Plasma assays for detecting cns-derived tau peptides

Abstract

Provided herein are methods and assays for detecting central nervous system (CNS)-derived tau peptides in blood-based samples from a subject involving the use of a capture antibody that binds to a tau epitope and a detection antibody that binds to an epitope comprising amino acid residues spanning the junction of exon 4 and exon 5 of CNS-derived tau.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 533,337, filed August 17, 2023, and U.S. Provisional Application No. 63 / 545,344, filed October 23, 2023, the entire contents of each of which are incorporated herein by reference.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically submitted in XML format, and is incorporated herein by reference in its entirety. The XML copy was created on August 6, 2024, named JAB7215WOPCT1_SL, and is 41,646 bytes in size. Background Technology

[0005] 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. In the United States, more than 5 million people have AD, and this number continues to grow as the population ages, as 10% of people over the age of 65 have AD, and it is the fifth leading cause of death in that population. It is estimated that more than 50 million people worldwide have AD or related dementia, further demonstrating that AD continues to be a major public health problem.

[0006] 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.

[0007] Neurofibrillary tangles are primarily composed of aggregates of hyperphosphorylated tau protein. The main physiological functions of tau are microtubule polymerization and stabilization. Tau binding to microtubules occurs through ionic interactions between the positive charge in the tau microtubule-binding region and the negative charge on the microtubule network (Butner and Kirschner 1991). Tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with tau's primary function. Tau bound to the axonal microtubule network is hypophosphorylated, while aggregated tau in AD is hyperphosphorylated, thus providing unique epitopes distinct from a repertoire of physiologically active tau (Iqbal et al., 2010).

[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., 2009; Clavaguera et al., 2009). It is believed that tau disease can spread from one brain region to the next in a prion-like manner. This spread process involves the externalization of tau seeds, which can be absorbed by nearby neurons and induce further tau disease.

[0009] The National Institute on Aging and Alzheimer's Association (NIA-AA) Research Framework provides a protocol for diagnosing AD based on measurements associated with underlying pathological processes, β-amyloid (A), pathological tau (T), and neurodegeneration (N), termed "AT(N)" (Jack, Jr. et al., 2018). While plasma amyloid-β and phosphorylated tau (p-tau) have shown promise as reliable biomarkers for A and T, respectively, within the AT(N) framework (Nakamura et al., 2018; Schindler et al., 2019; Palmqvist et al., 2020; Thijssen et al., 2021), reliable biomarkers for N remain elusive. For example, plasma neurofilament light chains (NFL) have been shown to be effective in identifying Alzheimer's disease compared to controls, but they cannot distinguish AD from other neurodegenerative diseases (Ashton et al., 2021; Bridel et al., 2019).

[0010] Total tau (t-tau) has shown promise as a biomarker for neurodegeneration. T-tau from CSF has been shown to reliably reflect neurodegeneration in Alzheimer's disease, but not in other neurodegenerative diseases such as Parkinson's disease, Lewy body dementia, and frontotemporal dementia (Grothe et al., 2021; Sjögren et al., 2001). However, the use of CSF in diagnosis can be challenging because CSF retrieval requires an invasive lumbar puncture procedure involving a physician inserting a needle into the spinal canal to collect a CSF sample for measurement; this procedure is uncomfortable and cumbersome, and therefore not desirable for frequent repetition and is not suitable for regular monitoring of a patient's disease status. Furthermore, t-tau has not yet demonstrated good diagnostic utility in plasma assays (Barthélemy et al., 2020; Frank et al., 2022) because plasma t-tau concentrations are not correlated with CSF t-tau, and plasma assays may detect tau from tissues outside the central nervous system (CNS) (Barthélemy et al., 2020; Dugger et al., 2016). Therefore, while t-tau may have the potential to serve as a biomarker for neurodegeneration, there is currently no practical and reliable t-tau assay. Summary of the Invention

[0011] Some key aspects of the present invention are summarized below. Further aspects are described in the detailed description, embodiments, drawings, and claims of this disclosure. The descriptions in each part of this disclosure are intended to be read in conjunction with the other parts. Furthermore, the various embodiments described in each part of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the invention.

[0012] In one aspect, this article provides an assay method for detecting CNS-derived tau peptides in a blood-based sample from a subject, the method comprising: (a) contacting the blood-based sample with a capture antibody that binds to a tau epitope to capture tau peptides in the blood-based sample; and (b) contacting the captured tau peptides with a detection antibody that binds to an epitope comprising an amino acid residue comprising the linker of exon 4 and exon 5 of CNS-derived tau.

[0013] On the other hand, this article provides a method for measuring total central nervous system (CNS)-derived tau (t-tau) peptides in a blood-based sample from a subject, the method comprising: (a) contacting the blood-based sample with a capture antibody that binds to a tau epitope to capture tau peptides in the blood-based sample; (b) contacting the captured tau peptides with a detection antibody that binds to an epitope comprising an amino acid residue comprising the link between exon 4 and exon 5 of CNS-derived tau; and (c) detecting the detection antibody to determine the amount of CNS-derived t-tau peptides in the blood-based sample.

[0014] In some implementations, the solid phase is a magnetic bead.

[0015] In some implementations, the blood-based sample is a plasma sample.

[0016] In some embodiments, the method further includes obtaining a sample from a subject. In other embodiments, the method further includes washing the captured tau peptide before contacting it with a detection antibody. The captured tau peptide may be washed, for example, with a stringent buffer.

[0017] In some embodiments, the concentration of tau or t-tau detected in a blood-based sample is correlated with the concentration of p217+tau in a blood-to-blood sample. In some embodiments, the concentration of tau or t-tau detected in a blood-based sample is correlated with the concentration of NFL in a blood-to-blood sample.

[0018] On the other hand, this article provides a method for detecting amyloid status in a subject, the method comprising: (a) contacting a blood-based sample from the subject with a capture antibody that binds to a tau epitope to capture tau peptides in the blood-based sample; (b) contacting the captured tau peptides with a detection antibody that binds to an epitope comprising an amino acid residue comprising the link between exon 4 and exon 5 of CNS-derived tau; and (c) detecting the detection antibody to determine the amount of CNS-derived t-tau peptides in the blood-based sample. If the amount of CNS-derived t-tau peptides in the blood-based sample is above a predetermined threshold, the amyloid status is positive, and if the amount of CNS-derived t-tau peptides in the blood-based sample is below a predetermined threshold, the amyloid status is negative.

[0019] In another aspect, this article provides a kit comprising: (i) a capture antibody that binds to a tau epitope; and (ii) a detection antibody that binds to an epitope comprising an amino acid residue at the junction of exon 4 and exon 5 of central nervous system (CNS)-derived tau.

[0020] In some embodiments, the capture antibody binds to epitopes comprising amino acid residues 7-20 of human tau protein, wherein the amino acid residues are numbered according to the amino acid sequence of SEQ ID NO:1. In some embodiments, the capture antibody comprises heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO:5, 6, and 7, respectively, and light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO:8, 9, and 10, respectively. In some embodiments, the capture antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:11 and a light chain variable region containing the amino acid sequence of SEQ ID NO:12. In a preferred embodiment, the capture antibody is hT43.

[0021] In some embodiments, the detection antibody binds to epitopes comprising amino acid residues 116-127 of human tau protein, wherein the amino acid residues are numbered according to the amino acid sequence of SEQ ID NO:1. In some embodiments, the detection antibody comprises HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively, and LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively. In some embodiments, the detection antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:19 and a light chain variable region containing the amino acid sequence of SEQ ID NO:20. In a preferred embodiment, the detection antibody is pT82.

