Blood-based screening of subjects for clinical trials for treatment of tauopathies or amyloidosis diseases

Abstract

A method of pre-screening a human subject for a clinical trial for the treatment of tauopathy or amyloidosis disease. The method includes obtaining a plasma sample from the subject and determining a concentration of p217 + tau present in the plasma sample. The method further includes instructing the subject to further screen for the clinical trial when the concentration of p217 + tau present in the plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold. The minimum threshold corresponds to the amount of p217 + tau in plasma over which the subject exhibits mild cognitive impairment (MCI) as well as increased accumulation of tau tangles in the brain compared to cognitive normal patients. The maximum threshold value corresponds to the amount of p217 + tau present in plasma beyond which the subject exhibits a pathology of extensive accumulation of tau entanglement in multiple regions of the brain.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 366,449, filed June 15, 2022, and U.S. Provisional Application No. 63 / 437,260, filed January 5, 2023, the entire contents of each of which are incorporated herein by reference.

[0003] Reference sequence list submitted electronically

[0004] The sequence list of this application was filed electronically as an XML-formatted sequence list with the United States Patent and Trademark Center (USPTC) under the filename "JAB7165WOPCT1SEQLIST.xml", created on June 5, 2023, and is 17 kilobytes (KB) in size. This sequence list is an integral part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] This application relates to a blood-based screening method for subjects in clinical trials for the treatment of tau proteinopathy or amyloidogenic disease. Specifically, this application relates to a method and its use for identifying patients suitable for clinical trials for the treatment of tau proteinopathy or amyloidogenic disease based on measuring the amount of monophosphorylated or polyphosphorylated p217+ tau protein species in blood-based samples. Background Technology

[0006] Alzheimer's disease (AD) is a degenerative brain disorder characterized by progressive loss of memory, cognition, reasoning, judgment, and emotional stability, which gradually leads to severe mental decline and eventual death. AD is a very common cause of progressive mental disorder (dementia) in older adults. More than 5 million people in the United States have AD, and this number is increasing with the aging population. In fact, 10% of people over 65 years of age have AD, and it is the fifth leading cause of death in this population. Overall, AD is the sixth leading cause of death in the United States (one in three older adults dies from AD or other dementia), and the disease was estimated to cost $305 billion in 2020. AD is also observed in racial groups worldwide and represents a major public health problem now and in the future.

[0007] Individuals with Alzheimer's disease (AD) exhibit characteristic lesions in their brains called senile (or amyloid) plaques, amyloid angiopathy (the deposition of amyloid protein in blood vessels), and neurofibrillary tangles. These lesions are typically found in large numbers in several brain regions important for memory and cognitive function in patients with AD, particularly amyloid plaques and neurofibrillary tangles with paired spiral filaments.

[0008] The development of tau disease in the AD brain follows a distinct spreading pattern. Hypotheses regarding tau disease propagation and spread have been described based on the Braak stage of tau disease development in the human brain and the spread of tau disease following injection of tau aggregates in preclinical tau models (Frost et al., J Biol Chem. 284:12845-52, 2009; Clavaguera et al., Nat Cell Biol. 11:909-13, 2009). It is believed that tau disease can spread from one brain region to the next in a prion-like manner. This spread involves the externalization of tau seeds, which can be absorbed by nearby neurons and induce further tau disease.

[0009] There is a significant unmet need for clinically effective and safe treatments for tau disease or amyloidosis (such as Alzheimer's disease). However, any investigational medicine intended to treat tau disease or amyloidosis requires clinical trials in appropriate subjects to demonstrate its efficacy in improving or slowing the progression of tau disease or amyloidosis. Summary of the Invention

[0010] An exemplary embodiment of this application relates to a method for pre-screening human subjects in a clinical trial for the treatment of tau proteinopathy or amyloidosis. The method includes obtaining a plasma sample from the subject and determining the concentration of p217+tau present in the plasma sample. For example, the concentration of p217+tau present in the plasma sample can be determined by the following steps: contacting the plasma sample with a capture antibody targeting the p217+tau epitope, such that the capture antibody binds to the p217+tau peptide in the plasma sample to form an antibody-peptide complex; contacting the antibody-peptide complex with a detection antibody, such that the detection antibody binds to the antibody-peptide complex; and detecting the detection antibody to determine the amount of p217+tau peptide in the plasma sample. In one example, an assay with an LLOQ < 0.04 pg / ml is used to determine the concentration of p217+tau.

[0011] The method also includes instructing subjects to undergo further screening for clinical trials when the concentration of p217+tau in a plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold. The minimum threshold corresponds to the amount of p217+tau in plasma exceeding which subjects exhibit mild cognitive impairment (MCI) and increased accumulation of tau tangles in the brain compared to cognitively normal patients. In one example, the minimum threshold is from about 0.075 pg / ml to about 0.125 pg / ml. The maximum threshold corresponds to the amount of p217+tau in plasma exceeding which subjects exhibit pathology of widespread accumulation of tau tangles in multiple areas of the brain. In one example, the maximum threshold is from about 0.225 pg / ml to about 0.275 pg / ml. More specifically, the instruction step includes instructing subjects to undergo further screening for clinical trials when the concentration of p217+tau in a plasma sample is ≥0.1 pg / ml and ≤0.25 pg / ml.

[0012] In another aspect of this application, a method is provided for screening human subjects for clinical trials to treat tau proteinopathy or amyloidosis. The method includes obtaining a plasma sample from the subject and determining the concentration of p217+tau present in the plasma sample. The method also includes obtaining PET data of the subject's brain generated using a tau-specific or amyloid-specific radiotracer when the concentration of p217+tau present in the plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold. The minimum threshold corresponds to the amount of p217+tau in the plasma exceeding which the subject exhibits mild cognitive impairment (MCI) and increased accumulation of tau tangles in the brain compared to cognitively normal patients. In one example, the minimum threshold is from about 0.075 pg / ml to about 0.125 pg / ml. The maximum threshold corresponds to the amount of p217+tau present in the plasma exceeding which the subject exhibits pathology of widespread accumulation of tau tangles in multiple areas of the brain. In one example, the maximum threshold is from about 0.225 pg / ml to about 0.275 pg / ml. More specifically, the indication step includes instructing subjects to undergo further screening for clinical trials when the concentration of p217+tau in a plasma sample is ≥0.1 pg / ml and ≤0.25 pg / ml. The method also includes analyzing the PET data to determine whether a subject is tau-positive or amyloid-positive, and whether the subject has extensive tau tangles or extensive amyloid fibrils. The method further includes indicating a subject's suitability for inclusion in a clinical trial if: (i) the concentration of p217+tau in the plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold; (ii) PET data indicates that the subject is tau-positive or amyloid-positive; and (iii) PET data indicates that the subject does not have extensive tau tangles or extensive amyloid fibrils.

[0013] These and other aspects of the invention will become apparent to those skilled in the art after reading the following detailed description of the invention (including the accompanying drawings and claims). Attached Figure Description

[0014] Figure 1 An exemplary method is shown for pre-screening or screening human subjects for clinical trials intended to treat tau proteinosis or amyloidosis.

[0015] Figure 2 Another exemplary method for pre-screening or screening human subjects for clinical trials intended to treat tau proteinosis or amyloidosis is shown.

[0016] Figure 3aExemplary p217+tau assays across different concentrations are shown, from plasma samples collected from 832 patients screened for clinical trials.

[0017] Figure 3b The concentration distribution of p217+tau detected in plasma samples collected from 831 patients screened for clinical trials is shown by an exemplary p217+tau assay.

[0018] Figure 4 The Braak ROI is shown.

[0019] Figure 5 A significant correlation (p<10) was shown between plasma p217+tau and MK6240 across all six (6) Braak ROIs. -15 ).

