Blood-based screening of subjects for clinical trials for the treatment of tauopathy or amyloid-forming diseases

Abstract

A method for pre-screening human subjects for a clinical trial for the treatment of tauopathy or amyloid-forming diseases. The method includes obtaining a plasma sample from the subject and determining the concentration of p217+ tau present in the plasma sample. The method further includes instructing the subject to undergo further screening for the clinical trial when the concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold. The minimum threshold corresponds to the amount of p217+ tau in plasma such that above it, the subject exhibits an increased accumulation of tau changes in the brain compared to subjects with mild cognitive impairment (MCI) and cognitively normal patients. The maximum threshold corresponds to the amount of p217+ tau present in plasma such that above it, the subject exhibits the pathology of extensive accumulation of tau changes in multiple regions of the brain.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 366,449, filed on June 15, 2022, and U.S. Provisional Patent Application No. 63 / 437,260, filed on January 5, 2023, each of which is hereby incorporated by reference in its entirety.

[0002] (Reference to Electronically Filed Sequence Listing) The sequence listing for this application is submitted electronically via the Patent Center of the United States Patent and Trademark Office as an XML - formatted sequence listing with the file name "JAB7165WOPCT1SEQLIST.xml", created on June 5, 2023, and having a size of 17 kilobytes (KB). This submitted sequence listing is part of this specification and is hereby incorporated by reference in its entirety.

[0003] (Field of the Invention) This application relates to a method for blood - based screening of subjects for clinical trials for the treatment of tauopathy or amyloid - forming diseases. In particular, this application relates to a method for identifying patients suitable for clinical trials for the treatment of tauopathy or amyloid - forming diseases based on measuring the amount of single or multiple phosphorylated p217 + tau protein species in a blood - based sample and its use.

Background Art

[0004] Alzheimer's disease (AD) is a degenerative brain disorder characterized clinically by the progressive loss of memory, cognition, logical thinking, judgment, and emotional stability, which leads to a gradually severe decline in mental function and ultimately death. AD is a very common cause of progressive mental impairment (dementia) in the elderly. In the United States, more than 5 million people live with AD, and the number is increasing with the aging population. In fact, 10% of people over 65 have AD, which is the fifth leading cause of death in this group. Overall AD is the sixth leading cause of death in the United States (one in three elderly people die of AD or another dementia), and it is estimated to cost $305 billion in 2020. AD has also been observed in ethnic groups around the world and presents a major current and future public health problem.

[0005] The brains of individuals with AD exhibit characteristic lesions called senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels), and neurofibrillary changes. The majority of these lesions, particularly amyloid plaques and the paired helical filament neurofibrillary changes, are generally seen in several regions of the human brain that are important for memory and cognitive function in patients with AD.

[0006] The progression of tauopathy in the AD brain follows different spreading patterns. The transmission and spreading hypothesis of tauopathy based on the Braak stages of the progression of tauopathy in the human brain and the spreading of tauopathy after injection of tau aggregates in preclinical tau models have been described (Frost et al., J Biol Chem. 284:12845 - 52, 2009, Clavaguera et al., Nat Cell Biol. 11:909 - 13, 2009). Tauopathy is thought to spread like a prion from one brain region to the next. This spreading process involves the externalization of tau seeds that can be taken up by neighboring neurons and cause further tauopathy.

[0007] Currently, there is an important unmet need for clinically effective and safe treatments for tauopathy or amyloid-forming diseases such as Alzheimer's disease. However, any investigational pharmaceutical for the treatment of tauopathy or amyloid-forming diseases requires clinical trial studies in suitable subjects to demonstrate its effectiveness in improving or delaying the progression of tauopathy or amyloid-forming diseases. SUMMARY OF THE INVENTION

[0008] One exemplary embodiment of the present application relates to a method for pre-screening human subjects for a clinical trial for the treatment of tauopathy or amyloid-forming diseases. 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 contacting the plasma sample with a capture antibody directed against the p217+ tau epitope to bind the capture antibody to the p217+ tau peptide in the plasma sample to form an antibody-peptide complex, contacting the antibody-peptide complex with a detection antibody to bind the detection antibody to the antibody-peptide complex, detecting the detection antibody, and determining the amount of p217+ tau peptide in the plasma sample. In one example, the concentration of p217+ tau is determined using an assay with a lower limit of quantitation (LLOQ) of <0.04 pg / ml.

[0009] The method further includes instructing further screening for clinical trials in a subject when the concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold. The minimum threshold corresponds to the amount of p217+ tau in the plasma in which the subject exhibits an increased accumulation of tau changes in the brain as compared to subjects with mild cognitive impairment (MCI) and 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, above which the subject exhibits the pathology of extensive accumulation of tau changes in multiple regions 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 step of instructing includes instructing further screening for clinical trials in a subject when the concentration of p217+ tau present in the plasma sample is ≧0.1 pg / ml and ≦0.25 pg / ml.

[0010] In another aspect of the present application, a method for screening human subjects for a clinical trial for the treatment of tauopathy or amyloid-forming disease is provided. 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 radioactive tracer when the concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold. The minimum threshold corresponds to the amount of p217+ tau in the plasma that exhibits an increased accumulation of tau changes in the brain compared to subjects with mild cognitive impairment (MCI) and 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 is the amount of p217+ tau present in the plasma, above which the subject exhibits the pathology of extensive accumulation of tau changes in multiple regions 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 instructing step includes instructing the subject to undergo further screening 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. The method further includes analyzing the PET data to determine whether the subject is tau positive or amyloid positive and whether the subject has extensive tau changes or extensive amyloid fibrils. The method further includes indicating that the subject is suitable for inclusion in the clinical trial when (i) the concentration of p217+ tau present in the plasma sample is above the minimum threshold and below the maximum threshold, (ii) the PET data indicates that the subject is tau positive or amyloid positive, and (iii) the PET data indicates that the subject does not have extensive tau changes or extensive amyloid fibrils.

[0011] These and other aspects of the invention will become apparent to those skilled in the art upon reading the following detailed description of the invention, which includes the drawings and the appended claims.

Brief Description of the Drawings

[0012]

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[0013] 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 belongs. If not, the specific terms used herein have the meaning set forth herein. All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein. Note that when used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0014] Unless otherwise indicated, all numerical values such as concentrations or concentration ranges described in this specification should be understood as being modified by the term "about" in all cases. Thus, the numerical values typically include ±10% of the recited 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). As used herein, the use of a numerical range, unless otherwise clearly indicated in the context, includes all possible sub-ranges, including integers and fractional values within that range, and explicitly includes all individual numerical values within that range.

[0015] As used herein, the terms "antibody" or "immunoglobulin" refer to a specific protein capable of binding to an antigen or a portion thereof. These terms are used herein in a broad sense and include immunoglobulins or antibody molecules, including polyclonal antibodies, monoclonal antibodies (including mouse, human, human - adapted, humanized, and chimeric monoclonal antibodies), and antibody fragments.

