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US-20260126434-A1 - METHODS AND KITS FOR MEASURING THREE OR MORE TAU EPITOPES

US20260126434A1US 20260126434 A1US20260126434 A1US 20260126434A1US-20260126434-A1

Abstract

The disclosure relates to methods and kits for detecting, quantifying, or both, phosphorylated tau (p-tau) in a biological sample. The disclosure further provides methods for detecting brain-associated tau polypeptide, a brain injury or a neurodegenerative disease in a subject, determining the eligibility of individuals for participation in clinical trials for brain injury and/or neurodegenerative disease treatments, distinguishing between individuals with brain injury and/or neurodegenerative disease and without brain injury and/or neurodegenerative disease, and monitoring response to treatment of the same.

Inventors

  • John H. Kenten
  • George Sigal
  • Galina Nikolenko
  • Ryan Connelly

Assignees

  • MESO SCALE TECHNOLOGIES, LLC.

Dates

Publication Date
20260507
Application Date
20250613

Claims (20)

  1. 1 . A method of detecting, quantifying, or both, phosphorylated tau (p-tau) in a biological sample, wherein the p-tau is phosphorylated at two or more sites, comprising: (a) contacting the biological sample with: (i) a capture reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; (ii) a first detection reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; and (iii) a second detection reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; (b) forming a complex comprising the capture reagent, the p-tau, the first detection reagent and the second detection reagent; and (c) detecting the complex, thereby detecting the p-tau; or (d) measuring an amount of the complex, thereby quantifying an amount of the p-tau, wherein at least two of the capture reagent, first detection reagent, and second detection reagent binds a phosphorylated tau site.
  2. 2 . The method of claim 1 , wherein at least one of the capture reagent, first detection reagent, or second detection reagent binds a brain-associated tau polypeptide.
  3. 3 - 8 . (canceled)
  4. 9 . The method of claim 1 , wherein the capture reagent, the first detection reagent, and the second detection reagent comprise an antibody or antigen-binding fragment thereof, antigen, ligand, receptor, oligonucleotide, hapten, epitope, mimotope, or an aptamer.
  5. 10 - 12 . (canceled)
  6. 13 . The method of claim 1 , wherein (i) the capture reagent binds pTau181, pTau205, pTau212, pTau217 or pTau231, and does not bind non-phosphorylated tau; or (ii) either the first or the second detection reagent binds pTau181, pTau205, pTau212, pTau217, or pTau231, and does not bind non-phosphorylated tau; or (iii) any combination of (i) and (ii).
  7. 14 - 22 . (canceled)
  8. 23 . The method of claim 1 , wherein the capture reagent binds a surface, thereby forming the complex on the surface comprising the capture reagent, the p-tau, the first detection reagent and the second detection reagent, wherein the first detection reagent or second detection reagent comprises a detectable label.
  9. 24 - 29 . (canceled)
  10. 30 . The method of claim 1 , wherein the biological sample comprises whole blood, blood serum plasma, cerebrospinal fluid (CSF), urine, saliva, or an extraction or purification therefrom, or dilution thereof.
  11. 31 . A method of detecting a brain injury, brain injury severity, or a neurodegenerative disease in a subject, the method comprising obtaining measured levels of phosphorylated tau (p-tau) in a sample from the subject according to claim 1 , wherein the sample is whole blood, cerebrospinal fluid (CSF), serum, plasma, or a combination thereof.
  12. 32 . The method of claim 31 , wherein the method detects (i) brain injury or brain injury severity and the brain injury is concussive injury, subconcussive injury, acute concussive injury, impact head injury, acceleration or deceleration head trauma, closed-skull neurotrauma, traumatic brain injury, stroke, seizure, status epilepticus, chronic traumatic encephalopathy (CTE), or a combination thereof; or (ii) a neurodegenerative disease and the neurodegenerative disease is Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, Creutzfeldt-Jakob disease, Neuronal Synuclein Disease (NSD), motor neuron disease, or any combination thereof.
  13. 33 . (canceled)
  14. 34 . (canceled)
