-conjugated Fab fragment digested from α-N-terminal Tau recombinant ADx204/RD-073 (detector). Analytical performance evaluated specificity, LLOQ and precision. Performance in patient samples was evaluated using a prospective memory cohort of 42 biomarker confirmed AD patients in the prodromal or dementia stage as well as 35 healthy spouse controls. LUMIPULSE G results were compared with results from the Quanterix homebrew assays designed by ADx using the same antibody configuration. Robust LUMIPULSE G1200 measurements (mean intra-run CV 3%) distinguished AD patients from spouse controls with an area under the curve (AUC) of 0.81 and 0.88 for pTau181 and pTau217, respectively. The Quanterix homebrew assays demonstrated higher AUC of 0.98 for pTau181 (pDeLong = 0.002) and of 0.95 for pTau217 (Figure 1A). Addition of age, sex and ApoE4 status to the LUMIPULSE G-based models increased the performance of both pTau181 and pTau217 to predict AD diagnosis – relatively to univariate models - with AUCs of respectively 0.91 and 0.95 (Figure 1B, both pDeLong < 0.05), approximating Simoa-based pTau performance. PTau values measured on both platforms correlated significantly (p<0.0001) for pTau181 (r = 0.66) and pTau217 (r = 0.77). The LUMIPULSE G platform is a scalable, automated, real-time-testing platform that is globally available. These data demonstrate that two assays on this platform, pTau181 and pTau217, are capable of distinguishing AD from controls in plasma. The sensitivity and specificity are slightly less on LUMIPULSE G when compared to an assay using the same antibodies developed on the Simoa instrument. Thus, additional experiments are required on the LUMIPULSE G to optimize analytical performance of these assays using an IVD-grade automated system.

The International CTAD Task Force (TF) addressed challenges related to designing clinical trials for agitation in dementia, presenting accomplishments from the two previous TFs on neuropsychiatric symptoms (NPS). In addition, this TF proposed a paradigm shift in NPS assessment and management, presenting Mild Behavioral Impairment (MBI) as a clinical syndrome. MBI is marked by later-life emergent and persistent NPS in dementia-free older persons (ranging from cognitively unimpaired to subjective cognitive decline to mild cognitive impairment), which facilitates earlier detection and better prognostication of Alzheimer's disease (AD). The TF has made the following recommendations for incorporation of NPS into AD preventative trials: (1) clinical trials targeting improvement in MBI symptoms should be undertaken; (2) treatment trials for MBI should be disease specific and confirm the diagnosis of participants using biomarkers; trials should include measures sensitive to cognitive changes in preclinical AD, which can serve as outcome measures, in addition to changes in biomarker levels; (3) as a first step, pharmacotherapeutic trials should address the full MBI complex as well as the specific symptoms/domains that constitute MBI; (4) clinical trials using problemadaptation psychotherapy to target affective MBI should be considered; and (5) MBI should be considered in AD trials of disease modifying therapies. The well-validated and widely-used MBI Checklist (MBI-C) is an appropriate symptom rating scale for these studies, as it was developed specifically to identify and measure MBI in dementia-free persons. Other scales such as the Neuropsychiatric Inventory (NPI) may be used, although administration at two timepoints may be necessary to operationalize the MBI criterion of symptom persistence.

In randomized clinical trials (RCTs) for Alzheimer’s Disease (AD), cerebrospinal fluid (CSF) and positron emission tomography (PET) biomarkers are currently used for the detection and monitoring of AD pathological features. The use of less resource-intensive plasma biomarkers could decrease the burden to study volunteers and limit costs and time for study enrollment. Blood-based markers (BBMs) could thus play an important role in improving the design and the conduct of RCTs on AD. It remains to be determined if the data available on BBMs are strong enough to replace CSF and PET biomarkers as entry criteria and monitoring tools in RCTs.