[0022] In other embodiments, the detection antibody binds to epitopes containing amino acid residues 119-126 of human tau protein, wherein the amino acid residues are numbered according to the amino acid sequence of SEQ ID NO:1. In some embodiments, the detection antibody comprises (i) HCDR1 containing the amino acid sequence of SEQ ID NO:21, HCDR2 containing the amino acid sequence of SEQ ID NO:22, HCDR3 containing the amino acid sequence of SEQ ID NO:23, LCDR1 containing the amino acid sequence of SEQ ID NO:24, LCDR2 containing the amino acid sequence of SEQ ID NO:25, and LCDR3 containing the amino acid sequence of SEQ ID NO:26; or (ii) HCDR1 containing the amino acid sequence of SEQ ID NO:27, HCDR2 containing the amino acid sequence of SEQ ID NO:28, HCDR3 containing the amino acid sequence of SEQ ID NO:29, LCDR1 containing the amino acid sequence of SEQ ID NO:24, LCDR2 containing the amino acid sequence of SEQ ID NO:25, and LCDR3 containing the amino acid sequence of SEQ ID NO:26; or (iii) HCDR1 containing the amino acid sequence of SEQ ID NO:30, HCDR2 containing the amino acid sequence of SEQ ID NO:31, HCDR3 containing the amino acid sequence of SEQ ID NO:32, and LCDR3 containing the amino acid sequence of SEQ ID NO:26. LCDR1 containing the amino acid sequence NO:33, LCDR2 containing the amino acid sequence LVS, and LCDR3 containing the amino acid sequence SEQ ID NO:26; or (iv) HCDR1 containing the amino acid sequence SEQ ID NO:35, HCDR2 containing the amino acid sequence SEQ ID NO:36, HCDR3 containing the amino acid sequence SEQ ID NO:23, LCDR1 containing the amino acid sequence SEQ ID NO:24, LCDR2 containing the amino acid sequence SEQ ID NO:25, and LCDR3 containing the amino acid sequence SEQ ID NO:26; or (v) HCDR1 containing the amino acid sequence SEQ ID NO:37, HCDR2 containing the amino acid sequence SEQ ID NO:38, HCDR3 containing the amino acid sequence SEQ ID NO:39, LCDR1 containing the amino acid sequence SEQ ID NO:40, LCDR2 containing the amino acid sequence SEQ ID NO:41, and LCDR3 containing the amino acid sequence SEQ ID NO:42. In some embodiments, the detection antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 43 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 44. In a preferred embodiment, the detection antibody is hT36.

[0023] On another aspect, this document provides an antibody or antigen-binding fragment thereof comprising (i) HCDR1 containing the amino acid sequence of SEQ ID NO:21, HCDR2 containing the amino acid sequence of SEQ ID NO:22, HCDR3 containing the amino acid sequence of SEQ ID NO:23, LCDR1 containing the amino acid sequence of SEQ ID NO:24, LCDR2 containing the amino acid sequence of SEQ ID NO:25, and LCDR3 containing the amino acid sequence of SEQ ID NO:26; or (ii) HCDR1 containing the amino acid sequence of SEQ ID NO:27, HCDR2 containing the amino acid sequence of SEQ ID NO:28, HCDR3 containing the amino acid sequence of SEQ ID NO:29, LCDR1 containing the amino acid sequence of SEQ ID NO:24, LCDR2 containing the amino acid sequence of SEQ ID NO:25, and LCDR3 containing the amino acid sequence of SEQ ID NO:26; or (iii) HCDR1 containing the amino acid sequence of SEQ ID NO:30, HCDR2 containing the amino acid sequence of SEQ ID NO:31, and LCDR3 containing the amino acid sequence of SEQ ID NO:26. HCDR3 containing the amino acid sequence of SEQ ID NO:32, LCDR1 containing the amino acid sequence of SEQ ID NO:33, LCDR2 containing the amino acid sequence LVS, and LCDR3 containing the amino acid sequence of SEQ ID NO:26; or (iv) HCDR1 containing the amino acid sequence of SEQ ID NO:35, HCDR2 containing the amino acid sequence of SEQ ID NO:36, HCDR3 containing the amino acid sequence of SEQ ID NO:23, LCDR1 containing the amino acid sequence of SEQ ID NO:24, LCDR2 containing the amino acid sequence of SEQ ID NO:25, and LCDR3 containing the amino acid sequence of SEQ ID NO:26; or (v) HCDR1 containing the amino acid sequence of SEQ ID NO:37, HCDR2 containing the amino acid sequence of SEQ ID NO:38, HCDR3 containing the amino acid sequence of SEQ ID NO:39, LCDR1 containing the amino acid sequence of SEQ ID NO:40, LCDR2 containing the amino acid sequence of SEQ ID NO:41, and LCDR3 containing the amino acid sequence of SEQ ID NO:42. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain region containing the amino acid sequence of SEQ ID NO: 43 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 44.This document also provides an antibody or antigen-binding fragment thereof comprising (i) HCDR1, HCDR2, and HCDR3 contained in the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:43, and (ii) LCDR1, LCDR2, and LCDR3 contained in the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:44. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO:45 and a light chain containing the amino acid sequence of SEQ ID NO:46. Attached Figure Description

[0024] Figure 1 Dilution linearity plots of hT43xpT82 and pT82xhT43 sandwich enzyme-linked immunosorbent assays (ELISA) as described in Example 1 are provided.

[0025] Figure 2 The standard curve (Figure A), dilution linearity (Figure B), and recovery rate (Figure C) of the hT43xpT82 sandwich ELISA using the three-step protocol as described in Example 1 are provided.

[0026] Figure 3 A schematic diagram of the structure of peptide 1 as described in Example 2 (Figure A) is provided, along with surface plasmon resonance (SPR) changes of antibodies hT36 (Figure B) and pT82 (Figure C) as measurements of antibody-peptide 1 binding.

[0027] Figure 4 A schematic diagram of the structure of peptide 2 as described in Example 2 (Figure A) is provided, along with surface plasmon resonance (SPR) changes of antibodies hT36 (Figure B) and pT82 (Figure C) as measurements of antibody-peptide 2 binding.

[0028] Figure 5 A schematic diagram of the structure of peptide 3 as described in Example 2 (Figure A) is provided, along with surface plasmon resonance (SPR) changes of antibodies hT36 (Figure B) and pT82 (Figure C) as measurements of antibody-peptide 3 binding.

[0029] Figure 6 The graphs showing the p217+tau concentration versus plasma CNS-derived t-tau concentration in samples from cohort 1 (Fig. A) and cohort 2 (Fig. B) as described in Example 3, and the graphs showing the NFL concentration versus plasma CNS-derived t-tau concentration in samples from cohort 1 (Fig. C) and cohort 2 (Fig. D) are shown.

[0030] Figure 7The figure shows the plasma CNS-derived t-tau concentrations in samples from the Longitudinal Cohort Study of Novel Alzheimer's Disease Therapeutics (NTAD) in cohort 1 (Figure A) and cohort 2 (Figure B) as described in Example 3, in amyloid-negative (A-), NTAD amyloid-positive (A+), and healthy control (HC) samples. Detailed Implementation

[0031] While some key embodiments of the invention have been described in the Summary of the Invention, Embodiments and Claims sections of this patent application, this Detailed Description section provides certain additional descriptions in relation to the invention and is intended to be read in conjunction with all other parts of this patent application.