[0020] Figure 6 The study showed that plasma p217+tau could predict Braak stage 3 or lower in vivo, with an AUC of 82%. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings set forth in this specification. All patents, published patent applications, and publications referenced herein are incorporated herein by reference as if fully set forth herein. It is noteworthy that, unless the context clearly determines otherwise, the singular forms “a,” “an,” “the,” and “described” used herein and in the appended claims include plural references.

[0022] Unless otherwise indicated, any numerical value, such as concentrations or concentration ranges as described herein, is to be understood in all cases to be modified by the term “about”. Therefore, numerical values ​​generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, numerical ranges explicitly include all possible subranges, all individual numerical values ​​within that range, including both integers and fractions within such ranges.

[0023] As used herein, the terms “antibody” or “immunoglobulin” refer to specific proteins capable of binding to an antigen or a portion thereof. These terms are used broadly herein and include immunoglobulin or antibody molecules, including polyclonal antibodies, monoclonal antibodies (including murine, human, human-adapted, humanized, and chimeric monoclonal antibodies), and antibody fragments.

[0024] Generally, antibodies are proteins or peptide chains that exhibit binding specificity to a particular antigen. Antibody structures are well-known. Immunoglobulins can be designated as five major classes based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subclassed into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Therefore, the antibody of this application can be any of the five major classes or their corresponding subclasses. Preferably, the antibody of this application is IgG1, IgG2, IgG3, or IgG4. Based on the amino acid sequence of its constant domain, the light chain of an antibody from any vertebrate species can be designated as one of two completely different types, κ and λ. Therefore, the antibody of this application can contain either a κ or λ light chain constant domain. According to a specific embodiment, the antibody of this application comprises the heavy chain and / or light chain constant regions derived from mouse or human antibodies.

[0025] In addition to the constant heavy and light chain domains, antibodies also contain variable light and heavy chain regions. These variable regions of immunoglobulins consist of "framework" regions interrupted by "antigen-binding sites." Antigen-binding sites are defined using various terms and numbering schemes:

[0026] (i) Kabat: “Complementarity-determining region” or “CDR” is based on sequence variability (Wu and Kabat, J Exp Med. 132:211-50, 1970). Generally, antigen-binding sites have three CDRs in each variable region (e.g., HCDR1, HCDR2, and HCDR3 in the heavy chain variable region (VH), and LCDR1, LCDR2, and LCDR3 in the light chain variable region (VL);

[0027] (ii) Chothia: The terms “hypervariant region” or “HVR” refer to regions of the antibody variable domains that are structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk, J Mol Biol. 196:901-17, 1987). Generally, the antigen binding site has three hypervariable regions in each VH (H1, H2, H3) and VL (L1, L2, L3).

[0028] The numbering system and annotations for CDRs and HVRs have been developed by Abhinandan and Martin.

[0029] (Abhinandan and Martin, Mol Immunol. 45:3832-9, 2008);

[0030] (iii)IMGT: Another definition of the region that forms the antigen-binding site has been adopted by Lefranc.

[0031] (Lefranc et al., Dev Comp Immunol. 27:55-77, 2003) proposed a comparison based on the V domains of immunoglobulins and T-cell receptors. The International Immunogenetics (IMGT) database (http: / / www.imgt.org) provides standardized numbers and definitions for these regions. The correspondence between CDR, HVR, and IMGT classifications is described in Lefranc et al., 2003 (ibid.).

[0032] (iv) Antigen binding sites can also be identified based on “specifically determined residues used” (SDRU) (Almagro, Mol Recognit. 17: 132-43, 2004), where SDR refers to the amino acid residues of immunoglobulins that are directly involved in antigen contact.

[0033] The “framework” or “framework sequence” is the remaining sequence within the variable region of an antibody, excluding those sequences defined as antigen-binding sites. Because the precise definition of an antigen-binding site can be determined through various partitions as described above, the exact framework sequence depends on the definition of the antigen-binding site. The frame region (FR) is the more highly conserved portion of the variable domain. The variable domains of the natural heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), which typically adopt a β-sheet configuration and are linked by three hypervariable loops. The hypervariable loops in each chain are tightly bound to the hypervariable loops of the other chain via the FRs and contribute to the formation of the antibody's antigen-binding site. Structural analysis of antibodies has revealed a relationship between the sequence and shape of the binding site formed by the complementarity-determining region (Chothia et al., J. Mol. Biol. 227:799-817, 1992; Tramontano et al., J. Mol. Biol. 215:175-182, 1990). Despite their high sequence variability, five of the six rings adopt only small-scale main-chain conformations, known as "canonical structures." These conformations are determined first by the length of the rings and secondly by key residues present at specific locations within the rings and frame regions. These key residues determine the conformation by their stacking, hydrogen bonding, or ability to present a unique main-chain conformation.

[0034] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin, antibody, or its antigen-binding fragment specifically binds. Epitopes can be formed from either consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by GEMorris (1996).

[0035] As used herein, the term “tau” or “tau protein” refers to a rich variety of central and peripheral nervous system proteins with multiple isotypes. In the human central nervous system (CNS), six major tau isotypes exist, ranging in length from 352 to 441 amino acids, due to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9, 2009). These isotypes are distinguished from each other by regulatory inclusions of 0 to 2 N-terminal insertion sequences and 3 or 4 tandemly arranged microtubule-binding repeat sequences, and are designated ON3R, 1N3R, 2N3R, ON4R, 1N4R, and 2N4R. As used herein, the term “control tau” refers to the tau isotype of SEQ ID NO:1, which lacks phosphorylation and other post-translational modifications. As used herein, the term “tau” includes proteins containing mutations of full-length wild-type tau, such as point mutations, fragments, insertions, deletions, and splice variants. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0036] Unless otherwise specified, as used herein, the amino acid numbers in tau protein or fragments thereof refer to the amino acid sequence shown in SEQ ID NO:1.

[0037] As used herein, the terms “p217+tau peptide,” “p217+tau,” or “p217+tau protein” refer to human tau protein or tau fragments that are phosphorylated at residue 217 (pT217) of tau protein and may or may not be further phosphorylated at other residues such as, for example, residue 212 (pT212) of tau protein, wherein these sites are numbered according to the numbers in SEQ ID NO:1.

[0038] As used herein, the term "p217+tau epitope" refers to a tau epitope containing at least one of phosphorylated T217 and phosphorylated T212, wherein these positions are numbered according to the numbers in SEQ ID NO:1. Examples of p217+tau epitopes include, for example, the pT3 epitope. As used herein, the term "pT3 epitope" refers to an epitope containing amino acids 210 to 220 of human tau protein, which is phosphorylated at residue 217 and may or may not be further phosphorylated at other residues such as, for example, residue 212, wherein these positions are numbered according to the numbers in SEQ ID NO:1.

[0039] As used herein, the term "capture antibody" refers to an antibody that binds to an antigen of interest and is directly or indirectly linked to a solid-phase carrier. Examples of solid-phase carriers include, but are not limited to, microparticles or beads, such as magnetic or paramagnetic beads. Examples of capture antibodies include, but are not limited to, monoclonal antibodies that bind to the p217+tau epitope.

[0040] As used herein, the term "detection antibody" refers to an antibody that binds to an antigen of interest and has a detectable tag or is linked to an auxiliary detection system. Examples of detectable tags include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of detection antibodies include, but are not limited to, monoclonal antibodies that bind to tau protein, preferably containing epitopes of amino acids 7 to 20 or 116 to 127 of human tau protein, wherein these positions are numbered according to the numbers in SEQ ID NO:1. When a monoclonal antibody that binds to tau protein at an epitope containing amino acids 7 to 20 is used as a detection antibody for the captured p217+tau peptide, a long tau fragment is detected. When a monoclonal antibody that binds to tau protein at an epitope containing amino acids 116 to 127 is used as a detection antibody for the captured p217+tau peptide, both short and long tau fragments are detected.