INTRODUCTION:We assessed the use of cerebrospinal fluid (CSF) biomarkers as an alternative to positron emission tomography (PET) for brain amyloid beta (Aβ) pathology confirmation in the EMERGE and ENGAGE clinical trials.METHODS:EMERGE and ENGAGE were randomized, placebo-controlled, Phase 3 trials of aducanumab in participants with early Alzheimer's disease. Concordance between CSF biomarkers (Aβ42, Aβ40, phosphorylated tau 181, and total tau) and amyloid PET status (visual read) at screening was examined.RESULTS:Robust concordance between CSF biomarkers and amyloid PET visual status was observed (for Aβ42/Aβ40, AUC: 0.90; 95% CI: 0.83-0.97; p < 0.0001), confirming CSF biomarkers as a reliable alternative to amyloid PET in these studies. Compared with single CSF biomarkers, CSF biomarker ratios showed better agreement with amyloid PET visual reads, demonstrating high diagnostic accuracy.DISCUSSION:These analyses add to the growing body of evidence supporting CSF biomarkers as reliable alternatives to amyloid PET imaging for brain Aβ pathology confirmation.HIGHLIGHTS:CSF biomarkers and amyloid PET concordance were assessed in Ph3 aducanumab trials. Robust concordance between CSF biomarkers and amyloid PET was observed. CSF biomarker ratios increased diagnostic accuracy over single CSF biomarkers. CSF Aβ42/Aβ40 demonstrated high concordance with amyloid PET. Results support CSF biomarker testing as a reliable alternative to amyloid PET.

BACKGROUND:The amyloid probability score (APS) is the model read-out of the analytically validated mass spectrometry-based PrecivityAD® blood test that incorporates the plasma Aβ42/40 ratio, ApoE proteotype, and age to identify the likelihood of brain amyloid plaques among cognitively impaired individuals being evaluated for Alzheimer's disease.PURPOSE:This study aimed to provide additional independent evidence that the pre-established APS algorithm, along with its cutoff values, discriminates between amyloid positive and negative individuals.METHODS:The diagnostic performance of the PrecivityAD test was analyzed in a cohort of 200 nonrandomly selected Australian Imaging, Biomarker & Lifestyle Flagship Study of Aging (AIBL) study participants, who were either cognitively impaired or healthy controls, and for whom a blood sample and amyloid PET imaging were available.RESULTS:In a subset of the dataset aligned with the Intended Use population (patients aged 60 and older with CDR ≥0.5), the pre-established APS algorithm predicted amyloid PET with a sensitivity of 84.9% (CI: 72.9-92.1%) and specificity of 96% (CI: 80.5-99.3%), exclusive of 13 individuals for whom the test was inconclusive.INTERPRETATION:The study shows individuals with a high APS are more likely than those with a low APS to have abnormal amounts of amyloid plaques and be on an amyloid accumulation trajectory, a dynamic and evolving process characteristic of progressive AD pathology. Exploratory data suggest APS retains its diagnostic performance in healthy individuals, supporting further screening studies in the cognitively unimpaired.

There is a need for automated random access plasma phosphorylated Tau (pTau) methods that reflect AD pathology with consistent performance. LUMIPULSE (LP) assays could offer a solution. Initial prototype pTau181 and pTau217 LP assays showed lower clinical performance versus Quanterix Homebrew assays (Poster #79533 – P4-06). This new study presents optimized pTau181 and pTau217 LP assays, showing data on their analytical and clinical performance compared to the initial prototype method (Ref) and an in-house pTau181 Quanterix Homebrew assay. Plasma pTau181 and pTau217 LP assays were developed using recombinant pTau181 mAb RD-070, resp. pTau217 mAb RD-085, combined with an ALP-Fab conjugate from recombinant N-terminal Tau mAb RD-073. In the optimized automated protocol, an assay specific diluent (ASD) is added (20%) to the sample to increase signal specificity. Analytical and clinical performance of the reference versus ASD-treated prototypes to distinguish AD from controls was evaluated using 40 CSF-confirmed AD-dementia and 40 age-matched healthy volunteer plasma samples. Analytical performance: Both optimized pTau181 and pTau217 LP tests demonstrated high precision comparable to their reference assays (inter-run %CV on 3 neat plasma samples < 7.4). LLOQ was not affected by ASD-treatment. Clinical performance: pTau181 LP assays demonstrated comparable AUC and median fold change with (AUC 0.828, fold change 1.9) and without ASD (AUC 0.823, fold change 1.5) while the SIMOA assay demonstrated a higher AUC (0.866) and fold change (2.9). For pTau217, the performance improved from an initial AUC of 0.918 to 0.935, and a fold change of 3.72 instead of initially 2.76. (Figure 1). All assays correlated strongly (p<0.00001) (Table 1); highest correlations among them were observed with the pTau217 ASD-treated assay. Using an assay specific diluent resulted in improved clinical performance for the pTau217 prototype LUMIPULSE assay, in line with other published pTau217 assays. We did not achieve improved performance of the pTau181 LP assay with the ASD addition. Most important to emphasize, a performant pTau217 assay prototype is under development on an fully automated, scalable platform. Next steps include verification of performance in additional well-characterized AD cohorts and characterization of lot consistency and robustness of the pTau181 and pTau217 N-terminal LUMIPULSE assays.