[0016] Generally, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. The structure of an antibody is well - known. Immunoglobulins can be assigned to five main classes, namely IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of the heavy - chain constant domain. IgA and IgG are further subclassified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, the antibodies of the present application can be of any of the five main classes or the corresponding subclasses. Preferably, the antibodies of the present application are IgG1, IgG2, IgG3, or IgG4. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, namely κ or λ, based on the amino acid sequence of their constant domain. Thus, the antibodies of the present application can contain a κ or λ light - chain constant domain. According to certain embodiments, the antibodies of the present application include the heavy - chain and / or light - chain constant regions of mouse antibodies or human antibodies.

[0017] In addition to the heavy and light chain constant domains, an antibody contains light and heavy chain variable regions. An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by an "antigen-binding site". The antigen-binding site is defined using various terms and numbering schemes as follows. (i) Kabat: "Complementarity Determining Region" or "CDR (Complementarity Determining Region)" is based on sequence variability (Wu and Kabat, J Exp Med. 132:211-50, 1970). Generally, an antigen-binding site has 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)). (ii) Chothia: The terms "hypervariable region", "HVR" refer to regions of antibody variable domains that are hypervariable in structure, as defined by Chothia and Lesk (Chothia and Lesk, J Mol Biol. 196:901-17, 1987). Generally, an antigen-binding site has three hypervariable regions in each VH (H1, H2, H3) and VL (L1, L2, L3). The numbering system and annotation of CDRs and HVRs have been revised by Abhinandan and Martin (Abhinandan and Martin, Mol. Immunol. 45:3832-9, 2008). (iii) IMGT: Another definition of the region forming the antigen-binding site has been proposed by Lefranc based on the comparison of V domains from immunoglobulins and T cell receptors (Lefranc et al., Dev Comp Immunol. 27:55-77, 2003). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides the standard numbering and definition of these regions. The correspondence between the descriptions of CDRs, HVRs, and IMGT is described in Lefranc et al., 2003, ibid. (iv) The antigen-binding site can also be described based on the "Specificity Determining Residue Usage" (SDRU) (Almagro, Mol Recognit. 17:132-43, 2004), where SDR refers to the amino acid residues of the immunoglobulin that are directly involved in antigen contact.

[0018] The "framework" or "framework sequence" is the remaining sequence within the variable region of the antibody other than that defined as the sequence of the antigen-binding site. Since the exact definition of the antigen-binding site can be determined by various descriptions as mentioned above, the exact framework sequence depends on the definition of the antigen-binding site. The framework region (FR) is the more highly conserved part of the variable domain. The variable domains of the native heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) that generally use a β-sheet structure connected by three hypervariable loops. The hypervariable loops of each chain are tightly folded together by the FRs and, together with the hypervariable loops of the other chain, contribute to the formation of the antigen-binding site of the antibody. Structural analysis of antibodies has revealed the relationship between the sequence and shape of the binding site formed by the complementarity-determining regions (Chothia et al., J. Mol. Biol. 227:799-817, 1992; Tramontano et al., J. Mol. Biol. 215:175-182, 1990). Despite these high sequence variabilities, only 5 out of the 6 loops adopt the main-chain structures of a small repertoire called the "canonical structure". These structures are first determined by the length of the loop and then by the presence of key residues at specific positions in the loop and framework regions, where the structure is determined by the ability to predict folding, hydrogen bonding, or abnormal main-chain structures.

[0019] As used herein, the term "epitope" refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof specifically binds. Epitopes can be formed from either contiguous amino acids juxtaposed by the tertiary folding of a protein or from non-contiguous amino acids. Epitopes formed from contiguous amino acids are typically retained upon exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are typically lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids within a unique spatial structure. Methods for determining the spatial structure of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0020] As used herein, the term "tau" or "tau protein" refers to a protein of the central and peripheral nervous systems that has multiple isoforms. In the human central nervous system (CNS), six major tau isoforms in the size range of 352 - 441 amino acids in length exist due to alternative splicing (Hanger et al., Trends Mol Med. 15:112 - 9, 2009). These isoforms differ from each other by having 0 - 2 N-terminal insertions and being controlled to contain 3 or 4 tandemly arranged microtubule-binding repeat sequences, and are designated 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R. As used herein, the term "control tau" refers to the tau isoform of SEQ ID NO:1 that is not phosphorylated and not otherwise post-translationally modified. As used herein, the term "tau" includes proteins that are variants 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.

[0021] Unless otherwise indicated, when used herein, the numbering of amino acids in a tau protein or fragment thereof refers to the amino acid sequence set forth in SEQ ID NO:1.

[0022] As used herein, the term "p217+ tau peptide", "p217+ tau", or "p217+ tau protein" means a human tau protein or tau fragment that is phosphorylated at residue 217 (pT217) of the tau protein, and may or may not be further phosphorylated at additional residues such as residue 212 (pT212) of the tau protein, where the numbering of positions follows the numbering of SEQ ID NO:1.

[0023] As used herein, the term "p217+ tau epitope" refers to a tau epitope containing at least one of phosphorylated T217 and phosphorylated T212, where the numbering follows that of 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 - 220 of the human tau protein phosphorylated at residue 217, which may or may not be further phosphorylated at additional residues such as residue 212, where the numbering follows that of SEQ ID NO: 1.

[0024] As used herein, the term "capture antibody" refers to an antibody that binds to an antigen of interest and is directly or indirectly bound to a solid support. Examples of solid supports include, but are not limited to, microparticles or beads, such as magnetic beads or paramagnetic beads. Examples of capture antibodies include, but are not limited to, monoclonal antibodies that bind to the p217+ tau epitope.

[0025] As used herein, the term "detection antibody" refers to an antibody that binds to an antigen of interest and has a detectable label or is conjugated to a secondary detection system. Examples of detectable labels include, but are not limited to, various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of detection antibodies include, but are not limited to, monoclonal antibodies that bind to epitopes containing amino acids 7 - 20 or 116 - 127 of the tau protein, preferably the human tau protein, where the numbering follows that of SEQ ID NO: 1. When a monoclonal antibody that binds to the tau protein in the epitope containing amino acids 7 - 20 is used as the detection antibody for the captured p217+ tau peptide, long tau fragments are detected. When a monoclonal antibody that binds to the tau protein in the epitope containing amino acids 116 - 127 is used as the detection antibody for the captured p217+ tau peptide, both short tau fragments and long tau fragments are detected.

[0026] As used herein, the term "subject" refers to an animal, preferably a mammal. According to certain embodiments, the subject is a mammal including non - primates (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, marmoset, or mouse), or primates (e.g., monkey, chimpanzee, or human). In certain embodiments, the subject is a human.

[0027] As used herein, "tauopathy" encompasses any neurodegenerative disease associated with pathological aggregation of tau in the brain. In addition to familial and sporadic AD, other exemplary tauopathies are frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP - 17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle - only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis - parkinsonism dementia complex, Down syndrome, Gerstmann - Straussler - Scheinker disease, Hallervorden - 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 - Guamanian motor neuron disease with neurofibrillary tangles, post - encephalitic parkinsonism, and chronic traumatic encephalopathy such as pugilistic dementia (boxer's disease) (Morris et al., Neuron, 70:410 - 26, 2011).