[0041] As used herein, the term "subject" refers to an animal, and preferably a mammal. According to a particular embodiment, the subject of treatment is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, marmosets, or mice) or primates (e.g., monkeys, chimpanzees, or humans). In a particular embodiment, the subject is a human.

[0042] As used in this article, "tau proteinopathy" encompasses any neurodegenerative disease involving the pathological accumulation of tau within the brain. Besides familial and sporadic AD, other exemplary tau protein diseases include frontotemporal dementia and Parkinson's syndrome associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangles-only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic granular dementia, amyotrophic lateral sclerosis Parkinson's syndrome-dementia complex, Down syndrome, Gershevsky-Schwarz disease, Hallewarden-Scholes disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, post-encephalitis Parkinson's syndrome, and chronic traumatic encephalopathy such as boxing dementia (boxing disease) (Morris et al., Neuron, 70:410-26, 2011).

[0043] As used herein, the term "amyloid-forming disease" includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils. Exemplary amyloid-forming diseases include, but are not limited to, systemic amyloidosis, Alzheimer's disease, early-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-associated infectious spongiform encephalopathy (kuru and Creutzfeldt-Jacob disease in humans, and scrapie and BSE in sheep and cattle). Different amyloid-forming diseases are defined or characterized by the nature of the polypeptide components of the deposited fibrils. For example, in subjects or patients with Alzheimer's disease, β-amyloid (e.g., wild-type, variant, or truncated β-amyloid) is the characteristic polypeptide component of amyloid deposition. Thus, Alzheimer's disease is an example of a "disease characterized by Aβ deposition" or "disease associated with Aβ deposition" in the brain of, for example, a subject or patient. The terms “β-amyloid protein,” “β-amyloid peptide,” “β-amyloid,” “Aβ,” and “Aβ peptide” are used interchangeably in this document.

[0044] As used herein, the terms “determine,” “measure,” “evaluate,” and “determine” are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement and include determining the presence of a property, trait, or characteristic. Evaluations can be relative or absolute. “Evaluating presence” includes determining the quantity of something present, as well as determining its existence.

[0045] As used herein, the term “diagnosis” means the detection of a disease or disorder or the determination of a stage or extent of a disease or disorder (such as tau proteinopathy or amyloidosis). Typically, the diagnosis of a disease or disorder is based on the assessment of one or more factors and / or symptoms indicative of that disease. A diagnosis may be made based on the presence, absence, or quantity of factors (e.g., p217+tau) indicative of the presence or absence of a disease or disorder. Each factor or symptom considered indicative of a particular disease need not be solely related to that particular disease; i.e., differential diagnoses may exist that can be inferred from the diagnostic factors or symptoms. Similarly, factors or symptoms indicative of a particular disease may exist in individuals who do not have that particular disease. The term “diagnosis” also encompasses determining the effectiveness of a drug therapy (e.g., anti-p217+tau antibody therapy) or predicting a pattern of response to a drug therapy (e.g., anti-p217+tau antibody therapy). Diagnostic methods may be used independently or in combination with other diagnostic and / or staging methods known in the medical field for a particular disease or disorder (e.g., Alzheimer's disease).

[0046] As used herein, the terms “increase” and “decrease” refer to a difference in the amount of a specific biomarker in a sample compared to a control or reference level. For example, the amount of a specific peptide may be increased or decreased compared to a reference level in a sample from a patient with a disease. In one embodiment, “increase” or “decrease” may be a difference in the level of a biomarker present in a sample compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, or more. In one embodiment, “increase” or “decrease” may be a statistically significant difference in the level of a biomarker present in a sample compared to a control. For example, a difference may be statistically significant if the measured level of a biomarker falls outside approximately 1.0, 1.5, 2.0, or 2.5 standard deviations from the mean of any control or reference group. A reference or control may be, for example, a sample from a healthy individual or a sample taken from the same individual at an earlier time point (such as before administration of the treatment agent or at an earlier time point during the treatment regimen).

[0047] As used herein, the term "isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, isolated, or purified from other biological components of the organism in which the component is naturally present (i.e., other chromosomes and extrachromosomal DNA and RNA, as well as proteins). Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. "Isolated" nucleic acids, peptides, and proteins may be part of a composition, and are still considered isolated if such a composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also includes nucleic acids, peptides, and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0048] As used herein, "isolated antibody bound to tau protein" or "isolated anti-tau antibody" is intended to refer to an antibody that specifically binds to tau protein and is substantially free of other antibodies with different antigen specificities (e.g., isolated anti-tau detection antibody is substantially free of antibodies that specifically bind to antigens other than tau). However, isolated anti-tau detection antibodies may be cross-reactive with other relevant antigens, such as those from other species (e.g., tau species homologs).

[0049] As used herein, the terms "specific binding" or "specific binding" refer to the anti-tau antibody of this application binding at approximately 1 × 10⁻⁶. -6 M or lower, for example, about 1×10 -7 M or lower, approximately 1×10 -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10 -10 M or lower, approximately 1×10 -11 M or lower, approximately 1×10 -12 M or lower, or approximately 1×10 -13 M or a lower dissociation constant (K) D The ability to bind with a predetermined target. D The ratio of Kd to Ka (i.e., Kd / Ka) is obtained and expressed as molar concentration (M). According to this disclosure, the K of the antibody... D The value can be determined using methods in the art. For example, the K value of anti-tau antibodies. D Values ​​can be obtained by using surface plasmon resonance, such as through the use of biosensor systems, for example... The system, Proteon instrument (BioRad), KinExA instrument (Sapidyne), ELISA, or competitive binding assays known to those skilled in the art are used to determine this. Typically, the anti-tau antibody binds to the predetermined target (i.e., tau) via K... D Compared to its K for non-specific targets DThe difference is at least ten times smaller, as measured by surface plasmon resonance using instruments such as the Proteon instrument (BioRad). However, anti-tau antibodies that specifically bind to tau may exhibit cross-reactivity with other relevant targets, such as those from other species (homologs), such as the same pre-determined targets from mice, rats, marmosets, dogs, or pigs.

[0050] As used in this article, the term "accuracy" refers to how close a value is to the true value that was measured.

[0051] As used herein, the term "accuracy" refers to the degree of consistency among a series of measurements obtained from multiple samplings of the same homogeneous sample.

[0052] As used herein, the term “sensitivity” refers to the lowest concentration of analyte in a sample that can be measured with acceptable accuracy and precision in an assay.

[0053] When conducting clinical trials for the treatment of tau disease or amyloidosis, it is necessary to screen human subjects seeking participation and select those who require such treatment, such as those who have already developed tau disease or amyloidosis and / or are at risk of developing tau disease or amyloidosis, for inclusion in the clinical trial. Therefore, subjects need to meet a minimum level of disease progression in tau disease and / or amyloidosis to be included in the clinical trial. Additionally, it is believed that anti-tau therapy and anti-amyloid therapy are most effective in treating tau disease and amyloidosis, respectively, when treatment begins early in disease progression. Therefore, it may also be desirable to select subjects who are not in late-stage disease progression of tau disease or amyloidosis, making treatment more likely to be effective and / or making any effect of slowing the progression of tau disease or amyloidosis through treatment in a clinical trial readily observable. To identify subjects who require treatment for tau disease or amyloidosis but are not in advanced disease progression of tau disease and / or amyloidosis, it may be desirable to select subjects above the lowest level of disease progression of tau disease and / or amyloidosis and below the highest level of disease progression of tau disease and / or amyloidosis for further screening tests or inclusion in clinical trials.