[0032] To facilitate a better understanding of the invention, certain terms are first defined. Further definitions are set forth throughout this disclosure. 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 related to this invention.

[0033] Any headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be obtained by referring to the specification as a whole. Therefore, the terms defined thereafter are defined more fully by referring to the entire specification.

[0034] All references cited in this disclosure are incorporated herein by reference in their entirety. Furthermore, any manufacturer's specifications or catalogues of any products referenced or mentioned herein are incorporated by reference. Documents incorporated herein by reference, or any teachings therein, may be used in the practice of this invention. Documents incorporated herein by reference are not acknowledged as prior art.

[0035] definition

[0036] The wording or terminology used in this disclosure is for descriptive purposes and not for limitation, and the terminology or terminology in this specification should be interpreted by those skilled in the art based on the teachings and instructions.

[0037] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural references. The term “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably.

[0038] As used herein, the terms “comprising” and “including” are used interchangeably. The terms “comprising” and “including” should be construed as specifying the presence of the mentioned feature or component, but do not exclude the presence or addition of one or more features, components, or combinations thereof. Furthermore, the terms “comprising” and “including” are intended to include examples covered by the term “consisting of”. Therefore, the term “consisting of” can be used instead of the terms “comprising” and “including” to provide more specific embodiments.

[0039] As used herein, the term "or" should be interpreted as inclusive "or," meaning any one or any combination thereof. Therefore, "A, B, or C" means any of the following: A; B; C; A and B; A and C; B and C; A, B, and C. Exceptions to this definition will only occur if the combination of elements, functions, steps, or actions is inherently mutually exclusive in some way.

[0040] Furthermore, "and / or" is considered to be a specific disclosure of each of two specified features or components with or without the other. Therefore, the term "and / or," as used in phrases such as "A and / or B," is intended to include A and B, A or B, A (alone) and B (alone). Similarly, the term "and / or," as used in phrases such as "A, B, and / or C," is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0041] Units, prefixes, and symbols are represented in their International System of Units (SI) accepted form. A numerical range includes the number that defines the range, and any single value provided herein can be used as an endpoint of a range that includes other single values ​​provided herein. For example, a set of values ​​such as 1, 2, 3, 8, 9, and 10 is also a disclosure of numerical ranges from 1 to 10, from 1 to 8, from 3 to 9, etc. Similarly, a disclosed range is a disclosure of each individual value (i.e., intermediate value) covered by that range, including integers and fractions. For example, the specified range of 5 to 10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, as well as 5.2, 7.5, 8.7, etc., individually.

[0042] Unless otherwise stated, the terms "at least" or "about" preceding a series of elements should be understood to refer to each element in the series. The term "about" preceding a numerical value includes ±10% of the stated value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of about 1% (w / v) to 10% (w / v) includes 0.9% (w / v) to 11% (w / v).

[0043] Amino acids are represented in this paper by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides are represented by their generally accepted single-letter codes.

[0044] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site within the variable region of an immunoglobulin molecule. The terms "antibody" or "immunoglobulin" are used interchangeably herein.

[0045] A typical antibody consists of at least two heavy chains and two light chains linked by disulfide bonds. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region and a light chain constant region (CL). The light chain constant region consists of one domain, Cl. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0046] Antibodies can belong to any of the five major classes of immunoglobulins—IgA, IgD, IgE, IgG, and IgM—or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2)—based on the identity of their heavy chain constant domains (designated α, δ, ε, γ, and μ, respectively). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Two classes of mammalian light chains exist: λ and κ.

[0047] The heavy chain variable region and light chain variable region can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework (FW) regions. The CDRs in each chain bind tightly to the CDRs of the other chain via the FW regions and contribute to the formation of antigen-binding sites for antibodies. Each heavy chain variable region and light chain variable region consists of three CDRs: CDR1, CDR2, and CDR3.

[0048] At least two techniques exist for determining CDRs: (1) methods based on cross-species sequence variability (Kabat et al., 1991); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997). Furthermore, combinations of these two methods are sometimes used in the field to determine CDRs.

[0049] The amino acid position numbering in Kabat refers to a numbering system used for variable domains in the heavy or light chain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortened or inserted FW or CDRs of the variable domain. For example, a variable domain in the heavy chain may include a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and inserted residues after FW residue 82 of the heavy chain (e.g., residues 82a, 82b, and 82c, etc., according to Kabat).

[0050] The Kabat residue number of a given antibody can be determined by comparing the homologous region of the antibody sequence with the "standard" Kabat numbering sequence. Chothia refers to the location of the structural loop (Chothia & Lesk, 1987). When using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme inserts at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the AbM antibody modeling software from Oxford Molecular.

[0051] IMGT (ImMunoGeneTics) also provides a numbering system for variable regions, including CDRs (see, for example, Lefranc et al., 2003). The IMGT numbering system is based on alignments, structural data, and characterization of hypervariable loops from over 5,000 sequences and allows for easy comparison of variable regions and CDRs across all species. According to the IMGT numbering scheme, heavy chain variable region CDR-1 is located at positions 26–35, heavy chain variable region CDR-2 at positions 51–57, heavy chain variable region CDR-3 at positions 93–102, light chain variable region CDR-1 at positions 27–32, light chain variable region CDR-2 at positions 50–52, and light chain variable region CDR-3 at positions 89–97.

[0052] Furthermore, variable regions can be partitioned based on the "Specific Determining Residue Use" (SDRU) (Almagro 2004), where SDRs refer to amino acid residues of immunoglobulins that are directly involved in antigen contact. This SDRU concept was used to develop a "contact" approach to define CDRs, which renamed SDRs as "contact residues" (MacCallum et al., 1996).

[0053] As used herein, the term “antibody” encompasses polyclonal antibodies; monoclonal antibodies; multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies; humanized antibodies; human antibodies; chimeric antibodies; fusion proteins containing the antigenic determinant portion of an antibody; and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity.

[0054] A monoclonal antibody (mAb) is a homogeneous group of antibodies that participate in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically consist of different antibodies targeting different antigenic determinants. The term "monoclonal" can be applied to both full-length and complete monoclonal antibodies, and also to antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to any number of such antibodies prepared in any manner, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0055] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that has been engineered to contain a minimal amount of non-human (e.g., mouse) sequence. Typically, a humanized antibody is a human immunoglobulin in which residues from the CDR (corresponding receptor) are replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that possess the desired specificity, affinity, and capability (Jones et al., 1986; Riechmann et al., 1998; Verhoeyen et al., 1988). In some cases, the Fv and FW residues of the human immunoglobulin are replaced with corresponding residues from an antibody derived from a non-human species that possess the desired specificity, affinity, and capability.

[0056] Humanized antibodies can be further modified by substituting additional residues in the Fv framework region and / or within the replaced non-human residues to improve and optimize antibody specificity, affinity, and / or potency. Generally, humanized antibodies will contain at least one and typically two or three variable domains containing all or substantially all of the CDR regions corresponding to non-human immunoglobulins, and all or substantially all of the FR regions being those FR regions of the common sequence of human immunoglobulins. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region or domain (Fc), typically at least a portion of the constant region or domain of human immunoglobulins. Examples of methods for generating humanized antibodies are described in U.S. Patent Nos. 5,225,539 and 5,639,641.

[0057] The term "human antibody" refers to an antibody produced by a human being or an antibody having an amino acid sequence corresponding to a human-produced antibody prepared using any technique known in the art. The definition of a human antibody includes full-length or complete antibodies comprising at least one human heavy chain and / or light chain polypeptide, such as antibodies comprising mouse light chain and human heavy chain polypeptides.