[0028] As used herein, the term "amyloid-forming disease" includes any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibers. Exemplary amyloid-forming diseases include, but are not limited to, systemic amyloidosis, Alzheimer's disease, adult-onset diabetes, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-related transmissible spongiform encephalopathies (Creutzfeldt-Jakob disease in humans, and scrapie and BSE in sheep and cattle, respectively). Different amyloid-forming diseases are defined or characterized by the nature of the polypeptide constituent of the deposited protofibrils. For example, in a subject or patient having Alzheimer's disease, the β-amyloid protein (e.g., wild-type, variant, or truncated β-amyloid protein) is the characteristic polypeptide constituent of the amyloid deposits. Thus, Alzheimer's disease is, for example, an example of a "disease characterized by Aβ deposition" or a "disease associated with Aβ deposition" in the brain of a subject or patient. The terms "β-amyloid protein", "β-amyloid peptide", "β-amyloid", "Aβ", and "Aβ peptide" are used interchangeably herein.

[0029] As used herein, the terms "determine", "measure", "evaluate", and "assay" are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement and include determining whether a property, trait, or characteristic is present. An evaluation may be relative or absolute. "Evaluating the presence of" includes determining the amount of something present, as well as determining whether it is present or absent.

[0030] As used herein, the term "diagnosis" means detecting a disease or disorder, or determining the stage or degree of a disease or disorder such as tauopathy or amyloid-forming disease. Usually, the diagnosis of a disease or disorder is based on the evaluation of one or more factors and / or symptoms indicative of the disease. The diagnosis can be made based on factors indicating the presence, absence, or amount of a disease or condition, such as the presence of p217+ tau. Each factor or symptom considered indicative of the diagnosis of a particular disease need not be associated only with that particular disease, i.e., there may be different diagnoses that can be inferred from the diagnostic factor or symptom. Similarly, there may be instances where a factor or symptom indicative of a particular disease is present in an individual who does not have that particular disease. The term "diagnosis" also encompasses determining the therapeutic effect of a drug therapy, such as anti-p217+ tau antibody therapy, or predicting the pattern of response to a drug therapy, such as anti-p217+ tau antibody therapy. The diagnostic method can be used independently or in combination with other diagnostic methods and / or staging methods known in the medical field for a particular disease or disorder, such as Alzheimer's disease.

[0031] As used herein, the terms "increasing" and "decreasing" refer to the difference in the amount of a particular biomarker in a sample as compared to a control or reference level. For example, the amount of a particular peptide may be present in an increased amount or a decreased amount in a sample from a patient having a disease as compared to a reference level. In one embodiment, an "increase in level" or "decrease in level" can be that the difference between the levels of a biomarker present in a sample is 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 when compared to a control. In one embodiment, an "increase in level" or "decrease in level" can be that the difference between the levels of a biomarker present in a sample is statistically significant when compared to a control. For example, the difference can be statistically significant if the measured level of a biomarker is outside of about 1.0 standard deviation, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 standard deviations of the mean of any control group or reference group. The reference or control can be, for example, a sample from a healthy individual, or a sample taken from the same individual at an earlier time point such as prior to administration of a therapeutic agent or an earlier time point during a treatment regimen.

[0032] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) is substantially separated from other biological components of the organism in which it naturally occurs, i.e., other chromosomes and extrachromosomal DNA and RNA, and proteins, or is produced apart from or purified from these other components. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition and are isolated even if such composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as nucleic acids chemically synthesized.

[0033] As used herein, the term "isolated antibody that binds to tau protein" or "isolated anti-tau antibody" is intended to refer to an antibody that specifically binds to tau protein and substantially does not contain other antibodies having different antigen specificities (e.g., an isolated anti-tau detection antibody substantially does not contain antibodies that specifically bind to antigens other than tau). However, an isolated anti-tau detection antibody may have cross-reactivity, for example, with other related antigens from other species (such as homologs of tau species).

[0034] As used herein, the terms "specifically binds" or "specific binding" mean that the anti-tau antibodies of the present application bind to a given target with a dissociation constant (K -6 M) or tighter, e.g., about 1×10 -7 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, about 1×10 -12 M or less, or about 1×10 -13 M or less. D ). Dis obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The K D value of the antibody can be determined using methods in the art in view of the present disclosure. For example, the K D value of an anti-tau antibody can be determined by using surface plasmon resonance, such as a biosensor system, e.g., a Biacore® system, a Proteon instrument (BioRad), a KinExA instrument (Sapidyne), ELISA, or by using a competitive binding assay known to those skilled in the art. Typically, an anti-tau antibody binds to a given target (i.e., tau) with a K D that is at least 10-fold lower than the K D for a non-specific target when measured by surface plasmon resonance using, for example, a Proteon instrument (BioRad). However, an anti-tau antibody that specifically binds to tau may have cross-reactivity to a given identical target from other related targets, e.g., other species (homologs), e.g., from mouse, rat, marmoset, dog, or pig.

[0035] As used herein, the term "accuracy" refers to the degree of closeness of a value to the true value of an assay.

[0036] As used herein, the term "precision" refers to the closeness of agreement between a series of measurements obtained from multiple samplings of the same homogeneous sample of an assay.

[0037] As used herein, the term "sensitivity" refers to the lowest analyte concentration in a sample that can be measured with acceptable accuracy and precision in an assay.

[0038] When conducting a clinical trial for the treatment of tauopathy or amyloid-forming diseases, it is necessary to screen human subjects who are seeking to enroll in the clinical trial and select subjects in need of such treatment, such as subjects who have developed and / or are at risk of developing tauopathy or amyloid-forming diseases, for inclusion in the clinical trial. Thus, the subject needs to meet a minimum level of disease progression of tauopathy and / or amyloid-forming diseases for inclusion in the clinical trial. Additionally, when treatment is initiated early in disease progression, anti-tau therapy and anti-amyloid are each thought to be most effective in treating tauopathy and amyloid-forming diseases, respectively. Thus, it may be desirable to select subjects who are not in the late stage of disease progression of tauopathy or amyloid-forming diseases so that the treatment is more likely to be effective and / or any effect of the treatment for tauopathy or amyloid-forming diseases under clinical trial can be easily observed to slow the progression of tauopathy or amyloid-forming diseases. Before proceeding to further screening tests or for inclusion in a clinical trial, it may be desirable to select subjects who exceed the minimum level of disease progression of tauopathy and / or amyloid-forming diseases and are below the maximum level of disease progression of tauopathy and / or amyloid-forming diseases to identify subjects in need of treatment for tauopathy or amyloid-forming diseases and who are not in the late stage of disease progression of tauopathy and / or amyloid-forming diseases.