[0054] This application provides a method for pre-screening or screening human subjects for clinical trials aimed at treating tau proteinopathy or amyloidosis using an assay for detecting monophosphorylated or polyphosphorylated p217+tau peptide in blood-based samples. Accurate, sensitive, and precise measurement of pTau isoforms in plasma is considered a promising non-invasive method for detecting abnormal amyloid and tau processes. Longer fragments of pT217-containing polyphosphorylated tau are considered among the most associated isoforms with tau proteinopathy and amyloidosis (particularly Alzheimer's disease pathology) and may begin accumulating in CSF and plasma 10–20 years prior to cognitive decline. Phosphorylated tau (particularly p217+tau) measured in CSF and plasma is considered one of the most sensitive and specific biomarkers for tau proteinopathy and amyloidosis (particularly Alzheimer's disease pathology) and is believed to predict “tau-positive” or “amyloid-positive” pathologies (both further defined below) and cognitive decline. It is believed that the amount of monophosphorylated or polyphosphorylated p217+tau peptide measured from blood-based samples from subjects can be used as an indicator of the progression of tau disease and / or amyloidosis in subjects. Lower concentrations of p217+tau peptide measured from samples correspond to early disease progression of tau disease and / or amyloidosis, while higher concentrations correspond to late disease progression of tau disease and / or amyloidosis. Therefore, the amount of monophosphorylated or polyphosphorylated p217+tau peptide measured from blood-based samples from subjects can be used as a pre-screening or screening criterion to determine whether subjects are suitable for inclusion in clinical trials for the treatment of tau disease or amyloidosis.

[0055] The pre-screening or screening methods described in this application can be used to identify suitable subjects for clinical trials for the treatment of tau proteinopathy or amyloidosis. Treatment for tau proteinopathy can be any type of protocol or pharmaceutical active agent used to treat or slow the progression of tau proteinopathy or amyloidosis. For example, treatment for tau proteinopathy in a clinical trial can be a pharmaceutical composition containing an active agent with the potential to treat or slow the progression of tau proteinopathy, such as anti-tau antibodies, anti-p217+tau antibodies, small interfering RNA (siRNA) against human tau, anti-p217+tau siRNA, etc. In another example, treatment for amyloidosis in a clinical trial can be a pharmaceutical composition containing an active agent with the potential to treat or slow the progression of amyloidosis, such as anti-amyloid antibodies, β-secretase inhibitors, γ-secretase inhibitors, small interfering RNA (siRNA) against human β-amyloid, etc. In a specific example, the treatment in a clinical trial is an active agent with the potential to treat or slow the progression of Alzheimer's disease.

[0056] Collection of blood-based samples (such as plasma) is rapid and readily available, and does not cause significant discomfort or burden to the subject. In contrast, other currently available tools for determining the progression of tau proteinopathy and / or amyloidosis (such as Alzheimer's disease) in subjects are expensive and cumbersome. For example, positron emission tomography (PET) using tau-specific radiotracers (Tau PET) has been used to measure and localize tau neurofibrillary tangles (NFT) pathology in the brains of patients. Similarly, PET using amyloid-specific radiotracers (amyloid PET) (e.g., Aβ40 or Aβ42-specific radiotracers) has been used to measure and localize amyloid fibrillary pathology in the brains of patients. However, both Tau PET and amyloid PET are expensive and cumbersome procedures, and the availability of suitable tracers is limited. Furthermore, the procedures used for imaging can be uncomfortable and therefore more cumbersome than collecting a blood sample from a patient. Another exemplary tool for determining the progression of tau proteinopathy and / or amyloidosis in a subject is by measuring the amount of phosphorylated tau peptides (e.g., monophosphorylated or polyphosphorylated p217+ tau peptides) in a sample of CSF retrieved from the subject. However, CSF retrieval requires the patient to undergo an invasive lumbar puncture procedure, which involves a physician inserting a needle into the spinal canal to collect a CSF sample for measurement. Such invasive procedures can be painful and expose subjects to the risk of spinal canal infection. Therefore, using blood-based assays (more specifically, plasma-based assays) to detect monophosphorylated or polyphosphorylated p217+tau peptide in samples for pre-screening or screening human subjects for clinical trials targeting the treatment of tau proteinopathy or amyloidosis (particularly Alzheimer's disease) provides an improved approach to rapidly and efficiently identify candidates most likely to fall within the expected clinical criteria (e.g., having minimal levels of disease progression in tau proteinopathy and / or amyloidosis, but below the maximum levels of disease progression in tau proteinopathy and / or amyloidosis) for inclusion in clinical trials.

[0057] In one aspect of this application, a method 100 is provided for pre-screening or screening human subjects for clinical trials of tau proteinosis or amyloidosis. Method 100 includes a first step 102 of obtaining a blood-based sample from the human subject. The blood-based sample may be blood, serum, or plasma. Preferably, the sample is a plasma sample. More preferably, the plasma sample has not been immunoprecipitated to concentrate the p217+tau peptide contained therein. In a particular embodiment, the sample is a crude plasma sample.

[0058] In step 104, the sample may be further processed by any suitable method to concentrate the p217+tau peptide contained therein, and then the concentrated sample may be analyzed using an assay to determine the amount or concentration of p217+tau present in the sample. Preferably, in step 104, the sample may be directly analyzed using an assay to determine the amount or concentration of p217+tau present in the sample (step 104) without further concentration of the p217+tau peptide prior to the assay. Any suitable assay may be used to analyze blood-based samples to determine the amount or concentration of p217+tau present in the sample. More specifically, the assay is a highly sensitive and accurate assay for measuring p217+tau in plasma. The amount or concentration of p217+tau may be determined using any suitable technique known in the art, including ELISA and single-molecule array platforms. Depending on a specific aspect, the method of this application may use a high-sensitivity array platform (such as Quanterix Simoa or MSD S-plex) to measure the amount or concentration of p217+tau peptide in blood-based samples (particularly plasma samples). Specifically, the blood-based sample is a plasma sample, and the amount or concentration of p217+tau present in the plasma sample is determined using an assay capable of providing an accurate, sensitive, and precise quantitative measurement of p217+tau present in plasma samples obtained from cognitively normal subjects (including those who are not amyloid-positive) and those with tau disease or amyloidosis or at risk of developing tau disease or amyloidosis (particularly those with mild cognitive impairment (MCI) within an acceptable and reliable sensitivity level). For example, the lower limit of quantitation (LLOQ) of the assay may be 0.05 pg / ml, 0.045 pg / ml, 0.04 pg / ml, 0.038 pg / ml, or 0.036 pg / ml.

[0059] Specifically, the blood-based sample is a plasma sample, and the assay for determining the amount or concentration of p217+tau present in the plasma sample includes a capture antibody that binds to the p217+tau epitope and a detection antibody labeled with a detectable marker (e.g., a fluorescent molecule, biotin, etc.), which is either directly detectable or detectable by a secondary reaction (e.g., a reaction with streptavidin). The capture antibody can be immobilized to a solid phase such that it selectively binds to the solid phase and immobilizes the p217+tau peptide present in the sample to the solid phase. The capture antibody binds to the p217+tau epitope and forms an antibody-peptide complex with the p217+tau peptide. This antibody-peptide complex is then contacted with a detection antibody to allow the detection antibody to bind to the antibody-peptide complex. In one example, the detection antibody is biotinylated. The detectable marker on the detection antibody is then detected to determine the amount or concentration of p217+tau peptide in the sample.

[0060] In one example, the capture antibody is a monoclonal antibody comprising immunoglobulin heavy chains HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO:2, 3, and 4, respectively, and immunoglobulin light chains LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO:5, 6, and 7, respectively. In a particular embodiment, the capture antibody is pT3. As used herein, the term "pT3" refers to an antibody that binds to the p217+tau epitope and has the amino acid sequence of the heavy chain variable region of SEQ ID NO:8 and the amino acid sequence of the light chain variable region of SEQ ID NO:9. In one embodiment, the pT3 monoclonal antibody is expressed from a mouse hybridoma.