[0058] The term "chimeric antibody" refers to an antibody whose amino acid sequence of an immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are sequence-homogeneous with those in an antibody derived from another mammalian species (usually human) to avoid triggering an immune response in that species.

[0059] The term "antigen-binding fragment" refers to the portion of a complete antibody that contains the antibody's complementarity-determining variable region. Examples of antibody fragments that can constitute an "antigen-binding fragment" include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single-chain antibodies (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0060] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K). D ( ) indicates affinity. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies typically bind to antigens more quickly and tend to maintain the binding for longer.

[0061] The affinity or cohesion of an antibody to an antigen can be determined using any suitable method (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)) or kinetics (e.g., KINEXA). ® Or BIACORE ™ Or OCTET ® (Analysis) is determined experimentally. Direct binding assays and competitive binding assays can be readily employed (see, for example, Berzofsky et al., 1984; Kuby, 1992). The affinity of a specific antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen-binding parameters (e.g., K0) will affect the measurement. D or Kd K on K off The measurement results were obtained using standard solutions of antibodies and antigens, as well as standard buffer solutions (as known in the art).

[0062] 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 by 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, 1996).

[0063] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject for diagnosis, prognosis, or treatment, particularly mammalian subjects. Mammal subjects include humans, livestock, farm animals, sporting animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, etc.

[0064] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also covers amino acid polymers that have been naturally modified or modified by intervention; natural or interventional modifications include, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. In some embodiments, the polypeptide may exist as a single chain or an associated chain.

[0065] "Isolated" peptides, antibodies, or antigen-binding fragments are those that do not exist in nature. Isolated peptides, antibodies, or antigen-binding fragments include those that have been purified to the point that they are no longer in the form found in nature. In some embodiments, the isolated peptides, antibodies, or antigen-binding fragments are substantially pure. When used herein, the term "substantially pure" means a purity greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0066] As used herein, the term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," means any polynucleotide or polydeoxynucleotide that may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in various ways; therefore, "polynucleotides" include chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides that are normally found naturally, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often referred to as oligonucleotides.

[0067] As used in this article, the term "vector" is a replicon in which another nucleic acid segment can be operatively inserted to induce replication or expression of that segment.

[0068] As used herein, the term "host cell" refers to a cell containing the nucleic acid molecules of the present invention. A "host cell" can be any type of cell, such as a primary cell, a cultured cell, or a cell derived from a cell line. In one embodiment, a "host cell" is a cell transfected with the nucleic acid molecules of the present invention. In another embodiment, a "host cell" is a progeny or potential progeny of such transfected cells. The progeny of a cell may or may not be identical to the parent cell, for example, due to mutations or environmental influences that may occur in the progeny, or due to the integration of the nucleic acid molecules into the host cell genome.

[0069] As used herein, the term "expression" refers to the biosynthesis of a gene product. This term encompasses transcription from a gene to RNA. It also encompasses translation from RNA to one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The detection antibody or its antigen-binding fragment that binds to expressed tau may be located in the cytoplasm of the host cell, in an extracellular environment such as a growth medium for cell cultures, or anchored to the cell membrane.

[0070] As used herein, the term “tau” or “tau protein” or “tau peptide” refers to a diverse array 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., 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.

[0071] As used herein, the term "central nervous system-derived tau" or "CNS-derived tau" refers to tau that is produced or generated in the CNS. Examples of CNS-derived tau are tau present in the CSF or tau found in the brain. CNS-derived tau contrasts with tau that is not in the CNS (e.g., tau produced by the peripheral nerves or present in other tissues such as the liver, kidneys, or heart).

[0072] As used herein, the terms “total tau” or “t-tau” refer to multiple tau species that can be in any phosphorylation state; for example, multiple tau species may include non-phosphorylated tau species, phosphorylated tau species, or both non-phosphorylated and phosphorylated tau species. Measurements of t-tau in a sample refer to measurements of tau species unaffected by phosphorylation state. A “phosphorylated tau species” refers to a tau protein that is phosphorylated at at least one site. The phosphorylation site can be any site described in Hanger et al. (2009).

[0073] Unless otherwise specified, as used herein, the amino acid numbers in tau protein or fragments thereof are referenced to the amino acid sequence shown in SEQ ID NO: 1.

[0074] 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 beads or paramagnetic beads.

[0075] 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 the tau protein.

[0076] As used in this article, a “sandwich ELISA” refers to a type of ELISA involving two antigen-binding molecules (usually antibodies) that target different epitopes of an antigen. Typically, one of the antigen-binding molecules is attached to a solid-phase carrier and used to “capture” the antigen, i.e., to facilitate antigen immobilization. The other antigen-binding molecule is conjugated and facilitates the detection of the antigen.

[0077] 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 with 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-Guan Island motor neuron disease with neurofibrillary tangles, post-encephalitis Parkinson's syndrome, and chronic traumatic encephalopathy such as boxing dementia (boxing disease) (Morris et al., 2011).

[0078] As used herein, the term "amyloid status" refers to whether a subject is amyloid-positive or amyloid-negative. Amyloid status can be determined by methods known in the art, such as by measuring CSF Aβ42 levels or by positron emission tomography (PET) scans of amyloid; or by the methods of the present invention described herein.

[0079] As used in this article, "amyloid-positive" or "amyloid-positive" refers to the accumulation of amyloid-β in the brain associated with symptoms of Alzheimer's disease or other amyloid-related conditions. As used in this article, "amyloid-negative" or "amyloid-negative" refers to the absence of amyloid-β accumulation in the brain or extremely low levels of amyloid-β accumulation that are insufficient to trigger symptoms of Alzheimer's disease or other amyloid-related conditions.

[0080] 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.

[0081] As used herein, the term “diagnosis” means the detection of a disease or disorder or the determination of the stage or extent of a disease or disorder (such as tau protein lesions). 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., 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-tau antibody therapy) or predicting a pattern of response to a drug therapy (e.g., anti-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).

[0082] As used herein, the terms “increase” and “decrease” refer to a difference in quantity compared to a control or reference level, such as a difference in the quantity of a specific biomarker in a sample. For example, the quantity 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 about 1.0, about 1.5, about 2.0, or about 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 the administration of the treatment agent or at an earlier time point during the treatment regimen).

[0083] Other terms are defined elsewhere in this patent disclosure or used in accordance with their common meaning in the art.

[0084] Determination and Methods

[0085] This application provides a assay and method for detecting and measuring CNS-derived tau peptides in blood-based samples, particularly plasma. Compared to lumbar puncture for collecting CSF, blood sample collection is rapid and easy to perform, and offers a reduced risk of infection or other complications.

[0086] The assay and method of this invention are based in part on the use of a detection antibody that binds to CNS-derived tau peptides and will not bind to non-CNS-derived tau peptides (e.g., tau peptides produced by peripheral nerves or from tissues such as the liver, kidney, or heart). The dominant form of tau in peripheral tissues can be distinguished from CNS-derived tau by the presence of an extra exon, exon 4a, between exon 4 and exon 5 in the MAPT gene (Gonzalez-Ortiz et al., 2023; Couchie et al., 1992; Georgieff et al., 1991). In the MAPT gene for CNS-derived tau, exon 4 and exon 5 are adjacent. Therefore, a detection protein that at least partially binds to the amino acid residues at the junction of exon 4 and exon 5 will target CNS-derived tau peptides and will not target tau peptides from other tissues.