[0039] This application provides a method for pre-screening or screening human subjects for clinical trials of treatments for tauopathy or amyloid-forming diseases using an assay for detecting single or multiple phosphorylated p217+ tau peptides in a blood-based sample. Accurate, sensitive, and precise measurement of p-tau isoforms in plasma is considered a promising non-invasive method for detecting abnormal amyloid and tau processes. Longer fragments of multiply phosphorylated tau containing pT217 are considered to be one of the isoforms most relevant to the pathology of tauopathy and amyloid-forming diseases, specifically Alzheimer's disease, and can begin to accumulate in CSF and plasma 10 - 20 years prior to cognitive decline. Phosphorylated tau, particularly p217+ tau, measured in CSF and plasma is considered to be one of the most sensitive and specific biomarkers for the pathology of tauopathy and amyloid-forming diseases, specifically Alzheimer's disease, and is thought to predict "tau-positive" pathology or "amyloid-positive" pathology (both further defined below), as well as a decline in cognitive status. The amount of single or multiple phosphorylated p217+ tau peptides measured from a blood-based sample from a subject can serve as an indicator of the progression of tauopathy and / or amyloid-forming diseases in the subject. Lower concentrations of p217+ tau peptides measured from the sample correspond to the early stages of disease progression of tauopathy and / or amyloid-forming diseases, while higher concentrations of p217+ tau peptides measured from the sample correspond to the later stages of disease progression of tauopathy and / or amyloid-forming diseases. Thus, the amount of single or multiple phosphorylated p217+ tau peptides measured from a blood-based sample from a subject can be useful as a pre-screening or screening criterion for determining whether the subject is suitable for inclusion in a clinical trial of a treatment for tauopathy or amyloid-forming diseases.

[0040] The prescreening or screening methods described in this application are useful for identifying suitable subjects for clinical trials of treatments for tauopathy or amyloid-forming diseases. Treatments for tauopathy can be any type of procedure or pharmaceutically active agent for treating tauopathy or amyloid-forming diseases or slowing their progression. For example, a treatment for tauopathy under clinical trial can be a pharmaceutical composition containing an active agent having the potential to treat tauopathy or slow its progression, such as an anti-tau antibody, an anti-p217+ tau antibody, small interfering RNA (siRNA) against human tau, siRNA against p217+ tau, etc. In another example, a treatment for amyloid-forming disease under clinical trial can be a pharmaceutical composition containing an active agent having the potential to treat amyloid-forming disease or slow its progression, such as an anti-amyloid antibody, a beta-secretase inhibitor, a gamma-secretase inhibitor, small interfering RNA (siRNA) against human β-amyloid, etc. In a specific example, the treatment under clinical trial is an active agent having the potential to treat Alzheimer's disease or slow its progression.

[0041] The collection of blood-based samples, such as plasma, is carried out quickly and easily and does not impose a significant discomfort or burden on the subject. In contrast, other currently available tools for determining the progression of a subject's tauopathy and / or amyloid-forming disease, such as Alzheimer's disease, are costly and time-consuming. For example, positron emission tomography using a tau-specific radiotracer (tau PET) has been used to measure and localize tau neurofibrillary change pathology in a patient's brain. Similarly, positron emission tomography using an amyloid-specific radiotracer (amyloid PET), such as an Aβ40- or Aβ42-specific radiotracer, has also been used to measure and localize amyloid fibril pathology in a patient's brain. However, both tau PET and amyloid PET are costly, involve cumbersome procedures, and have limited availability of suitable tracers. In addition, the process for imaging can be uncomfortable and thus more time-consuming than collecting a patient's blood sample. Another exemplary tool for determining the progression of a subject's tauopathy and / or amyloid-forming disease is the amount of phosphorylated tau peptide (e.g., single or multiple phosphorylated p217+ tau peptide) measured by an assay in a sample CSF recovered from the subject. However, the recovery of CSF requires the patient to undergo an invasive lumbar puncture procedure, which involves a physician inserting a needle into the spinal canal to collect a sample of CSF for use in the assay. Such invasive procedures can be painful and expose the subject to the risk of infection in the spinal canal. Thus, the use of a blood-based assay, more specifically a plasma-based assay, for detecting single or multiple phosphorylated p217+ tau peptides in a sample provides an improved method for quickly and effectively identifying the most likely candidates meeting the desired clinical criteria (e.g., having a minimum level of disease progression of tauopathy and / or amyloid-forming disease but less than a maximum level of disease progression of tauopathy and / or amyloid-forming disease) for pre-screening or screening human subjects for clinical trials for the treatment of tauopathy or amyloid-forming disease, particularly Alzheimer's disease.

[0042] In one aspect of the present application, a method 100 for pre-screening or screening human subjects for a clinical trial for the treatment of tauopathy or amyloid-forming diseases is provided. The method 100 includes a first step 102 of obtaining a blood-based sample from a human subject. The blood-based sample can be a blood, serum, or plasma sample. 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 certain embodiments, the sample is a crude plasma sample.

[0043] In step 104, the sample can be further processed by any suitable means of concentrating the p217+ tau peptide contained therein before analyzing the concentrated sample in an assay to determine the amount or concentration of p217+ tau present in the sample. Preferably, in step 104, the sample is analyzed directly in an assay to determine the amount or concentration of p217+ tau present in the sample without further concentrating the p217+ tau peptide prior to the assay (step 104). A blood-based sample can be analyzed using any suitable assay for determining the amount or concentration of p217+ tau present in the sample. More specifically, the assay is a sensitive and accurate assay for measuring p217+ tau in plasma. The amount or concentration of the p217+ tau peptide can be determined using any suitable technique known in the art, including ELISA and single molecule array platforms. According to certain embodiments, the methods of the present application can use a sensitive array platform, such as Quanterix Simoa or MSD S-plex, to measure the amount or concentration of the p217+ tau peptide in a blood-based sample (specifically, a plasma sample). 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 that can provide an accurate, sensitive, and precise quantitative measurement of p217+ tau present in plasma samples obtained from cognitively normal subjects (including subjects who are not amyloid positive), as well as subjects who have or are at risk of developing tauopathy or amyloid-forming disease, particularly subjects with mild cognitive impairment (MCI) within an acceptable and reliable level of sensitivity. For example, the lower limit of quantification (LLOQ) of the assay can be 0.05 pg / ml, 0.045 pg / ml, 0.04 pg / ml, 0.038 pg / ml, or 0.036 pg / ml or less.

[0044] Specifically, the blood-based sample is a plasma sample, and the assay used to determine 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 label (e.g., a fluorescent molecule, biotin, etc.) that is directly detectable or detectable via a secondary reaction (e.g., reaction with streptavidin). The capture antibody can be immobilized on a solid phase such that the capture antibody selectively binds to the p217+ tau peptide present in the sample and immobilizes it on the solid phase. The capture antibody binds to the p217+ tau epitope and forms an antibody-peptide complex with the p217+ tau peptide. The antibody-peptide complex is then contacted with the detection antibody to bind the detection antibody to the antibody-peptide complex. In one example, the detection antibody is biotinylated. Then, the detectable label on the detection antibody is determined to determine the amount or concentration of p217+ tau peptide in the sample.

[0045] In one example, the capture antibody is a monoclonal antibody comprising immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the polypeptide sequences of SEQ ID NOs: 2, 3, and 4, respectively, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 5, 6, and 7. 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 heavy chain variable region amino acid sequence of SEQ ID NO: 8 and the light chain variable region amino acid sequence of SEQ ID NO: 9. In one embodiment, the pT3 monoclonal antibody is expressed by a mouse-hybridoma.