  15. 35 . A method of determining eligibility of a subject to participate in a clinical trial of a therapeutic drug for preventing or delaying Alzheimer's disease, the method comprising: (a) obtaining a measurement of phosphorylated tau levels of a sample from the subject according to claim 1 ; and (b) determining the eligibility of the subject for the clinical trial based on the measurement of phosphorylated tau levels; wherein the subject has been diagnosed with dementia or mild cognitive impairment, or wherein the subject has subjective cognitive complaints, or wherein the subject does not have any cognitive impairment; or a method of conducting a clinical trial of a therapeutic drug or intervention for Alzheimer's disease, the method comprising: (a) obtaining a measurement of phosphorylated tau levels of a sample from a subject according to claim 1 : (b) determining eligibility of the subject for the clinical trial based on the measurement of phosphorylated tau levels; and (c) administering the therapeutic drug to the subject: or a method of distinguishing a subject afflicted with Alzheimer's disease from an individual afflicted with non-Alzheimer's dementia, the method comprising: (a) obtaining a measurement of phosphorylated tau levels of a sample from the subject according to claim 1 ; and (b) identifying, based on the measurement of phosphorylated tau levels, the subject as (i) afflicted with Alzheimer's disease or (ii) afflicted with non-Alzheimer's dementia; or a method of treating Alzheimer's disease in a subject in need thereof, the method comprising: (a) obtaining a measurement of phosphorylated tau levels of a sample from the subject according to claim 1 , wherein the measurement is obtained prior to administration of a treatment for Alzheimer's disease, (b) determining, based on the measurement of phosphorylated tau levels, that the subject is afflicted with Alzheimer's disease, and (c) administering a treatment regimen for Alzheimer's disease to the subject; or a method of monitoring response to treatment for Alzheimer's disease in a subject, the method comprising: (a) obtaining a first measurement of phosphorylated tau levels of a sample from the subject according to claim 1 , wherein the first measurement is obtained prior to administration of a treatment regimen for Alzheimer's disease, (b) obtaining a second measurement of phosphorylated tau levels of a sample from the subject at one or more time points after administration of the treatment regimen for Alzheimer's disease has been initiated, (c) determining, based on the first and second measurements of phosphorylated tau levels, that the subject is responding positively to the Alzheimer's treatment regimen, and (d) continuing to administer the treatment regimen for Alzheimer's disease to the subject.
  16. 36 - 39 . (canceled)
  17. 40 . A kit for detecting phosphorylated tau (p-tau) in a biological sample, wherein the p-tau is phosphorylated at two or more sites, comprising, in one or more vials, containers, or compartments, three reagents: (a) a capture reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; (b) a first detection reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; and (c) a second detection reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site, wherein at least two of the three reagents bind a phosphorylated tau site.
  18. 41 . The kit of claim 40 , wherein the p-tau comprises: T175 (pTau175), T181 (pTau181), T212 (pTau212), T205 (pTau205), S214 (pTau214), T217 (pTau217), cis or trans T231 (pTau231), S293 (pTau293), 5396 (pTau396), L243 (Tau243) or any combination thereof, with reference to SEQ ID NO: 1.
  19. 42 . The kit of claim 41 , comprising: (a) a capture reagent that binds pTau217; (b) a first detection reagent that binds pTau231; and (c) a second detection reagent that binds pTau181, Tau243, or a brain-associated tau polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (HVTQARMV) or SEQ ID NO: 3 (AGHVTQARMVSK); or (a) a capture reagent that binds pTau217; (b) a first detection reagent that binds pTau231; and (c) a second detection reagent that binds a brain-associated tau polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (HVTQARMV) or SEQ ID NO: 3 (AGHVTQARMVSK), or (a) a capture reagent that binds pTau217; (b) a first detection reagent that binds pTau181; and (d) a second detection reagent that binds pTau231.
  20. 43 . (canceled)