[0087] The assays and methods of this application measure CNS-derived t-tau peptides in blood-based samples with sufficient sensitivity, precision, and accuracy. Therefore, compared to CSF-based assays, this application provides an improved method for measuring and / or monitoring CNS-derived t-tau peptide levels in subjects, minimizing the burden of sample collection and thereby enabling more frequent measurement and monitoring of changes in CNS-derived t-tau peptide levels, which is particularly desirable for monitoring and evaluating response to treatment. The samples used in the assays and methods of this application can be blood, serum, or plasma samples. Preferably, the sample is a plasma sample. More preferably, the plasma sample has not been immunoprecipitated to concentrate the CNS-derived tau peptides contained therein. In a particular embodiment, the sample is a crude plasma sample.

[0088] The assay and method portion of this application relates to measuring CNS-derived t-tau peptides in blood-based samples by using a capture antibody that binds to tau peptides in the sample to form a capture antibody-tau peptide complex. Preferably, the capture antibody is immobilized on a solid phase such that it selectively binds to and immobilizes tau peptides present in the sample to the solid phase. The captured tau peptide (i.e., the capture antibody-tau peptide complex) is then contacted with a detection antibody labeled with a reporter element that allows the detection of captured CNS-derived tau species. The assays and methods described herein can be used for a variety of diagnostic purposes, such as diagnosing AD, other tau diseases, other diseases characterized by amyloid-β deposition, or other amyloid-forming disorders in subjects; monitoring the effectiveness of treatment; identifying subjects suitable for anti-tau therapy; pre-screening subjects for PET imaging and / or CSF assays for further detection of AD, other tau diseases, other diseases characterized by amyloid-β deposition, or other amyloid-forming disorders; identifying subjects for enrollment in clinical trials associated with AD, other tau diseases, other diseases characterized by amyloid-β deposition, or other amyloid-forming disorders; and so on.

[0089] In some embodiments, the assay and method of this application include the following steps: contacting a blood-based sample with a capture antibody targeting a tau epitope, such that the capture antibody binds to tau peptides in the sample and forms a capture antibody-tau peptide complex. The capture antibody-tau peptide complex can then be contacted with a detection antibody to bind the capture antibody-tau peptide complex containing CNS-derived tau peptides. The detection antibody can then be detected to determine the amount of CNS-derived tau peptides in the sample.

[0090] In some embodiments, the capture antibody-tau peptide complex can be washed before contact with the detection antibody. For example, the capture antibody-tau peptide complex can be washed with any suitable solution that does not interfere with the assay, such as a buffer solution (e.g., phosphate-buffered saline (PBS) solution). In some embodiments, the buffer is capable of reducing and / or eliminating significant matrix interference from blood or plasma. In a preferred embodiment, the buffer is a harsh buffer.

[0091] As described herein (see the Background section below), historically, plasma t-tau measurements have performed poorly as a diagnostic tool for Alzheimer's disease. In contrast, the assay and method of this application surprisingly rely on t-tau as a diagnostic agent, measuring CNS-derived t-tau by means of at least part of a capture antibody that recognizes an amino acid sequence absent in peripherally derived tau. Therefore, this application provides an improved assay and method that enhances the use of t-tau as a diagnostic agent.

[0092] Therefore, in one aspect, the assay and method of the present invention measures CNS-derived t-tau peptide from a blood-based sample of a subject, and subsequently determines that the subject has tau disease and / or amyloidosis or is at risk of developing tau disease and / or amyloidosis when the amount of CNS-derived t-tau peptide measured from the blood-based sample is greater than a predetermined threshold. The predetermined threshold can be any suitable threshold used to distinguish those subjects who have tau disease and / or amyloidosis or are at risk of developing tau disease and / or amyloidosis from those subjects who are healthy and not at risk of developing tau disease and / or amyloidosis. The predetermined threshold can be determined as the plasma CNS-derived t-tau peptide concentration, which is used to: distinguish patients with tau levels greater than or less than those measured by PET imaging in the brain or brain region; distinguish patients with tau levels greater than or less than those in the CSF (e.g., phosphorylated tau, such as p181 or p217+tau); distinguish patients with amyloid-β levels greater than or less than those in the CSF or plasma (e.g., Aβ40 or Aβ42); distinguish patients with Aβ42 to Aβ40 ratio greater than or less than that ratio in the CSF or plasma; and distinguish patients with normal cognition from those with dementia.

[0093] Subjects identified as having tau disease and / or amyloidosis, or at risk of developing tau disease and / or amyloidosis, may be directed to undergo further clinical testing, such as CSF collection and / or PET imaging, to further evaluate their brain pathology. In other embodiments, subjects identified as having tau disease and / or amyloidosis, or at risk of developing tau disease and / or amyloidosis, may be administered an active agent for the treatment of cognitive decline or tau disease and / or amyloidosis (e.g., AD). Active agents for the treatment of tau disease may include anti-tau antibodies, anti-p217+tau antibodies, small interfering RNA (siRNA) targeting human tau, siRNA targeting p217+tau, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, etc. Active agents targeting amyloidosis may include anti-amyloid antibodies, β-secretase inhibitors, γ-secretase inhibitors, small interfering RNA (siRNA) targeting human β-amyloid, cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) antagonists, etc.

[0094] In some implementations, the predetermined threshold may correspond to a baseline value or a value significantly higher than the baseline value. As used herein, "significantly higher" means a higher value that is statistically significant and not merely due to chance, having a p-value of 0.05 or less. In cases where the p-value is less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc., "significantly higher" can be at least about 1%, 2%, 5%, or 10% higher than that present in healthy volunteers. The baseline value may correspond to the average level in a population of healthy individuals. The baseline value may also correspond to the average of previous levels measured in the same subject.

[0095] According to embodiments of this application, values ​​associated with CNS-derived t-tau peptides in a sample, such as the amount of CNS-derived t-tau peptides, can be used for one or more diagnostic purposes. In some embodiments, if the amount of CNS-derived t-tau peptides is significantly higher than the corresponding baseline amount of CNS-derived t-tau peptides, the subject is determined to have tau proteinopathy. In some embodiments, if the ratio associated with CNS-derived t-tau peptides is significantly higher than the corresponding baseline ratio, the subject is determined to have tau proteinopathy.

[0096] In one aspect, the method of the present invention includes: (i) contacting a blood-based sample (preferably a plasma sample) with a capture antibody targeting a tau epitope to capture tau peptides in the sample; (ii) contacting the captured tau peptides with a detection antibody targeting an epitope containing an amino acid residue at the junction of exon 4 and exon 5 of CNS-derived tau; (iii) detecting the detection antibody to determine the amount of CNS-derived t-tau peptides in the sample; and (iv) determining whether a subject has tau proteinopathy or is at risk of developing tau proteinopathy based on the amount of CNS-derived t-tau peptides or a ratio associated with the amount of CNS-derived t-tau peptides. Diagnosis can be performed by comparing the amount or concentration of CNS-derived t-tau peptides in a sample from a subject with a corresponding predetermined threshold level. Diagnosis can also be performed by comparing a ratio associated with the amount of CNS-derived t-tau peptides with a corresponding baseline ratio.

[0097] On the other hand, the effectiveness of treatment in subjects can be determined by monitoring the amount of CNS-derived t-tau peptide or the ratio associated with the amount of CNS-derived t-tau peptide before, during, or after treatment. A decrease in values ​​relative to baseline signals a positive response to treatment. Values ​​can also temporarily increase in biological fluids when the half-life of pathological tau in circulation increases and / or when pathological tau is cleared from the brain.