[0046] An exemplary detection antibody is a monoclonal antibody that binds to an epitope comprising amino acids 7-20 of the human tau protein, where the numbering follows that of SEQ ID NO: 1. The detection antibody can be a monoclonal antibody that comprises, respectively, immunoglobulin heavy chain HCDR1, HCDR2, and HCDR3 having the polypeptide sequences of SEQ ID NO: 10, 11, and 12, and immunoglobulin light chain LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NO: 13, 14, and 15. In certain embodiments, the detection antibody is hT43. As used herein, the term "hT43" refers to a monoclonal antibody that binds to an epitope comprising amino acids 7-20 of the human tau protein, where the numbering follows that of SEQ ID NO: 1, and 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.

[0047] An exemplary assay for determining the amount of p217+ tau present in a plasma sample is described, for example, in International Publication No. WO 2022 / 013286, the entire disclosure of which is incorporated herein by reference.

[0048] In Project 106, the amount or concentration of p217+ tau present in a plasma sample is used to determine whether a subject proceeds to further screening for a clinical trial or is otherwise selected for or shown to be suitable for inclusion in a clinical trial. If the amount or concentration of p217+ tau present in a subject's plasma sample is above a minimum threshold, the subject is identified as one in need of treatment for tauopathy or an amyloidogenic disease. The minimum threshold is the amount or concentration of p217+ tau present in plasma, above which a subject exhibits a "tau positive" pathology, i.e., an accumulation of tau changes in the brain, more specifically in the bilateral inferior temporal cortex (e.g., accumulation of singly or multiply phosphorylated p217+ tau), increased compared to subjects with mild cognitive impairment (MCI) and cognitively normal subjects. Such an accumulation may correspond to a tau PET image having a SUVR Z-score >1 in the bilateral inferior temporal cortex of the subject. 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 is the amount or concentration of p217+ tau present in plasma, above which a subject tends to correspond to an "amyloid positive" pathology, i.e., an accumulation of β-amyloid (e.g., Aβ40 and / or Aβ42) in the bilateral inferior temporal cortex increased compared to subjects with mild cognitive impairment (MCI) and cognitively normal subjects. The amount or concentration of p217+ tau in the plasma sample, determined in Project 104, provides a good ability to predict the tau positive and / or amyloid positive status of a subject using a minimum threshold of 0.1 pg / ml.

[0049] In addition, when the amount or concentration of p217+ tau present in the plasma sample of the subject is below a maximum threshold, the subject is identified as not being in the late stage of disease progression of tauopathy or amyloid-forming disease, such that the treatment is more likely to be effective and / or any effect by the treatment for tauopathy or amyloid-forming disease under clinical trials to slow the progression of tauopathy or amyloid-forming disease can be easily observed. The maximum threshold corresponds to the amount or concentration of p217+ tau present in the plasma, and when it is exceeded, the subject tends to have a pathology of extensive accumulation of tau changes in multiple regions of the brain. For example, such extensive accumulation of tau changes may correspond to tau PET imaging having a SUVR Z-score > 5 in each of the Braak 4, 5, and 6 regions of the subject's brain. In one example, the maximum threshold is 0.225 pg / ml, 0.25 pg / ml, 0.275 pg / ml, 0.3 pg / ml. In another example, the maximum threshold is 0.25 pg / ml ± 0.025 pg / ml.

[0050] In one embodiment, if the amount or concentration of p217+ tau present in the plasma sample is ≧ the minimum threshold and ≦ the maximum threshold, the subject proceeds to further screening for the clinical trial, or alternatively, is selected for inclusion in the clinical trial or is shown to be suitable for inclusion in the clinical trial (step 108). If the amount or concentration of p217+ tau present in the plasma sample is < the minimum threshold and > the maximum threshold, the subject is excluded from the clinical trial (step 110). For example, if the amount or concentration of p217+ tau present in the plasma sample is ≧ 0.1 ± 0.025 pg / ml and ≦ 0.25 ± 0.025 pg / ml, the subject proceeds to further screening for the clinical trial, or alternatively, is selected for inclusion in the clinical trial or is shown to be suitable for inclusion in the clinical trial (step 108). If the amount or concentration of p217+ tau present 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 between 0.075 pg / ml and 0.275 pg / ml, between 0.075 pg / ml and 0.25 pg / ml, between 0.075 pg / ml and 0.225 pg / ml, between 0.1 pg / ml and 0.275 pg / ml, between 0.1 pg / ml and 0.25 pg / ml, between 0.1 pg / ml and 0.225 pg / ml, between 0.125 pg / ml and 0.275 pg / ml, between 0.125 pg / ml and 0.25 pg / ml, or between 0.125 pg / ml and 0.225 pg / ml, the subject proceeds to further screening for the clinical trial, or alternatively, is selected for inclusion in the clinical trial or is shown to be suitable for inclusion in the clinical trial (step 108). If the amount or concentration of p217+ tau present in the plasma sample is outside the range specified above, the subject is excluded from the clinical trial (step 110).

[0051] In another aspect of the present application, a method 200 for pre-screening or screening human subjects for a clinical trial for the treatment of tauopathy or amyloid-forming diseases is provided. Method 200 includes a first step 202 of obtaining a blood-based sample, more specifically a plasma sample, from a 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 can be assayed once or divided into aliquots and assayed multiple times (e.g., 2 times, 3 times, etc.) to generate multiple measurements of the amount or concentration of p217+ tau detected from the same sample.

[0052] In step 206, when the sample is assayed 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 is assayed with sufficient accuracy. For example, the coefficient of variation percentage (CV%) can be determined for the multiple measurements obtained from the same sample. When the sample is assayed only once, the CV% is not determined for the sample. If the CV% is less than the threshold CV, method 200 proceeds to step 208. If the CV% determined for the multiple measurements is greater than the threshold CV or the sample is assayed only once, method 200 proceeds to step 210. The threshold CV can be 25 CV%, 20 CV%, or 15 CV%. In one specific example, if the CV% is <20%, method 200 proceeds to step 208. If the CV% is ≧20% or the sample is assayed only once, method 200 proceeds to step 210.

[0053] In step 208, when the average amount or concentration of p217+ tau over multiple measurements of a sample is ≥ the minimum threshold and ≤ the maximum threshold, the subject proceeds to further screening for a clinical trial, or alternatively, is selected for inclusion in the clinical trial or shown to be suitable for inclusion in the clinical trial. When the average amount or concentration of p217+ tau over multiple measurements of a sample is < the minimum threshold and > the maximum threshold, the subject is excluded from the clinical trial. The minimum threshold and the maximum threshold are defined in the same manner as described above for step 106. In one example, when the average amount or concentration of p217+ tau over multiple measurements of a plasma sample is ≥ 0.1 ± 0.025 pg / ml and ≤ 0.25 ± 0.025 pg / ml, the subject proceeds to further screening for a clinical trial, or alternatively, is selected for inclusion in the clinical trial or shown to be suitable for inclusion in the clinical trial. When the average amount or concentration of p217+ tau over multiple measurements of a plasma sample is < 0.1 ± 0.025 pg / ml or > 0.25 ± 0.025 pg / ml, the subject is excluded from the clinical trial. In another example, when the amount or concentration of p217+ tau over 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 proceeds to further screening for a clinical trial, or alternatively, is shown to be suitable for inclusion in the clinical trial (step 210). When the amount or concentration of p217+ tau over multiple measurements of a plasma sample is outside the ranges specified above, the subject is excluded from the clinical trial (step 212).