[0061] An exemplary detection antibody is a monoclonal antibody that binds to epitopes containing amino acids 7 to 20 of human tau protein, wherein these positions are numbered according to the numbers in SEQ ID NO:1. The detection antibody may be a monoclonal antibody comprising immunoglobulin heavy chains HCDR1, HCDR2, and HCDR3 having polypeptide sequences of SEQ ID NO:10, 11, and 12, respectively, and immunoglobulin light chains LCDR1, LCDR2, and LCDR3 having polypeptide sequences of SEQ ID NO:13, 14, and 15, respectively. In a particular embodiment, the detection antibody is hT43. As used herein, the term "hT43" refers to a monoclonal antibody that binds to epitopes containing amino acids 7 to 20 of human tau protein, wherein these positions are numbered according to the numbers in SEQ ID NO:1, and that the antibody has the heavy chain variable region amino acid sequence of SEQ ID NO:16 and the light chain variable region amino acid sequence of SEQ ID NO:17.

[0062] Exemplary assays for determining the amount of p217+tau present in a plasma sample are described, for example, in WO2022 / 013286, the entire disclosure of which is incorporated herein by reference.

[0063] In step 106, the amount or concentration of p217+tau present in the plasma sample is used to determine whether a subject should continue further screening for a clinical trial, or alternatively be selected for inclusion in a clinical trial or indicated as suitable for inclusion in a clinical trial. If the amount or concentration of p217+tau present in a subject's plasma sample is greater than or equal to a minimum threshold, the subject is identified as a subject requiring treatment for tau proteinopathy or amyloidosis. The minimum threshold corresponds to the amount or concentration of p217+tau present in the plasma, exceeding which the subject exhibits a "tau-positive" pathology, i.e., those subjects exhibiting mild cognitive impairment (MCI) and an increased accumulation of tau tangles in the brain (more specifically, in the bilateral inferior temporal cortex) compared to cognitively normal patients (e.g., accumulation of monophosphorylated or polyphosphorylated p217+tau). Such accumulation may correspond to tau PET images with an SUVR Z score >1 in the subject's bilateral inferior temporal cortex. For example, the minimum threshold is 0.075 pg / ml, 0.1 pg / ml, or 0.125 pg / ml. In one example, the minimum threshold is 0.1 pg / ml ± 0.025 pg / ml. Alternatively, the minimum threshold corresponds to the amount or concentration of p217+tau present in the plasma, exceeding which subjects tend to correspond to an "amyloid-positive" pathology, i.e., those subjects exhibiting mild cognitive impairment (MCI) and increased accumulation of β-amyloid (e.g., Aβ40 and / or Aβ42) in the bilateral inferior temporal cortex compared to cognitively normal patients. As determined in step 104, the amount or concentration of p217+tau in the plasma sample provides a good ability to predict a subject's tau-positive and / or amyloid-positive status using a minimum threshold of 0.1 pg / ml.

[0064] Additionally, if the amount or concentration of p217+tau present in a subject's plasma sample is less than or equal to a maximum threshold, the subject is identified as not being in advanced disease progression of tau proteinopathy or amyloidosis, making treatment more likely to be effective and / or making any effect of treating tau proteinopathy or amyloidosis under clinical trials to slow its progression readily observable. The maximum threshold corresponds to the amount or concentration of p217+tau present in plasma exceeding which the subject is predisposed to having a pathology of widespread accumulation of tau tangles in multiple brain regions. For example, such widespread accumulation of tau tangles may correspond to tau PET imaging with an SUVR Z score >5 in each of Braak regions 4, 5, and 6 of the subject's brain. In one example, the maximum thresholds are 0.225 pg / ml, 0.25 pg / ml, 0.275 pg / ml, and 0.3 pg / ml. In another example, the maximum threshold was 0.25 pg / ml ± 0.025 pg / ml.

[0065] In one implementation, if the amount or concentration of p217+tau in the plasma sample is ≥ the minimum threshold and ≤ the maximum threshold, the subject continues to be screened for further clinical trials, or is alternatively selected for inclusion in a clinical trial or indicated as suitable for inclusion in a clinical trial (step 108). If the amount or concentration of p217+tau in the plasma sample is < the minimum threshold or > the maximum threshold, the subject is excluded from the clinical trial (step 110). For example, if the amount or concentration of p217+tau in the plasma sample is ≥ 0.1 ± 0.025 pg / ml and ≤ 0.25 ± 0.025 pg / ml, the subject continues to be screened for further clinical trials, or is alternatively selected for inclusion in a clinical trial or indicated as suitable for inclusion in a clinical trial (step 108). If the amount or concentration of p217+tau in the plasma sample is < 0.1 ± 0.025 pg / ml or > 0.25 ± 0.025 pg / ml, the subject is excluded from the clinical trial (step 110). In another example, if the concentration of p217+tau present in the plasma sample is 0.075 pg / ml to 0.275 pg / ml, 0.075 pg / ml to 0.25 pg / ml, 0.075 pg / ml to 0.225 pg / ml, 0.1 pg / ml to 0.275 pg / ml, 0.1 pg / ml to 0.25 pg / ml, 0.1 pg / ml to 0.225 pg / ml, 0.125 pg / ml to 0.275 pg / ml, 0.125 pg / ml to 0.25 pg / ml, or 0.125 pg / ml to 0.225 pg / ml, the subject continues to be further screened for clinical trials, or alternatively selected for inclusion in clinical trials or indicated as suitable for inclusion in clinical trials (step 108). If the amount or concentration of p217+tau in the plasma sample is outside the above range, the subject will be excluded from the clinical trial (step 110).

[0066] In another aspect of this application, a method 200 is provided for pre-screening or screening human subjects for clinical trials intended to treat tau proteinosis or amyloidosis. Method 200 includes a first step 202 of obtaining a blood-based sample (and more specifically, a plasma sample) from the human subject in the same manner as step 102, and a step 204 similar to step 104 for determining the amount or concentration of p217+tau present in the sample. However, in step 204, each sample obtained in step 202 may be measured once, or may be aliquoted and measured multiple times (e.g., twice, three times, etc.) to generate multiple measurements of the amount or concentration of p217+tau detected from the same sample.

[0067] In step 206, if the sample is measured multiple times, the multiple measurements of the amount or concentration of p217+tau detected from the same sample are analyzed to determine whether the sample was measured with sufficient precision. For example, the percentage coefficient of variation (%CV) can be determined for the multiple measurements obtained from the same sample. If the sample is measured only once, the %CV of the sample is not determined. If the %CV is less than a threshold %CV, method 200 proceeds to step 208. If the %CV determined for the multiple measurements is greater than the threshold CV, or if the sample was measured only once, method 200 proceeds to step 210. The threshold CV can be 25% CV, 20% CV, or 15% CV. In a particular example, if %CV < 20%, method 200 proceeds to step 208. If %CV ≥ 20%, or if the sample was measured only once, method 200 proceeds to step 210.