[0098] According to some implementation schemes, tau protein diseases include, but are not limited to, one or more of the following groups: Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), frontotemporal dementia associated with chromosome 17 with Parkinson's syndrome (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 (ALS) Parkinson's syndrome-dementia complex, Down syndrome, Gersh-Schwarz disease, Hallewarden-Schwarz 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, chronic traumatic encephalopathy, and boxing dementia (boxing disease).

[0099] Preferably, the tau protein disease is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease), FTDP-17, or progressive supranuclear palsy.

[0100] Most preferably, tau protein disease is Alzheimer's disease (including familial Alzheimer's disease and sporadic Alzheimer's disease).

[0101] According to another aspect, the method of the present invention includes: (i) contacting a blood-based sample (preferably a plasma sample) with a capture antibody targeting a tau epitope to capture tau peptides in the sample; (ii) contacting the captured tau peptides with a detection antibody targeting an epitope containing an amino acid residue at the junction of exon 4 and exon 5 of CNS-derived tau; (iii) detecting the detection antibody to determine the amount of CNS-derived t-tau peptides in the sample; and (iv) determining whether a subject is suitable for anti-tau antibody therapy based on the amount of CNS-derived t-tau peptides or a ratio associated with CNS-derived t-tau peptides.

[0102] According to certain implementation schemes, a subject is deemed suitable for anti-tau antibody therapy if the amount of CNS-derived t-tau peptide or the ratio associated with CNS-derived t-tau peptide in a blood-based sample (especially a plasma sample) is significantly higher than the corresponding baseline value.

[0103] In one aspect, the method of the present invention includes: (i) contacting a blood-based sample (preferably a plasma sample) with a capture antibody targeting a tau epitope to capture tau peptides in the sample; (ii) contacting the captured tau peptides with a detection antibody targeting an epitope containing an amino acid residue at the junction of exon 4 and exon 5 of CNS-derived tau; (iii) detecting the detection antibody to determine the amount of CNS-derived t-tau peptides in the sample; and (iv) determining, based on the amount of CNS-derived t-tau peptides, whether the subject has amyloidosis or is at risk of developing amyloidosis. Diagnosis can be performed by comparing the amount or concentration of CNS-derived t-tau peptides in a sample from the subject with a corresponding predetermined threshold level.

[0104] This invention also relates to the measurement of antibody-conjugated CNS-derived tau peptides and non-antibody-bound free CNS-derived tau peptides in blood-based samples (particularly plasma). In some embodiments, total antibodies are captured using affinity techniques, followed by denaturing conditions including dissociation agents, heat inactivation, or other protein destruction techniques. The CNS-derived tau peptides are separated from the antibodies using rpHPLC and measured using the methods of this application, thereby allowing for the quantification of antibody-bound CNS-derived tau peptides.

[0105] According to one aspect, the present invention relates to a method for monitoring treatment of tau proteinopathy (such as anti-tau antibody treatment) in a subject, the method comprising: (i) contacting a half-denatured sample containing CNS-derived tau from a blood-based sample obtained from the subject with a capture antibody targeting a tau epitope to capture tau peptides in the half-denatured sample; (ii) contacting the captured tau peptides with a detection antibody targeting an epitope containing an amino acid residue at the junction of exon 4 and exon 5 of CNS-derived tau; and (iii) detecting the detection antibody to determine the amount of CNS-derived t-tau peptides in the half-denatured sample. In some embodiments, the method further comprises obtaining a blood-based sample, particularly a plasma sample, from the subject, and / or obtaining a half-denatured sample from a blood-based sample containing CNS-derived tau peptides.

[0106] A semi-denatured sample can be prepared from a blood-based sample containing CNS-derived tau peptides by degrading antibodies and / or detection antibodies that interfere with the binding of capture antibodies and / or detection antibodies to CNS-derived tau peptides, or by interfering with the detection of detection antibodies that bind to CNS-derived tau peptides, but without degrading the CNS-derived tau peptides present in the blood-based sample. In some embodiments, the semi-denatured sample is prepared by heating the blood-based sample at a predetermined temperature for a predetermined amount of time to denature the antibody. The predetermined temperature can be 75°C to 100°C, 80°C to 90°C, or 85°C. The predetermined time can be 0.1 to 30 minutes, 1 to 15 minutes, 2 to 10 minutes, 3 to 9 minutes, or 7 minutes. After heat denaturation, the sample can optionally be cooled to a temperature that is appropriately stable to CNS-derived tau peptides (e.g., equal to or below 4°C) to stop further degradation of proteins in the semi-denatured sample. In some implementations, a semi-denatured sample is prepared by heating a blood-based sample to 85°C for 7 minutes and then cooling it in an ice bath at 4°C for 10 minutes.

[0107] In some implementations, monitoring of treatment is performed at one or more time points: before treatment administration, during treatment, after treatment, or a combination thereof. Treatment effectiveness can be determined by comparing the amount of CNS-derived t-tau peptide determined at different time points, wherein a decrease in the amount of CNS-derived t-tau peptide between earlier and later time points indicates a positive response to treatment, and an increase in the amount of CNS-derived t-tau peptide between earlier and later time points indicates a negative response to treatment.

[0108] In some embodiments, the method for monitoring treatment may further include determining the values ​​of antibody-free CNS-derived tau peptide and antibody-bound CNS-derived tau peptide. Treatment effectiveness can be determined by comparing the values ​​of antibody-free CNS-derived tau peptide and antibody-bound CNS-derived tau peptide determined at different time points, wherein a decrease in the amount of CNS-derived tau peptide at a later time point relative to a previous time point, or an increase in the value of antibody-bound CNS-derived tau peptide at a later time point relative to an earlier time point, and thus an increase in the ratio of antibody-bound CNS-derived tau peptide to antibody-free CNS-derived tau peptide at a later time point relative to an earlier time point, signals a positive response to treatment.

[0109] An indication of whether a response to treatment is positive or negative can be used for a variety of purposes, including as a decision-making tool to determine whether the dose level or dosing interval should be increased or decreased to ensure that an effective or safe level of drug is achieved or maintained; as an adjunct to the initiation of anti-tau drug therapy by providing evidence of achieving the lowest pharmacokinetic (pK) level; as an indication of whether a patient should be excluded from or included in a clinical trial; and as an adjunct to subsequent monitoring of compliance with clinical trial drug requirements.

[0110] In some embodiments, the concentration of tau or t-tau detected in the sample is correlated with the concentration of p217+tau in the blood-to-blood sample. In other embodiments, the concentration of tau or t-tau detected in the blood-based sample is correlated with the concentration of NFL in the sample.

[0111] According to one aspect, the present invention relates to a method for detecting the amyloid status of a subject. The method includes contacting a blood-based sample from the subject with a capture antibody that binds to a tau epitope to capture tau peptides in the blood-based sample; contacting the captured tau peptides with a detection antibody that binds to an epitope comprising an amino acid residue spanning the junction of exon 4 and exon 5 of CNS-derived tau; and detecting the detection antibody to determine the amount of CNS-derived t-tau peptide in the blood-based sample. If the amount of CNS-derived t-tau peptide in the blood-based sample is higher than a predetermined threshold, the subject's amyloid status is positive. If the amount of CNS-derived t-tau peptide in the blood-based sample is lower than a predetermined threshold, the amyloid status is negative.