Description

FIELD OF THE INVENTION The disclosure relates to methods and kits for detecting, quantifying, or both, phosphorylated tau (p-tau) in a biological sample. The disclosure further provides methods for detecting brain-associated tau polypeptide, a brain injury or a neurodegenerative disease in a subject, determining the eligibility of individuals for participation in clinical trials for brain injury and/or neurodegenerative disease treatments, distinguishing between individuals with and without a brain injury and/or neurodegenerative disease, and monitoring response to treatment of the same. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Jun. 10, 2025, is named “0076-0090WO1.xml” and is 15,464 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND Neurodegenerative diseases are conditions where brain cells progressively lose their function and die, leading to a variety of symptoms and impairments. Some examples include Alzheimer's disease (AD), Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). A traumatic brain injury (TBI) can predispose individuals to neurodegenerative diseases such as Chronic Traumatic Encephalopathy (CTE). Tau phosphorylation, a process where phosphate groups attach to the tau protein, is a key mechanism implicated in both TBI and neurodegenerative diseases. In a normal or non-disease state, tau phosphorylation helps regulate tau's interactions with microtubules. However, excessive phosphorylation (multi-phosphorylation) leads to tau detaching from microtubules and aggregating into neurofibrillary tangles (NFT). TBI can trigger this process by disrupting cellular homeostasis and interfering with tau phosphorylation, thereby increasing the risk of developing later-life neurodegenerative diseases. AD is a progressive neurodegenerative disorder that causes dementia in approximately 10% of individuals older than 65 years. One of AD's typical brain lesions is NFTs that are thought to consist of phosphorylated forms of the microtubule associated protein tau that is assembled into paired helical filaments. Tau expression is high in non-myelinated cortical axons, especially in the regions of the brain that are involved in memory consolidation such as the limbic cortex including the hippocampus. As noted above, multi-phosphorylation of tau is thought to cause the protein to detach from the microtubules, thereby destabilizing microtubules and compromising axonal transport. While tau phosphorylation promotes axonal and synaptic plasticity in the developing brain (Lovestone et al., “The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative processes,” Neurosciences 78(2):309-324 (1997)), it is pathological in the adult brain and specifically related to a group of disorders referred to as tauopathies, which includes AD and some forms of frontotemporal dementia (FTD) (Ballatore et al., “Tau-mediated neurodegeneration in Alzheimer's disease and related disorders,” Nature Reviews Neuroscience 8(9):663-672 (2007)). Understanding the mechanisms of tau phosphorylation and multi-phosphorylation in TBI and neurodegenerative diseases is important for developing therapies that can target and reverse the detrimental effects of tau aggregation. SUMMARY OF THE INVENTION In some aspects, the techniques described herein relate to a method of detecting, quantifying, or both, tau in a biological sample, wherein the tau comprises a plurality of detectable epitopes, the method comprising: (a) contacting the biological sample with a plurality of binding reagents, wherein the first binding reagent binds to a first epitope, the second binding reagent binds to a second epitope, and the third binding reagent binds to a third epitope; (b) forming a complex comprising the plurality of binding reagents and the tau; and (c) detecting the complex, thereby detecting the tau; or (d) measuring an amount of the complex, thereby quantifying an amount of the tau. In some aspects, the techniques described herein relate to a method of detecting, quantifying, or both, phosphorylated tau (p-tau) in a biological sample, wherein the p-tau is phosphorylated at two or more sites, comprising: (a) contacting the biological sample with: (i) a capture reagent that a phosphorylated tau site, or a non-phosphorylated tau site; (ii) a first detection reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; and (iii) a second detection reagent that binds a phosphorylated tau site, or a non-phosphorylated tau site; (b) forming a complex comprising the capture reagent, the p-tau, the first detection reagent and the second detection reagent; and (c) detecting the complex, thereby detecting the p-tau; or (d) me