[0054] In step 220, using the average amount or concentration of p217+ tau over multiple measurements of the sample, or a single measurement of the amount or concentration of p217+ tau in the plasma sample, it is determined which of the following three different steps the method 200 proceeds to next. First, if the average amount or concentration of p217+ tau over multiple measurements of the plasma sample or a single measurement of the amount or concentration of p217+ tau in the plasma sample is within the range of ±Δ of the minimum threshold or ±Δ of the maximum threshold, the 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.

[0055] In step 222, the sample is retested in the same manner as described in step 204 to obtain a further measurement of the amount or concentration of p217+ tau present in the sample. In step 224, if the retest data provides a quantitative result similar to the previous measurements (e.g., within ± standard deviation of the mean value), and the sample improves (i.e., the CV% is decreased), the average amount or concentration of p217+ tau over 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).

[0056] Next, if the average amount or concentration of p217+ tau over multiple measurements of the plasma sample or a single measurement of the amount or concentration of p217+ tau in the plasma sample is <LLOQ of the assay, the subject is excluded from the clinical trial (step 212). In this scenario, retesting is not necessary because the amount or concentration of p217+ tau in the plasma sample is below the detectable range of the assay and thus is likely to result in a poor CV%.

[0057] Finally, if the average amount or concentration of p217+ tau over multiple measurements of the plasma sample or a single measurement of the amount or concentration of p217+ tau in the plasma sample is (1) ≧LLOQ and < minimum threshold - Δ, (2) greater than the minimum threshold + Δ and less than the maximum threshold - Δ, or (3) greater than or equal to the maximum threshold + Δ, If Δ is as specified above, method 200 proceeds to step 208. If the amount or concentration of p217+ tau in the plasma sample meets any of the three criteria specified above, method 200 may optionally include a retest step (not shown) to improve the accuracy of the average amount or concentration of p217+ tau over multiple measurements and / or reduce the CV% for the measurements obtained for the sample.

[0058]

[0059] In certain embodiments, the minimum threshold is 0.1 pg / ml, the maximum threshold is 0.25 pg / ml, Δ is 0.025 pg / ml, and the LLOQ of the assay is 0.036 pg / ml. In step 208 of this embodiment, if the average concentration of p217+ tau over multiple measurements of the plasma sample is greater than or equal to 0.1 pg / ml and less than or equal to 0.25 pg / ml, the subject proceeds to further screening for a clinical trial or is otherwise considered suitable for inclusion in a clinical trial (step 210). In contrast, if the average concentration of p217+ tau over multiple measurements of the plasma sample is less than 0.1 pg / ml or greater than 0.25 pg / ml, the subject is excluded from the clinical trial (step 212).In step 220, using 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, it is determined which of the following three different steps the method 200 proceeds 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 - 0.125 pg / ml or 0.225 - 0.275 pg / ml, the 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, the subject is excluded from the clinical trial (step 212). 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 (1) ≧0.036 pg / ml and <0.075 pg / ml, (2) >0.125 pg / ml and <0.225 pg / ml, or (3) ≧0.275 pg / ml, the method 200 proceeds to step 208.

[0060] In some embodiments, the subjects identified in step 108 or 210 can be directed to additional clinical tests, such as PET imaging and / or CSF collection, to further evaluate the brain pathology of these subjects. The additional clinical trials can be used to confirm that the subject requires treatment for tauopathy or amyloidogenic disease (e.g., having mild cognitive impairment (MCI) and having an accumulation of increased tau changes in the brain compared to cognitively normal patients), and that the subject is not in the late stage of disease progression of tauopathy or amyloidogenic disease (e.g., presenting pathology of tau changes or extensive accumulation of amyloid fibrils in multiple regions of the brain).

[0061] For example, the subject identified in step 108 or 210 may be directed to tau PET imaging using a tau-specific tracer (e.g., p217+ tau-specific tracer), or amyloid PET using an amyloid-specific radiotracer (e.g., Aβ40 or Aβ42-specific radiotracer). The obtained PET data can be analyzed by a computing device to confirm that the subject is tau-positive or amyloid-positive. For example, the computing device analyzes the tau PET data to confirm that the subject has an accumulation of tau changes in the brain, more specifically in the bilateral inferior temporal cortices, that is increased compared to cognitively normal patients (e.g., accumulation of singly or multiply phosphorylated p217+ tau). More specifically, the tau PET data can be analyzed to confirm that the subject has a SURVR Z-score > 1 in the bilateral inferior temporal cortices. In another example, the amyloid PET data can be analyzed to confirm that the subject has an accumulation of amyloid fibrils in the brain, more specifically in the bilateral inferior temporal cortices, that is increased compared to cognitively normal patients.

[0062] The computing device can also analyze the obtained PET data to confirm that the subject is not in the late stage of disease progression of tauopathy or amyloidogenic disease (e.g., presenting a pathology of extensive tau changes or extensive accumulation of amyloid fibrils in multiple regions of the brain). For example, the tau PET data can be analyzed to confirm that the subject does not have extensive accumulation of tau changes in the brain. More specifically, the tau PET data can be analyzed to confirm that the subject does not have a SURVR Z-score > 5 in each of the Braak 4, 5, and 6 regions of the subject's brain. In another example, the amyloid PET data can be analyzed to confirm that the subject does not have extensive accumulation of amyloid fibrils in multiple regions of the subject's brain, such as the Braak 4, 5, and 6 regions of the subject's brain.

[0063] In one embodiment, a method for screening human subjects for a clinical trial for the treatment of tauopathy or amyloid-forming disease is provided. 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 in steps 102 and 104 above. When the amount or concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold, the method proceeds 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 is then analyzed to determine whether the subject is tau-positive or amyloid-positive and whether the subject has extensive tau changes or extensive amyloid fibrils. The subject is (i) the concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold, (ii) the PET data indicates that the subject is tau-positive or amyloid-positive, (iii) when the PET data indicates that the subject does not have extensive tau changes or extensive amyloid fibrils, is shown to be suitable for inclusion in a clinical trial.

[0064] Furthermore, subjects shown to be suitable for inclusion in a clinical trial can be further stratified into a high group and a low group based on the SUVR in the Braak4 region generated from the PET data. For example, a subject is stratified into the high group when the SUVR in the Braak4 region is > 4.62 and into the low group when the SUVR in the Braak4 region is ≦ 4.62.

[0065] One of ordinary skill in the art will understand that the exemplary computer-implemented embodiments described herein can be implemented in any number of ways, including as separate software modules, as a combination of hardware and software, and the like. For example, an exemplary method may be embodied in one or more programs stored on a non-transitory storage medium and including lines of code that, when compiled, can be executed by one or more processor cores or a separate processor. A system according to one embodiment includes a plurality of processor cores and a set of instructions executed on these plurality of processor cores to perform the exemplary method described above. The processor core or separate processor may be incorporated into or communicate with any suitable electronic device, such as an on-board processing device inside the device, or an external processing device of the device that can communicate with at least a portion of the device, such as a mobile computing device, a smartphone, a computing tablet, a computing device, and the like.