[0068] In step 208, if the average amount or concentration of p217+tau across multiple measurements of the sample is ≥ the minimum threshold and ≤ the maximum threshold, the subject continues to be screened for further clinical trials, or is alternatively selected for inclusion in a clinical trial or indicated as suitable for inclusion in a clinical trial. If the average amount or concentration of p217+tau across multiple measurements of the sample is < the minimum threshold or > the maximum threshold, the subject is excluded from the clinical trial. The minimum and maximum thresholds are defined in the same manner as described above for step 106. In one example, if the average amount or concentration of p217+tau across multiple measurements of the plasma sample is ≥ 0.1 ± 0.025 pg / ml and ≤ 0.25 ± 0.025 pg / ml, the subject continues to be screened for further clinical trials, or is alternatively selected for inclusion in a clinical trial or indicated as suitable for inclusion in a clinical trial. If the mean amount or concentration of p217+tau in a plasma sample across multiple measurements is <0.1±0.025 pg / ml or >0.25±0.025 pg / ml, the subject will be excluded from the clinical trial. In another example, if the average amount or concentration of p217+tau across multiple measurements of a plasma sample is 0.075 pg / ml to 0.275 pg / ml, 0.075 pg / ml to 0.25 pg / ml, 0.075 pg / ml to 0.225 pg / ml, 0.1 pg / ml to 0.275 pg / ml, 0.1 pg / ml to 0.25 pg / ml, 0.1 pg / ml to 0.225 pg / ml, 0.125 pg / ml to 0.275 pg / ml, 0.125 pg / ml to 0.25 pg / ml, or 0.125 pg / ml to 0.225 pg / ml, the subject continues to be screened for further clinical trials, or is otherwise deemed suitable for inclusion in a clinical trial (step 210). If the average amount or concentration of p217+tau from multiple measurements of a plasma sample is outside the range described above, the subject will be excluded from the clinical trial (step 212).

[0069] In step 220, the average amount or concentration of p217+tau from multiple measurements of the sample, or the amount or concentration of p217+tau in the plasma sample from a single measurement, is used to determine which of the three different steps method 200 will proceed to next. First, if the average amount or concentration of p217+tau from multiple measurements of the plasma sample, or the amount or concentration of p217+tau in the plasma sample from a single measurement, is within the range of a minimum threshold ± Δ or a maximum threshold ± Δ, then method 200 proceeds to step 222. For example, Δ is 0.01 pg / ml, 0.015 pg / ml, 0.02 pg / ml, 0.025 pg / ml, 0.03 pg / ml, or 0.035 pg / ml.

[0070] In step 222, the sample is retested in the same manner as described in step 204 to obtain further measurements of the amount or concentration of p217+tau present in the sample. In step 224, if the retest data provides quantitative results similar to the previous measurements (e.g., within ± standard deviation of the mean) and improves the sample (i.e., reduces %CV), the average amount or concentration of p217+tau across all measurements (including the retest) is further analyzed according to the criteria of step 208 to determine whether to proceed to step 210 or exclude the subject from the clinical trial (step 212).

[0071] Next, if the average amount or concentration of p217+tau across multiple measurements of the plasma sample, or a single measurement of the amount or concentration of p217+tau in the plasma sample, is less than the determined LLOQ, the subject is excluded from the clinical trial (step 212). Retesting is not necessary in this case because the amount or concentration of p217+tau in the plasma sample is below the detectable range and therefore unlikely to produce a superior CV.

[0072] Finally, if the average amount or concentration of p217+tau in a plasma sample across multiple measurements, or the amount or concentration of p217+tau in a single measurement of a plasma sample:

[0073] (1) ≥ LLOQ and < minimum threshold - Δ,

[0074] (2) > minimum threshold + Δ and < maximum threshold - Δ, or

[0075] (3) ≥ maximum threshold + Δ

[0076] Where Δ is as described above, method 200 then proceeds to step 208. If the amount or concentration of p217+tau in the plasma sample falls within any of the three criteria described above, method 200 may optionally include a retest step (not shown) to improve the accuracy of the average amount or concentration of p217+tau across multiple measurements and / or reduce the %CV of the measurements obtained for the sample.

[0077] In a specific implementation, the minimum threshold is 0.1 pg / ml, the maximum threshold is 0.25 pg / ml, Δ is 0.025 pg / ml, and the measured LLOQ is 0.036 pg / ml. In step 208 of this implementation, if the average concentration of p217+tau across multiple measurements of the plasma sample is ≥0.1 pg / ml and ≤0.25 pg / ml, the subject continues to be further screened for the clinical trial or is alternatively deemed suitable for inclusion in the clinical trial (step 210). Conversely, if the average concentration of p217+tau across multiple measurements of the plasma sample is <0.1 pg / ml or >0.25 pg / ml, the subject is excluded from the clinical trial (step 212).

[0078] In step 220 of this implementation scheme, the average concentration of p217+tau across multiple measurements of the plasma sample or a single measurement of the concentration of p217+tau in the plasma sample is used to determine which of the three different steps method 200 will proceed to next. Specifically, if the average concentration of p217+tau across multiple measurements of the plasma sample or a single measurement of the concentration of p217+tau in the plasma sample is within the range of 0.075 pg / ml to 0.125 pg / ml or 0.225 pg / ml to 0.275 pg / ml, then method 200 proceeds to step 222. If the average concentration of p217+tau across multiple measurements of the plasma sample or a single measurement of the concentration of p217+tau in the plasma sample is <0.036 pg / ml, then the subject is excluded from the clinical trial (step 212). If the average concentration of p217+tau in a plasma sample is from multiple measurements, or if the concentration of p217+tau in a plasma sample is from a single measurement:

[0079] (1) ≥0.036 pg / ml and <0.075 pg / ml,

[0080] (2) >0.125 pg / ml and <0.225 pg / ml, or

[0081] (3) ≥0.275 pg / ml,

[0082] Then method 200 proceeds to step 208.

[0083] In some implementations, subjects identified in step 108 or 210 may be directed to additional clinical tests, such as PET imaging and / or CSF collection, to further evaluate the subject's brain pathology. Additional clinical trials may be used to confirm that the subject requires treatment for tau disease or amyloidosis (e.g., having mild cognitive impairment (MCI) and increased accumulation of tau tangles in the brain compared to cognitively normal patients), and that the subject is not in advanced disease progression of tau disease or amyloidosis (e.g., exhibiting pathology showing extensive accumulation of tau tangles or amyloid fibrils in multiple areas of the brain).

[0084] For example, subjects identified in steps 108 or 210 can be directed to tau PET imaging using a tau-specific tracer (e.g., a p217+ tau-specific tracer), or to amyloid PET imaging using an amyloid-specific radiotracer (e.g., an Aβ40 or Aβ42-specific radiotracer). The acquired PET data can be analyzed using a computing device to confirm whether the subject is tau-positive or amyloid-positive. For example, the computing device can analyze tau PET data to confirm that, compared to cognitively normal patients, the subject has increased accumulation of tau tangles in the brain (particularly in the bilateral inferior temporal cortex) (e.g., accumulation of monophosphorylated or multiphosphorylated p217+ tau). More specifically, tau PET data can be analyzed to confirm that the subject has a SURVR Z score >1 in the bilateral inferior temporal cortex. In another example, amyloid PET data can be analyzed to confirm that subjects had increased amyloid fibrils in the brain (particularly in the bilateral inferior temporal cortex) compared to cognitively normal patients.

[0085] The computing device can also analyze the acquired PET data to confirm that the subject is not in the late stages of tau disease or amyloidosis (e.g., pathology showing extensive accumulation of tau tangles or amyloid fibrils in multiple brain regions). For example, tau PET data can be analyzed to confirm that the subject does not have extensive accumulation of tau tangles in the brain. More specifically, tau PET data can be analyzed to confirm that the subject does not have an SUVR Z score >5 in each of the subject's Braak 4, 5, and 6 brain regions. In another example, amyloid PET data can be analyzed to confirm that the subject does not have extensive accumulation of amyloid fibrils in multiple brain regions (such as the subject's Braak 4, 5, and 6 brain regions).