A previous comparative analysis of plasma Aβ42/40 immunoassays and mass spectrometry-based assays conducted in 2021 found that several assays had strong performance in predicting amyloid PET status (Zicha et al., 2022). In the spirit of enabling the National Institute on Aging and the Alzheimer’s Association framework for classifying Alzheimer’s disease (AD) utilizing measures of pathology for amyloid, tau, and neurodegeneration (ATN), we have added to this comparative analysis a newly developed immunoassay on an automated, scalable platform. Robustly validated plasma Aβ assays represent a non-invasive and cost-effective alternative to CSF or neuroimaging for early detection of Alzheimer’s disease pathology and screening participants for enrollment into clinical trials. The project team consists of pharmaceutical industry, patient advocacy, governmental, and academic representatives. The current study utilized an additional aliquot of the same 130 plasma samples provided by ADNI that were previously tested on three immunoassays and three mass spectrometry-based assays. The statistical analysis plan was performed as published previously to determine if the measurement of plasma Aβ with age and APOE ε4 status performs better than age and APOE ε4 status alone in determining amyloid PET status. In this sample cohort, the new automated immunoassay with age and APOE genotype had similar prediction of amyloid status compared to mass spectrometry-based assays, with an ROC AUC of 85.7 (95% CI: 79.1 – 92.4) for the immunoassay and 84.2 (95% CI: 77.0 – 91.3) for the best- performing mass spectrometry-based assay. The Spearman correlation of plasma Aβ42/40 with amyloid PET (florbetapir standardized uptake value ratio) for the new immunoassay was -0.567 (p < 0.001) compared to -0.536 (p < 0.001) for the best performing mass spectrometry-based assay. The new immunoassay significantly improved the prediction of amyloid positivity beyond age and APOE ε4 genotype (p = 0.003). Results from this comparative analysis together with the continued advancement in plasma assay technology identify a potential use for plasma Aβ42/40 measurement in addressing the amyloid component of the ATN framework. Further evaluation of plasma Aβ42/40 in addition to p-tau in plasma is planned using longitudinal samples to determine the ability of plasma biomarkers to detect amyloidosis.

Aducanumab is a human monoclonal antibody selective for aggregated forms of amyloid beta approved in the United States by the Food and Drug Administration (FDA) for the treatment of Alzheimer’s disease on June 7, 2021 under the accelerated approval pathway, which requires a randomized-controlled trial with an appropriate control to verify the clinical benefit of aducanumab. On March 25, 2022 the sponsor submitted the final protocol for this trial, named ENVISION, to the FDA. ENVISION (NCT05310071) is a multicenter, randomized, double-blind, placebo-controlled, parallel group study in participants with mild cognitive impairment due to Alzheimer’s disease (Stage 3) and mild Alzheimer’s disease dementia (Stage 4), designed to verify the clinical efficacy and safety of aducanumab. Key enrollment criteria include MMSE score of 22-30, age 60-85, CDR-G of 0.5 or 1.0, regardless of ApoE ε4 status. Participants will be randomized, to receive aducanumab or placebo in a 2:1 ratio and will be titrated to 10 mg/kg aducanumab or placebo by intravenous infusion every 4 weeks for a treatment duration of 24 months. The primary objective is to assess the clinical benefit of monthly doses of aducanumab in slowing cognitive and functional impairment as measured by change of CDR-SB score at 18 months as compared to placebo. Key secondary endpoints include change in ADCS-ADL-MCI, ADAS-Cog13, iADRS, MMSE, and NPI-10; other secondary endpoints include change in amyloid PET and tau PET signal. Safety endpoints include incidence of ARIA events and SAEs. Exploratory endpoints include CSF and plasma biomarkers. Approximately 1512 participants will be enrolled across 220 centers globally. A racially/ethnically representative population will be included in the United States, with an enrollment goal of 18% Black/African American and LatinX/Hispanic patients. ENVISION will begin in Q2 2022 and read out approximately 4 years after first patient in. Primary analysis read-out of clinical, biomarker, and safety data will be at 18 months, with additional longer-term data on aducanumab treatment effect up to 24 months. The confirmatory ENVISION trial will further explore clinical efficacy, biomarker profile, and safety of aducanumab in a racially/ethnically representative population.