Example

[0066] The following examples are for further explaining the essence of the present invention. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is defined by the appended claims.

[0067] Example 1: Example 1 reports data on the performance of the plasma 217+ tau assay as a prescreening tool for identifying subjects likely to be tau positive for inclusion in clinical trials for the treatment of tauopathies. The assay is performed using Quanterix's highly sensitive Simoa array platform with the pT3 antibody as the capture antibody and the hT43 antibody conjugated to a detectable label as the detection antibody. In addition, Example 1 describes how the assay can be used to enrich for low-tau PET layer patients for inclusion in clinical trials for treating or slowing the progression of tauopathies.

[0068] In Example 1, the plasma p217+ tau assay was performed on screened samples from subjects presenting with MCI in weekly batches. The technical performance over 58 batches was evaluated. Subjects presenting with plasma p217+ tau levels exceeding a pre-specified cut-off of 0.1 pg / ml proceeded to tau PET (using MK-6240 as tracer) screening. The concentration of plasma p217+ tau in this population and the prevalence of tau-positive status in subjects having a plasma p217+ tau concentration of ≧0.1 pg / ml were studied in Example 1.

[0069] A total of 55 batches of plasma samples were screened using the p217+ tau assay of Example 1 above. A panel of three peptide QCs (0.1, 0.4, and 1.6 pg / ml) was run in duplicate in each batch, revealing excellent within-run precision (average CV = 6.0, 4.5, and 5.5% respectively) and between-run precision (7.6, 6.6, and 13.3 CV% respectively). Among N = 725 plasma samples, the average CV was 7.9% (range 0 - 120%) and the estimated LLOQ was 0.030 pg / ml (based on the average concentration where CV > 20%), so the precision was acceptable even for endogenous samples.

[0070] Of the 787 MCI patients in whom plasma p217+ tau was measured, 72% had levels of ≧0.1 pg / ml. Patients having a plasma p217+ tau concentration of ≧0.1 pg / ml were then scheduled for tau PET imaging. Of the 346 patients imaged, 86% were tau-positive and 64% met the criteria for the test tau PET eligibility criteria of having SUVR Z-score > 1 in the bilateral inferior temporal cortices of the subjects but not having SUVR Z-score > 5 in each of the Braak 4, 5, and 6 regions of the subjects' brains.

[0071] Tau PET data was also used to stratify patients into high and low groups based on SUVR in the Braak stage 4 region. Assay screening was performed as expected, with 59% of eligible patients in the high stratum. ROC analysis showed that the plasma p217+ tau assay could predict patient strata with an AUC of 0.8. Therefore, to correct for stratification imbalance, one year after data collection, an upper threshold of plasma p217+ tau concentration (i.e., ≤0.25 pg / ml) was implemented to enrich patients with low to moderate tauopathy progression.

[0072] The plasma p217+ tau assay of Example 1 showed good precision within and between batches, demonstrating the ability to enrich the population for both tau positivity and tau stratification. The assay may serve as a tool to enable more rapid and efficient enrollment into clinical trials for the treatment of Alzheimer's disease or to identify early Alzheimer's disease in the general population.

[0073] Example 2: Example 2 provides additional data on the performance of the plasma p217+ tau assay for patients screened in the same clinical trial as Example 1. The data of Example 2 includes more samples than the data reported in Example 1. Figure 3a shows the precision (CV%) of the plasma p217+ tau assay across different concentrations of p217+ tau detected from plasma samples collected from 832 patients screened for the clinical trial. Figure 3b shows the distribution of the concentrations of p217+ tau detected by the p217+ tau assay from plasma samples collected from 831 patients screened for the clinical trial.

[0074] Example 3: Correlation between Simoa plasma p217+ tau and tau PET in participants screened for a clinical trial Example 3 provides a report on the correlation observed between plasma p217+ tau and 18F-MK-6240 tau PET uptake across brain regions. The Simoa-based plasma p217+ tau assay was performed in weekly batches (Triana-Baltzer et al., Alzheimers Dement(Amst). 2021). Participants with levels above 0.1 pg / ml were then scanned to confirm the presence of NFTs on tau PET. NFT levels were quantified using 18F-MK-6240 SUVR in Braak Regions of Interest (ROI) (Scholl et al. Neuron 2016) (reference region: cerebellar gray). The Braak ROI is shown in Figure 4. Figure 5 shows a significant (p<10 -15 ) correlation between plasma p217+ tau and MK6240 across all six Braak ROIs. Figure 6 shows that plasma p217+ tau can predict in vivo Braak stages of 3 or less with an AUC of 82%. Non-parametric Spearman correlations were obtained between plasma p217+ tau levels and mean SUVR across each of the six ROIs used for Braak staging. The results reported in Example 3 herein are from a first set of 355 participants in whom tau PET analysis was completed.

[0075] As shown herein, a significant correlation was found between plasma p217+ tau and tau PET in a clinical trial setting. The correlation was consistent with independent observational studies in individuals with cognitive impairment (Dore et al. Alzheimer’s Dement. 2022). The results demonstrate the consistent performance and utility of plasma p217+ tau as a prescreening in Alzheimer's disease (AD) trials. Simoa plasma p217+ tau levels are consistently correlated with tau PET and can be used as a prescreening in clinical trials to reduce participant burden and improve screening efficiency.

[0076] The specific embodiments disclosed herein are intended to illustrate some aspects of the present invention, and the invention described and claimed herein is not limited in scope by these embodiments. Any equivalent embodiments are intended to be within the scope of the present invention. Indeed, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

[0077] Sequence SEQ ID NO:1 - Tau protein 2N4R MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0078] SEQ ID NO:2 - PT3 mouse mAb HCDR1, Kabat numbering SYAMS

[0079] SEQ ID NO:3 - PT3 mouse mAb HCDR2, Kabat numbering SISKGGNTYYPNSVKG

[0080] Sequence number 4 - PT3 mouse mAb HCDR3, Kabat numbering GWGDYGWFAY

[0081] Sequence number 5 - PT3 mouse mAb LCDR1, Kabat numbering KASQDINRYLN

[0082] Sequence number 6 - PT3 mouse mAb LCDR2, Kabat numbering RANRLLD

[0083] Sequence number 7 - PT3 mouse mAb LCDR3, Kabat numbering LQYDEFPLT

[0084] Sequence number 8 - PT3 mouse mAb VH EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYAMSWVRQNPEKRLEWVASISKGGNTYYPNSVKGRFTISRDNARNILYLQMSSLRSEDTALYYCARGWGDYGWFAYWGQVTLVTVSA

[0085] Sequence number 9 - PT3 mouse mAb VL DIKMTQSPSSMYASLGERVTITCKASQDINRYLNWFQQKPGKSPKTLIYRANRLLDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCLQYDEFPLTFGDGTKLELK

[0086] Sequence number 10, HCDR1 of HT43 mAb GFTFRSYGMS

[0087] Sequence number 11, HCDR2 of HT43 mAb TINSDGSYTYYPDSVKG

[0088] Sequence number 12, HCDR3 of HT43 mAb SWDGAMDY

[0089] Sequence number 13, LCDR1 of HT43 mAb RSSQSILHSNGNTYFE

[0090] Sequence number 14, LCDR2 of HT43 mAb KVSNRFS

[0091] Sequence number 15, LCDR3 of HT43 mAb FQGSLVPWT

[0092] Sequence number 16, heavy chain variable region of HT43 mAb EVKLVESGGDLVKPGGSLKLSCAASGFTFRSYGMSWVRQTPDKRLEWVTTINSDGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCAVSWDGAMDYWGQGTSVTVSS

[0093] Sequence number 17, light chain variable region of HT43 mAb DVLVTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYFEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSLVPWTFGGGTKLEIK

Claims

1. A system for pre-screening human subjects for clinical trials for the treatment of tauopathy or amyloid-forming disorders, The system comprises one or more processors, and a non-temporary storage medium connected to the one or more processors and storing instruction sets executed by the one or more processors, wherein the instruction sets are executed by the one or more processors. To obtain data indicating the concentration of p217+ tau present in the plasma sample obtained from the aforementioned subject, When the concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold, the system outputs information indicating that the subject is eligible for further screening for the clinical trial. The minimum threshold is the amount of p217+ tau in plasma, and exceeding this threshold corresponds to the amount of p217+ tau in plasma that causes the subject to exhibit an accumulation of increased tau changes in the brain compared to patients with mild cognitive impairment (MCI) and cognitively normal patients. The system is such that the maximum threshold is the amount of p217+ tau present in the plasma, and exceeding this threshold results in the subject exhibiting a pathology of widespread accumulation of tau changes in multiple regions of the brain.

2. The system according to claim 1, wherein the minimum threshold is approximately 0.075 pg / ml to approximately 0.125 pg / ml.

3. The system according to claim 1, wherein the maximum threshold is approximately 0.225 pg / ml to approximately 0.275 pg / ml.

4. The system according to claim 1, wherein outputting the information includes outputting information indicating that the subject is eligible for further screening for the clinical trial when the concentration of the p217+ tau present in the plasma sample is ≥ 0.1 pg / ml and ≤ 0.25 pg / ml.

5. The data showing the concentration of p217 + tau mentioned above is The plasma sample is brought into contact with a capture antibody directed against the p217+ tau epitope, and the capture antibody is bound to the p217+ tau peptide in the plasma sample to form an antibody-peptide complex. The antibody-peptide complex is brought into contact with the detection antibody to bind the detection antibody to the antibody-peptide complex. The system according to claim 1, wherein the data is based on the amount of the p217+ tau peptide in the plasma sample, determined by detecting the detection antibody.

6. The system according to claim 5, wherein the capture antibody binds to an epitope containing amino acids 210 to 220 of human tau protein.

7. The system according to claim 5, wherein the detection antibody binds to an epitope containing amino acids 7-20 or 116-127 of human tau protein.

8. The system according to claim 6, wherein the capture antibody is pT3.

9. The system according to claim 7, wherein the detected antibody is hT43.

10. The system according to claim 1, wherein the concentration of p217+ tau is determined using an assay in which LLOQ is <0.04 pg / ml.

11. The aforementioned tauopathies include familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with chromosome 17-associated Parkinsonian syndrome (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle-dominant dementia, diffuse neurofibrillary tangle disease with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonian syndrome dementia complex, Down syndrome, and Gerstmann-Strouiss syndrome. The system according to claim 1, selected from the group consisting of Lar-Scheinker disease, Haller-Vorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid vascular disease, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guanian motor neuron disease due to neurofibrillary tangles, post-encephalitis parkinsonian syndrome, chronic traumatic encephalopathy, and boxer's dementia (boxer's disease).

12. The system according to claim 11, wherein the tauopathy is Alzheimer's disease.

13. A system for screening human subjects for clinical trials for the treatment of tauopathy or amyloid-forming disorders, The system comprises one or more processors, and a non-temporary storage medium connected to the one or more processors and storing instruction sets executed by the one or more processors, wherein the instruction sets are executed by the one or more processors. To obtain data indicating the concentration of p217+ tau present in the plasma sample obtained from the aforementioned subject, When the concentration of p217+ tau present in the plasma sample is above a minimum threshold and below a maximum threshold, PET data of the target brain generated using a tau-specific or amyloid-specific radiotracer is acquired. The PET data is analyzed to determine whether the subject is tau-positive or amyloid-positive, and whether the subject has extensive tau changes or extensive amyloid fibrils. (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, (vi) When the PET data indicates that the subject does not have extensive tau changes or extensive amyloid fibrils, the system outputs information indicating that the subject is suitable for inclusion in the clinical trial. The minimum threshold is the amount of p217+ tau in plasma, and exceeding this threshold corresponds to the amount of p217+ tau in plasma that causes the subject to exhibit an accumulation of increased tau changes in the brain compared to patients with mild cognitive impairment (MCI) and cognitively normal patients. The system is such that the maximum threshold is the amount of p217+ tau present in the plasma, and exceeding this threshold results in the subject exhibiting a pathology of widespread accumulation of tau changes in multiple regions of the brain.

14. The system according to claim 13, wherein the minimum threshold is approximately 0.075 pg / ml to approximately 0.125 pg / ml.

15. The system according to claim 13, wherein the maximum threshold is approximately 0.225 pg / ml to approximately 0.275 pg / ml.

16. The system according to claim 13, wherein outputting the aforementioned information includes outputting information indicating that the subject is suitable for inclusion in the clinical trial when the concentration of the p217+ tau present in the plasma sample is ≥ 0.1 pg / ml and ≤ 0.25 pg / ml.

17. The data showing the concentration of p217 + tau mentioned above is The plasma sample is brought into contact with a capture antibody directed against the p217+ tau epitope, and the capture antibody is bound to the p217+ tau peptide in the plasma sample to form an antibody-peptide complex. The antibody-peptide complex is brought into contact with the detection antibody to bind the detection antibody to the antibody-peptide complex. The system according to claim 13, wherein the data is based on the amount of the p217+ tau peptide in the plasma sample, determined by detecting the detection antibody.

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

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

20. The system according to claim 18, wherein the capture antibody is pT3.

21. The system according to claim 19, wherein the detection antibody is hT43.

22. The system according to claim 13, wherein the concentration of p217+ tau is determined using an assay in which LLOQ is <0.04 pg / ml.

23. The aforementioned tauopathies include familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia with chromosome 17-associated Parkinsonian syndrome (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle-dominant dementia, diffuse neurofibrillary tangle disease with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonian syndrome dementia complex, Down syndrome, and Gerstmann-Strouiss syndrome. The system according to claim 13, selected from the group consisting of Lar-Scheinker disease, Haller-Vorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid vascular disease, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guanian motor neuron disease due to neurofibrillary tangles, post-encephalitis parkinsonian syndrome, chronic traumatic encephalopathy, and boxer's dementia (boxer's disease).

24. The system according to claim 23, wherein the tauopathy is Alzheimer's disease.

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