[0086] In one embodiment, a method is provided for screening human subjects for clinical trials to treat tau proteinopathy or amyloidosis. The method includes obtaining a plasma sample from the subject and determining the amount or concentration of p217+tau present in the sample in the same manner as steps 102 and 104 described above. When the amount or concentration of p217+tau present in the plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold, the method continues to obtain PET data of the subject's brain generated using a tau-specific or amyloid-specific radiotracer. The minimum and maximum thresholds are defined in the same manner as described above for step 106. The PET data are then analyzed to determine whether the subject is tau-positive or amyloid-positive, and whether the subject has extensive tau tangles or extensive amyloid fibrils. Subjects are indicated as suitable for inclusion in the clinical trial if:

[0087] (i) The concentration of p217+tau in the plasma sample is greater than or equal to the minimum threshold and less than or equal to the maximum threshold.

[0088] (ii) PET data indicating that the subject was tau-positive or amyloid-positive, and

[0089] (iii) PET data indicated that the subjects did not have extensive tau tangles or extensive amyloid fibrils.

[0090] Furthermore, based on the SUVR in the Braak 4 region generated from PET data, subjects deemed suitable for inclusion in the clinical trial will be further stratified into "high" and "low" groups. For example, subjects stratified into the "high" group will have an SUVR > 4.62 in the Braak 4 region, and those stratified into the "low" group will have an SUVR ≤ 4.62 in the Braak 4 region.

[0091] Those skilled in the art will understand that the exemplary computer implementations described herein can be implemented in any number of ways, including as separate software modules, as a combination of hardware and software, etc. For example, an exemplary method may be an implementation in one or more programs stored in a non-transitory storage medium and containing lines of code that, when compiled, can be executed by one or more processor cores or a single processor. A system according to one embodiment includes multiple processor cores and a set of instructions executed on the multiple processor cores to perform the exemplary methods described above. Processor cores or individual processors may be integrated into or communicate with any suitable electronic device (e.g., on a board-based processing arrangement within a device or on a processing arrangement outside the device), such as mobile computing devices, smartphones, tablet computers, computing devices, etc., which may communicate with at least a portion of the device.

[0092] Example

[0093] The following embodiments are provided to further illustrate the essence of the present invention. It should be understood that the following embodiments do not limit the present invention, and the scope of the present invention is defined by the appended claims.

[0094] Example 1 :

[0095] Example 1 reports data on the performance of a plasma 217+ tau assay as a pre-screening tool to identify potentially tau-positive subjects for inclusion in clinical trials for the treatment of tau proteinopathy. The assay was performed using Quanterix's high-sensitivity Simoa array platform, with a pT3 antibody as the capture antibody and an attached detectable-labeled hT43 antibody as the detection antibody. Furthermore, Example 1 describes how this assay can be used to augment patients with low tau PET layers for inclusion in clinical trials for the treatment or slowing of tau proteinopathy progression.

[0096] In Example 1, plasma p217+tau was measured in screening samples from subjects exhibiting MCI, performed weekly in batches. Technical performance was evaluated across 58 batches. Subjects with plasma p217+tau levels above a pre-specified 0.1 pg / ml cutoff were screened using Tau PET (using MK-6240 as a tracer). Example 1 investigated the concentration of plasma p217+tau in this population and the prevalence of tau positivity in subjects with plasma p217+tau concentrations ≥0.1 pg / ml.

[0097] A total of 55 batches of plasma samples were screened using the p217+tau assay described in Example 1 above. A set of three peptide QCs (0.1 pg / ml, 0.4 pg / ml, and 1.6 pg / ml) was performed in duplicate within each batch, revealing excellent intra-batch precision (mean CVs of 6.0%, 4.5%, and 5.5%, respectively) and inter-batch precision (7.6%, 6.6%, and 13.3% CVs, respectively). Precision was also acceptable for endogenous samples, with a mean CV of 7.9% (0%–120%) and an estimated LLOQ of 0.030 pg / ml (based on an average concentration with a CV >20%) across N = 725 plasma samples.

[0098] Of the 787 MCI patients whose plasma p217+tau was measured, 72% had a level ≥0.1 pg / ml. Those patients with plasma p217+tau concentrations ≥0.1 pg / ml were then designated for tau PET imaging. Of the 346 patients imaged, 86% were tau positive, and 64% met the criteria for trial tau PET eligibility: an SUVR Z score >1 in the bilateral inferior temporal cortex and no SUVR Z score >5 in any of the Braak 4, 5, and 6 brain regions.

[0099] Tau PET data were also used to stratify patients into “high” and “low” groups based on SUVR in Braak 4 region. Despite assay screening as expected, 59% of eligible patients were in the high-level group. ROC analysis showed that plasma p217+tau assays predicted patient strata with an AUC of 0.8. Therefore, to correct for stratification imbalance, an upper threshold for plasma p217+tau concentration (i.e., ≤0.25 pg / ml) was implemented one year after data collection to enrich patients with low to moderate tau protein disease progression.

[0100] The plasma p217+ tau assay in Example 1 demonstrated good intra-batch and inter-batch accuracy and proved its ability to enrich both tau-positive and tau-stratified populations. This assay could serve as a tool to enable faster and more effective recruitment for clinical trials targeting Alzheimer's disease treatments, or as a tool to identify early-stage Alzheimer's disease in the general population.

[0101] Example 2 :

[0102] Example 2 provides additional data on the performance of plasma p217+tau assays in patients screened in the same clinical trial as in Example 1. The data in Example 2 include a larger sample size than those reported in Example 1. Figure 3aThe accuracy (%CV) of plasma p217+tau assays across different concentrations of p217+tau is shown in plasma samples collected from 832 patients screened for clinical trials. Figure 3b The concentration distribution of p217+tau, as determined by p217+tau assay, was shown in plasma samples collected from 831 patients screened for clinical trials.

[0103] Example 3: In participants screened for clinical trials, the difference between Simoa plasma p217+tau and tau PET. Correlation

[0104] Example 3 provides a report on the observed correlations between plasma p217+tau and 18F-MK-6240tau PET uptake across brain regions. Simoa-based plasma p217+tau assays were performed in weekly batches (Triana-Baltzer et al., Alzheimers Dement (Amst). 2021). Participants with levels exceeding 0.1 pg / ml were then scanned to confirm the presence of NFTs on tau PET. NFT levels were quantified using 18F-MK-6240SUVR in the Braak region of interest (ROI) (Scholl et al., Neuron 2016) (reference area: cerebellar gray). The Braak ROI is shown in... Figure 4 middle. Figure 5 A significant correlation (p<10) was shown between plasma p217+tau and MK6240 across all six (6) Braak ROIs. -15 ). Figure 6 Plasma p217+tau was shown to predict Braak stage 3 or lower in vivo, with an AUC of 82%. Nonparametric Spearman correlations were obtained between plasma p217+tau levels and the mean SUVR of each of the six ROIs used for Braak staging. The results reported in Example 3 are from the first cohort of 355 participants who completed the tauPET analysis.

[0105] As demonstrated in this paper, a significant correlation was found between plasma p217+tau and tau PET in a clinical trial setting. This correlation is consistent with independent observational studies in individuals with cognitive impairment (Dore et al., Alzheimer's Dement. 2022). These results demonstrate the consistent efficacy and utility of plasma p217+tau as a pre-screening method in Alzheimer's disease (AD) trials. Simoa's plasma p217+tau levels are consistently correlated with tau PET and can be used as a pre-screening method in clinical trials to reduce participant burden and improve screening efficiency.

[0106] The scope of the invention described herein and protected by the claims is not limited to the specific embodiments disclosed herein, as these embodiments are intended to illustrate several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. In fact, various modifications of the invention will become apparent to those skilled in the art from the foregoing description, in addition to those shown and described herein. Such modifications are also intended to fall within the scope of the appended claims. All publications cited herein are incorporated herein by reference in their entirety.