BACKGROUND:There is currently a lack of reliable and easily accessible biomarkers predicting cognitive decline in Alzheimer's disease (AD). Synaptic dysfunction and loss occur early in AD and synaptic loss measured in the brain tissue and by PET are closely linked to cognitive decline, rendering synaptic proteins a promising target for biomarker development.METHODS:We used novel Simoa assays to measure cerebrospinal fluid (CSF) levels of two synaptic biomarker candidates, postsynaptic density protein 95 (PSD-95/DLG4), and the presynaptically localized synaptosomal-associated protein 25 (SNAP-25), as well as neurogranin (Ng), an established postsynaptic biomarker. CSF samples from two well-characterized cohorts (n=178 and n=156) were selected from banked samples obtained from diagnostic lumbar punctures containing subjects with amyloid-ß (Aß) positive AD, subjects with non-AD neurodegenerative diseases, subjects with other neurological conditions, and healthy controls (HC).RESULTS:All subjects had detectable CSF levels of PSD-95, SNAP-25, and Ng. CSF levels of PSD-95, SNAP-25, and Ng were all correlated, with the strongest correlation between the presynaptic SNAP-25 and the postsynaptic neurogranin. AD subjects had on average higher concentrations of all three synaptic markers compared to those with non-AD neurodegenerative diseases, other neurological disorders, and HCs. Increased CSF levels of PSD-95, SNAP-25, and Ng were, however, not specific for AD and were present in sporadic cases with inflammatory or vascular disorders as well. High CSF levels of PSD-95 were also observed in a few subjects with other neurodegenerative disorders.CONCLUSION:The data establishes PSD-95 as a promising CSF marker for neurodegenerative disease synaptic pathology, while SNAP-25 and Ng appear to be somewhat more specific for AD. Together, these synaptic markers hold promise to identify early AD pathology, to correlate with cognitive decline, and to monitor responses to disease-modifying drugs reducing synaptic degeneration.

Recent technical performance improvements with the introduction of fully automated platforms have made cerebrospinal fluid (CSF) biomarkers attractive alternatives to positron emission tomography (PET) for brain amyloid pathology confirmation in the diagnosis of Alzheimer’s disease. Growing the body of evidence demonstrating high agreement between CSF biomarkers and amyloid PET across multiple automated platforms is critical to ensuring widespread access to and adoption of CSF biomarkers. Adding to previous data generated with Lumipulse® CSF immunoassays, this study assessed CSF biomarkers on the Elecsys® platform as valid alternatives to PET for amyloid confirmation by conducting a concordance analysis using data from EMERGE and ENGAGE, including performance evaluation of the Elecsys® Gen2 recommended cutoff. EMERGE and ENGAGE were randomized, double-blind, placebo-controlled, global Phase 3 studies of aducanumab. Participants were 50-85 years old and met clinical criteria for mild cognitive impairment due to AD or mild AD dementia. All enrolled participants had amyloid pathology detected by amyloid PET (visual read) during screening. Positive percent agreement (sensitivity), negative percent agreement (specificity), and overall percent agreement (OPA) were determined (with amyloid PET visual read as reference standard) for the following CSF biomarkers (and applicable ratios) using the Elecsys® immunoassay: using an internal cutoff value maximizing the Youden J index for Abeta40, Abeta42, phosphorylated tau (ptau181), tau; Abeta40/Abeta42, ptau181/Abeta42, tau/Abeta42; and for the ptau181/Abeta42 ratio using the Elecsys® Gen2 cutoff. N=349 participants (n=309 PET positive; n=40 PET negative) provided CSF at screening and were included in the analysis. Using internal cutoff values, tau, ptau 181 and Abeta 42 demonstrated OPA of 69%, 83% and 89% respectively, and CSF biomarker ratios showed increased diagnostic accuracy (OPA ≥95% for all ratios assessed). The Elecsys® Gen2 cutoff demonstrated excellent sensitivity(93%), specificity(82%), and OPA(92%). These analyses demonstrate robust concordance between CSF biomarkers and amyloid PET visual read in EMERGE/ENGAGE and align with results generated previously. This supports the interchangeability of these biomarker methods for determination of amyloid positivity and applicability of the Elecsys® recommended cutoff in similar patient populations, with significant potential to improve accessibility of this critical biomarker for AD diagnosis in clinical trials and clinical practice.