[0112] The predetermined threshold can be any suitable threshold used to distinguish between subjects who are amyloid-positive and those who are amyloid-negative. The predetermined threshold can be determined as plasma CNS-derived t-tau peptide concentration, used to: distinguish patients with levels greater than or less than amyloid levels in the brain or brain region as measured by PET imaging; distinguish patients with levels greater than, for example, amyloid-β (e.g., Aβ40 or Aβ42) in CSF or plasma; distinguish patients with levels greater than, for example, the Aβ42 to Aβ40 ratio in CSF or plasma; and distinguish cognitively normal patients from those with dementia.

[0113] In some implementations, the predetermined threshold may correspond to a value or average value of plasma CNS-derived t-tau peptide concentration in subjects identified as amyloid-positive or amyloid-negative via other diagnostic methods (e.g., by measuring Aβ42 in CSF or the ratio of Aβ42 to Aβ40 in CSF) or amyloid PET scans or combinations thereof.

[0114] In some implementations, the predetermined threshold may correspond to a baseline value or a value significantly higher than the baseline value. As used herein, "significantly higher" means a higher value that is statistically significant and not merely due to chance, having a p-value of 0.05 or less. In cases where the p-value is less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc., "significantly higher" can be at least about 1%, 2%, 5%, or 10% higher than that present in healthy volunteers. The baseline value may correspond to the average level in a population of healthy individuals. The baseline value may also correspond to the average of previous levels measured in the same subject.

[0115] In one aspect, methods for detecting a subject's amyloid status may include, or involve, in vivo methods, such as methods for treating a subject with amyloid-related conditions, or methods for preventing, improving, treating, and / or reducing amyloid-β deposition in amyloid-related conditions. In vivo methods may include administering treatment to a subject, wherein the subject is determined to have amyloid-related conditions by methods for detecting the subject's amyloid status as described herein; if the amyloid status is positive, the subject is determined to have amyloid-related conditions. Alternatively, in vivo methods may include detecting a subject's amyloid status, and if the amyloid status is positive, administering treatment to the subject. Amyloid-related conditions may be characterized by the formation of plaques containing β-amyloid. Such conditions include, but are not limited to, Alzheimer's disease, dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, or hereditary cerebral hemorrhage with Holland amyloidosis (HCHWA-D). Treatments for amyloid-related conditions are known in the art. In some embodiments, treatment may include an antibody or antigen-binding fragment thereof targeting amyloid-β, for example, the antibody or antigen-binding fragment thereof described in International Applications PCT / EP2020 / 058395 or PCT / IB2017 / 056831, each of which is incorporated herein by reference.

[0116] In some implementations, methods for detecting a subject's amyloid status may be included in or involved in methods for diagnosing Alzheimer's disease, for example, as part of the AT(N) framework for diagnosing Alzheimer's disease.

[0117] According to some embodiments, the capture antibody of the method of the present invention is first bound to a solid support (e.g., a microtiter, magnetic beads, etc.) before contacting the sample. The detection antibody may contain or be attached to any detectable tag (e.g., a fluorescent molecule, biotin, etc.), which can be detected directly or via a secondary reaction (e.g., reaction with streptavidin). Alternatively, a second reagent containing a detectable tag may be used, wherein the second reagent has binding specificity to the primary antibody. In one particular embodiment, the detection antibody is biotinylated.

[0118] According to certain embodiments of the present invention, the amount of CNS-derived t-tau peptide measured in the methods of this application can be determined using any suitable technique known in the art, including enzyme-linked immunosorbent assay (ELISA) and single-molecule array platforms. Depending on a particular aspect, the methods of this application use a high-sensitivity array platform (such as Quanterix Simoa or MSD S-plex) to measure the amount of CNS-derived t-tau peptide in blood-based samples (specifically plasma samples).

[0119] According to some embodiments, the assays and methods may further include measuring other biomarkers associated with or indicative of tau proteinopathy. Such biomarkers include, but are not limited to, amyloid-β (Aβ), phosphorylated (p)-tau181, p217+tau, p231+tau, and NFL. Exemplary assays and methods for measuring such biomarkers are disclosed in PCT Publications WO / 2018 / 083628, WO 2019 / 171258, and WO 2022 / 013286, which are incorporated herein by reference.

[0120] In some embodiments, the assays and methods of the present invention provide bead-based assays for measuring CNS-derived t-tau peptides in blood-based samples, for example, assays and methods in which a capture antibody binds to magnetic beads prior to contact with the blood-based sample. In some embodiments, the assays and methods involve using a sample diluent comprising a nonionic surfactant. In some embodiments, the nonionic surfactant comprises a hydrophilic polyethylene oxide chain and / or an aromatic hydrocarbon lipophilic or hydrophobic group. In some embodiments, the nonionic surfactant is Triton X-100. The sample diluent may also contain tris(hydroxymethyl)aminomethane (Tris). In certain embodiments, the sample diluent may further comprise other suitable components, such as NaCl, ethylenediaminetetraacetic acid (EDTA), heterophile blocking agents, and / or bovine serum albumin.

[0121] In some embodiments of the invention, CNS-derived t-tau peptide measurements obtained from blood-based samples are further analyzed in a computing device to detect and / or predict tau proteinopathy in subjects. Specifically, the computing device analyzes a combination of CNS-derived t-tau peptide measurements obtained from blood-based samples and corresponding measurements obtained from other biomarkers (which may also be detected from blood-based samples) to provide detection and / or prediction of further improvement in tau proteinopathy in subjects. The ability to detect and / or predict improvement in tau proteinopathy (particularly AD) using biomarkers that can be appropriately measured from blood-based samples can be used for various diagnostic purposes, such as diagnosing AD or other tau proteinopathy in subjects, monitoring the effectiveness of treatment, identifying subjects suitable for anti-tau therapy, pre-screening subjects for PET imaging and / or CSF assays for further detection of AD or other tau proteinopathy, identifying subjects suitable for inclusion in clinical trials involving AD or other tau proteinopathy, etc.

[0122] In some embodiments, the computing device obtains a measurement result of the detected CNS-derived t-tau peptide to generate tau data corresponding to the amount of CNS-derived t-tau peptide. The tau data may represent the amount of CNS-derived t-tau peptide detected by the measurement. Alternatively, the tau data may represent a binary state (yes / no) indicating whether its amount is greater than a predetermined threshold. As discussed above, the measurement is sensitive enough that the predetermined threshold is greater than the LLOQ of the measurement method. The computing device may also obtain medical data of the subject, such as demographic information (e.g., age, sex), medical history, electronic medical records (EMR), pharmacy data corresponding to the patient's medication records, etc. Specifically, the computing device may obtain biomarker data corresponding to a measurement result or binary state of at least one biomarker detected from the patient. The biomarker may be any suitable biomarker for tau proteinopathy. Preferably, the biomarker may be detected from a blood-based sample of the subject, particularly a plasma sample. For example, biomarkers can be selected from a group consisting of: amyloid-β (Aβ), p-tau181, p217+tau, p231+tau, NFL, lipofuscin, leptin, and other inflammatory or metabolic markers. More specifically, biomarkers are selected from NFL, lipofuscin, and leptin. The computing device uses a machine learning module to analyze tau and biomarker data to determine or predict whether a subject has tau disease or is at risk of developing tau disease. A set of reference data is used to train the machine learning module. The machine learning module compares the tau and biomarker data to the set of reference data to determine or predict whether a subject has tau disease or is at risk of developing tau disease. For patients in the reference group, the set of reference data includes tau and biomarker data, as well as data on the neuropathology corresponding to tau disease (e.g., disease stage, amount of tau detected in CSF, PET measurements of tau in brain tissue, etc.).