[0107] sequence

[0108] SEQ ID NO:1–tau protein 2N4R

[0109] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESP

[0110] LQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEI

[0111] PEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGA

[0112] DGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRS

[0113] GYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTA

[0114] PVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKD

[0115] NIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKL

[0116] DFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIV

[0117] YKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLSEQ ID NO:2–PT3 mouse mAb HCDR1, Kabat number

[0118] SYAMS

[0119] SEQ ID NO:3–PT3 mouse mAb HCDR2, Kabat number

[0120] SISKGGNTYYPNSVKG

[0121] SEQ ID NO:4–PT3 mouse mAb HCDR3, Kabat number

[0122] GWGDYGWFAY

[0123] SEQ ID NO:5–PT3 mouse mAb LCDR1, Kabat number

[0124] KASQDINRYLN

[0125] SEQ ID NO:6–PT3 mouse mAb LCDR2, Kabat number

[0126] RANRLLD

[0127] SEQ ID NO:7–PT3 mouse mAb LCDR3, Kabat number

[0128] LQYDEFPLT

[0129] SEQ ID NO:8–PT3 mouse mAb VH

[0130] EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYAMSWVRQNPEKRLEWV

[0131] ASISKGGNTYYPNSVKGRFTISRDNARNILYLQMSSLRSEDTALYYCARG

[0132] WGDYGWFAYWGQVTLVTVSA

[0133] SEQ ID NO:9–PT3 mouse mAb VL

[0134] DIKMTQSPSSMYASLGERVTITCKASQDINRYLNWFQQKPGKSPKTLIYR

[0135] ANRLLDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCLQYDEFPLTFGD

[0136] GTKLELK

[0137] SEQ ID NO:10, HCDR1 of HT43 mAb

[0138] GFTFRSYGMS

[0139] SEQ ID NO:11, HCDR2 of HT43 mAb

[0140] TINSDGSYTYYPDSVKG

[0141] SEQ ID NO:12, HCDR3 of HT43 mAb

[0142] SWDGAMDY

[0143] SEQ ID NO:13, LCDR1 of HT43 mAb

[0144] RSSQSILHSNGNTYFE

[0145] SEQ ID NO:14, LCDR2 of HT43 mAb

[0146] KVSNRFS

[0147] SEQ ID NO:15, LCDR3 of HT43 mAb

[0148] FQGSLVPWT

[0149] SEQ ID NO:16, Heavy chain variable region of HT43 mAb

[0150] EVKLVESGGDLVKPGGSLKLSCAASGFTFRSYGMSWVRQTPDKRLEWV

[0151] TTINSDGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCA

[0152] VSWDGAMDYWGQGTSVTVSS

[0153] SEQ ID NO:17, Light chain variable region of HT43 mAb

[0154] DVLVTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYFEWYLQRPGQSPK

[0155] LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSLVP

[0156] WTFGGGTCLAIK

Claims

1. A method for pre-screening human subjects for a clinical trial for treating tauopathy or amyloidosis, the method comprising: obtaining a plasma sample from the subject; determining the concentration of p217+tau present in the plasma sample; as well as when the concentration of p217+tau present in the plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold, instructing the subject to undergo further screening for the clinical trial, wherein the minimum threshold corresponds to an amount of p217+tau in plasma, above which the subject exhibits mild cognitive impairment (MCI) and increased accumulation of tau tangles in the brain compared to cognitively normal patients, and wherein the maximum threshold corresponds to an amount of p217+tau present in plasma, above which the subject exhibits a pathology of widespread accumulation of tau tangles in multiple regions of the brain.

2. The method of claim 1, wherein the minimum threshold is about 0.075 pg / ml to about 0.125 pg / ml.

3. The method of claim 1, wherein the maximum threshold is about 0.225 pg / ml to about 0.275 pg / ml.

4. The method of claim 1, wherein the indicating step comprises indicating that the subject is further screened for the clinical trial when the concentration of p217+tau present in the plasma sample is ≥ 0.1 pg / ml and ≤ 0.25 pg / ml.

5. The method of claim 1, wherein the determining step comprises: contacting the plasma sample with a capture antibody against a p217+tau epitope, so that the capture antibody binds to a p217+tau peptide in the plasma sample, thereby forming an antibody-peptide complex; contacting the antibody-peptide complex with a detection antibody so that the detection antibody binds to the antibody-peptide complex; as well as The detection antibody is detected to determine the amount of the p217+tau peptide in the plasma sample.

6. The method of claim 2, wherein the capture antibody binds to an epitope containing amino acids 210 to 220 of human tau protein.

7. The method of claim 2, wherein the detection antibody binds to an epitope comprising amino acids 7 to 20 or amino acids 116 to 127 of human tau protein.

8. The method of claim 3, wherein the capture antibody is pT3.

9. The method of claim 4, wherein the detection antibody is hT43.

10. The method of claim 1, wherein the concentration of p217+tau is determined using an assay with an LLOQ < 0.04 pg / ml.

11. The method of claim 1, wherein the tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaef disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (pugilistic disease).

12. The method of claim 11, wherein the tauopathy is Alzheimer's disease.

13. A method for screening human subjects for a clinical trial for treating tauopathy or amyloidosis, the method comprising: obtaining a plasma sample from the subject; determining the concentration of p217+tau present in the plasma sample; obtaining PET data of the subject's brain generated using a tau-specific or amyloid-specific radiotracer when the concentration of p217+tau present in the plasma sample is greater than or equal to a minimum threshold and less than or equal to a maximum threshold; analyzing the PET data to determine whether the subject is tau positive or amyloid positive, and whether the subject has extensive tau tangles or extensive amyloid fibrils; and The subject is indicated as suitable for inclusion in the clinical trial in the following cases: (iv) the concentration of p217+tau present in the plasma sample is greater than or equal to the minimum threshold and less than or equal to the maximum threshold, (V) the PET data indicates that the subject is tau positive or amyloid positive, and (vi) the PET data indicates that the subject does not have extensive tau tangles or extensive amyloid fibrils, wherein the minimum threshold corresponds to an amount of p217+tau in plasma, above which the subject exhibits mild cognitive impairment (MCI) and increased accumulation of tau tangles in the brain compared to cognitively normal patients, and wherein the maximum threshold corresponds to an amount of p217+tau present in plasma, above which the subject exhibits a pathology of widespread accumulation of tau tangles in multiple regions of the brain.

14. The method of claim 13, wherein the minimum threshold is about 0.075 pg / ml to about 0.125 pg / ml.

15. The method of claim 13, wherein the maximum threshold is about 0.225 pg / ml to about 0.275 pg / ml.

16. The method of claim 13, wherein the indicating step comprises indicating that the subject is further screened for the clinical trial when the concentration of p217+tau present in the plasma sample is ≥ 0.1 pg / ml and ≤ 0.25 pg / ml.

17. The method of claim 13, wherein the determining step comprises: contacting the plasma sample with a capture antibody against a p217+tau epitope, so that the capture antibody binds to a p217+tau peptide in the plasma sample, thereby forming an antibody-peptide complex; contacting the antibody-peptide complex with a detection antibody so that the detection antibody binds to the antibody-peptide complex; as well as The detection antibody is detected to determine the amount of the p217+tau peptide in the plasma sample.

18. The method of claim 17, wherein the capture antibody binds to an epitope containing amino acids 210 to 220 of human tau protein.

19. The method of claim 17, wherein the detection antibody binds to an epitope comprising amino acids 7 to 20 or amino acids 116 to 127 of human tau protein.

20. The method of claim 18, wherein the capture antibody is pT3.

21. The method of claim 19, wherein the detection antibody is hT43.

22. The method of claim 13, wherein the concentration of p217+tau is determined using an assay with an LLOQ < 0.04 pg / ml.

23. The method of claim 13, wherein the tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaefer disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilistica (pugilistic disease).

24. The method of claim 23, wherein the tauopathy is Alzheimer's disease.

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