This report details the approach taken to providing a dataset allowing for analyses on the performance of recently developed assays of amyloid beta (Aβ) peptides in plasma and the extent to which they improve the prediction of amyloid positivity.

The National Institute on Aging and the Alzheimer’s Association framework for classifying Alzheimer’s disease (AD) utilizes measures of pathology for amyloid, tau, and neurodegeneration (ATN), that can identify participants for clinical trials. Currently, amyloid pathology is determined by costly PET or invasive CSF measurements. When applied to participant selection, these measures are associated with high screen failure rates and contribute to the high costs and long duration of recruitment of AD clinical trials. Recent reports indicate plasma amyloid beta (Aβ) peptides correlate highly with PET and CSF amyloid measures. This study aims to characterize novel assays as a screening tool that can quantify plasma Aβ peptides in a robust and reproducible manner. The project team consists of representatives from pharmaceutical industry, nonprofit/patient advocacy, and academic institutions. Six plasma Aβ assays were selected to be part of the comparison: three liquid chromatography–mass spectrometry (LC-MS/MS) assays and three immunoassays. Plasma samples with corresponding amyloid PET data were selected from ADNI n =130 (50% Aβ+): cognitively normal n =54 (37% Aβ+), mild cognitive impairment n =54 (46% Aβ+), and AD n =22 (91% Aβ+). Each participant’s sample was tested in a blinded fashion on all six assays with analytical controls. Statistical tests were performed to identify which assays can significantly improve the Area Under the Receiver Operating Characteristic (AUROC) curve for predicting amyloid PET status compared to age and APOE ε4 status alone. The project tested 130 samples across assays with comparison to amyloid PET. Initial results from assay providers show AUROCs to predict amyloid status at levels consistent with performance in earlier publications. A comparison of results for the various assays will be presented with respect to their ability to classify amyloid PET status and whether they add significantly to the prediction of amyloid positivity beyond age and genotype. Measurement of Aβ in plasma contributes to addressing the amyloid component of the ATN framework and can be a pre-screen or a substitute for PET or CSF screening. Further clinical validation of these assays is planned to assess amyloidosis detection performance in a cognitively normal population.

Biofluid markers of neurodegeneration and neural injury have great potential to elucidate disease mechanisms and accelerate development of experimental therapeutics. Cerebrospinal fluid (CSF) tau and phosphorylated tau are established biomarkers for AD, while plasma (N-terminal) tau and phosphorylated tau (p-tau) are being evaluated as complementary markers in a more accessible biofluid. Tau is present in CSF and plasma in both full-length and truncated forms, with great interest in characterizing and advancing these different forms as biomarkers to support clinical development. To contribute to the emerging research on tau fragments as biomarker, novel ultra-sensitive immunoassays were developed targeting low-abundant, non-truncated full-length (FL) tau in CSF and a mid-region tau fragment in CSF and plasma. Simoa assays were developed using monoclonal antibodies (mAbs) targeting mid-region tau epitopes or epitopes on full-length tau. Resulting assays were qualified and partially validated for analytical performance parameters: measuring range, specificity, repeatability, reproducibility and parallelism were assessed. Reagent and analyte stability were investigated by assessing hold time and freeze/thaw (F/T) stability. Paired clinical routine CSF and plasma samples were analysed (20 controls and 20 AD based upon CSF Aβ 1-42 , t-tau and p-tau(T181) profile). FL- and mid-tau Simoa assays were analytically characterized, upscaled to bulk volumes and partially validated. Both assays had acceptable repeatability and reproducibility (%CV <20%) for plasma and/or CSF. Parallelism was within acceptance criteria (ACC of 75-125%; %CV <20%) for FL-tau in 4 CSF samples and for mid-tau in 4 CSF and 4 plasma samples. Analyte stability was demonstrated up to 5 F/T cycles and for a hold-time of 24h at 21°C with a %CV<20%. Testing of clinical samples showed an increased CSF FL- (p<0.005) and mid-Tau (p<0.0001) concentration in AD versus controls, whereas plasma mid-tau did not increase. A novel CSF assay for full length tau and a novel CSF/plasma assay for the mid-tau fragment was developed, qualified and partially validated. The generated data indicated well-performing tau assays available as RUO kits in pre-clinical and clinical use in drug development programs.