[0123] The machine learning module can be a supervised and / or unsupervised machine learning module. The machine learning module can be a machine learning classifier used to identify a dataset as associated with one of two categories. The machine learning module can include support vector machines, random forests, logistic regression, gradient boosting modules, or ensembles thereof. In some implementations, the machine learning module is an ensemble module including at least one of support vector machines, random forests, logistic regression, and / or gradient boosting modules.

[0124] 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 implementation 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, such as an onboard processing arrangement within a device or an external processing arrangement (e.g., a mobile computing device, smartphone, computing tablet, computing device, etc.), which may communicate with at least a portion of the device.

[0125] Capture and detect antibodies

[0126] The capture antibody used in the assays and methods of the present invention binds to a tau epitope. In some embodiments, the capture antibody is a monoclonal antibody that binds to an epitope between amino acid residues 7 and 20 or between amino acid residues 150 and 250 of human tau protein, wherein the amino acid numbers refer to the amino acid sequence of SEQ ID NO:1 (see Table 1). In some embodiments, the capture antibody binds to an epitope comprising amino acid residues 7-20 of human tau protein. In a preferred embodiment, the epitope comprises the amino acid sequence of SEQ ID NO:2 (see Table 1).

[0127] Table 1. Amino acid sequences of human tau protein and its epitopes .

[0128]

[0129] The exemplary capture antibody of the present invention may comprise the CDR of antibody hT43, namely, HCDR1, HCDR2, and HCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 5, 6, and 7, or respectively composed of the amino acid sequences thereof; and LCDR1, LCDR2, and LCDR3 respectively comprising the amino acid sequences of SEQ ID NO: 8, 9, and 10, or respectively composed of the amino acid sequences thereof; and / or the heavy chain variable region and light chain variable region of antibody hT43, namely, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, or respectively composed of the amino acid sequence thereof; and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, or respectively composed of the amino acid sequence thereof (see Table 2). In a preferred embodiment, the capture antibody is hT43.

[0130] Table 2. Amino acid sequences of hT43 CDR, heavy chain variable region, and light chain variable region. .

[0131]

[0132] The detection antibody used in the assay and method of this invention binds to an epitope containing amino acid residues spanning the junction of exon 4 and exon 5 in CNS-derived tau. The "junction of exon 4 and exon 5" refers to the point where exon 4 and exon 5 meet in CNS-derived tau. Therefore, the amino acid residues "spanning the junction of exon 4 and exon 5" contain at least the last amino acid residue of exon 4 and the first amino acid residue of exon 5 in the N-terminal to C-terminal direction.

[0133] In some embodiments, the detection antibody capture antibody is a monoclonal antibody that binds to the following epitopes: (a) amino acid residues 124, or 123 and 124, or 122-124, or 121-124, or 120-124, or 119-124, or 118-124, or 117-124, or 116-124, or 115-124, or 114-124, of SEQ ID NO: 1; and (b) amino acid residues 125, or 125 and 126, or 125-127, or 125-128, or 125-129, or 125-130, or 125-131, or 125-132, or 125-133, or 125-134, or 125-135.

[0134] In some embodiments, the detection antibody binds to an epitope comprising amino acid residues 116-127 of human tau protein. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 3 (see Table 1). Exemplary detection antibodies of the present invention may comprise the CDR of antibody pT82, i.e., comprising the amino acid sequences of SEQ ID NO: 13, 14, and 15, or HCDR1, HCDR2, and HCDR3 composed of those amino acid sequences, respectively; and comprising the amino acid sequences of SEQ ID NO: 16, 17, and 18, or LCDR1, LCDR2, and LCDR3 composed of those amino acid sequences, respectively; and / or the heavy chain variable region and light chain variable region of antibody pT82, i.e., comprising the amino acid sequence of SEQ ID NO: 19, or the heavy chain variable region composed of that amino acid sequence, and comprising the amino acid sequence of SEQ ID NO: 20, or the light chain variable region composed of that amino acid sequence (see Table 3).

[0135] Table 3. Amino acid sequences of pT82 CDR, heavy chain variable region, and light chain variable region .

[0136]

[0137] In some embodiments, the detection involves the antibody binding to an epitope comprising amino acid residues 119-126 of the human tau protein. In a preferred embodiment, the epitope comprises the amino acid sequence of SEQ ID NO: 4 (see Table 1). The exemplary detection antibody of the present invention may comprise the CDR of antibody hT36, namely, comprising the amino acid sequences of SEQ ID NO: 21, 22 and 23 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences, respectively; and comprising the amino acid sequences of SEQ ID NO: 24, 25 and 26 or LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences, respectively; or comprising the amino acid sequences of SEQ ID NO: 27, 28 and 29 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences, respectively; and comprising the amino acid sequences of SEQ ID NO: 24, 25 and 26 or LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences, respectively; or comprising the amino acid sequences of SEQ ID NO: 30, 31 and 32 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences, respectively; comprising the amino acid sequence of SEQ ID NO: 33 or LCDR1 composed of the amino acid sequence; comprising the amino acid sequence LVS or LCDR2 composed of the amino acid sequence; and comprising the amino acid sequence of SEQ ID NO: 23. The amino acid sequence of SEQ ID NO: 26 or LCDR3 composed of the amino acid sequence thereof; or the amino acid sequences of SEQ ID NO: 35, 36 and 23 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences thereof, respectively; and the amino acid sequences of SEQ ID NO: 24, 25 and 26 or LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences thereof, respectively; or the amino acid sequences of SEQ ID NO: 37, 38 and 39 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences thereof, respectively; and the amino acid sequences of SEQ ID NO: 40, 41 and 42 or LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences thereof, respectively. The exemplary detection antibody of the present invention may comprise a heavy chain variable region and a light chain variable region of antibody hT36, namely, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:43 or composed of the amino acid sequence thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO:44 or composed of the amino acid sequence thereof; and / or a heavy chain and a light chain of antibody hT36, namely, a heavy chain comprising the amino acid sequence of SEQ ID NO:45 or composed of the amino acid sequence thereof and a light chain comprising the amino acid sequence of SEQ ID NO:46 or composed of the amino acid sequence thereof (see Table 4).

[0138] Table 4. hT36 CDR, heavy chain variable region and light chain variable region, and amino acid sequences of the heavy chain and light chain.。

[0139]

[0140]

[0141]

[0142] One aspect of the invention also relates to an antibody or an antigen-binding fragment thereof that can bind to a CDR-derived tau peptide. In some embodiments, the antibody or its antigen-binding fragment comprises amino acid sequences comprising or composed of SEQ ID NO: 21, 22, and 23, respectively, or HCDR1, HCDR2, and HCDR3, respectively, and amino acid sequences comprising or composed of SEQ ID NO: 24, 25, and 26, respectively, or LCDR1, LCDR2, and LCDR3, respectively; or amino acid sequences comprising or composed of SEQ ID NO: 27, 28, and 29, respectively, or HCDR1, HCDR2, and HCDR3, respectively, and amino acid sequences comprising or composed of SEQ ID NO: 24, 25, and 26, respectively, or LCDR1, LCDR2, and LCDR3, respectively; or amino acid sequences comprising or composed of SEQ ID NO: 30, 31, and 32, respectively, or HCDR1, HCDR2, and HCDR3, respectively, and amino acid sequences comprising or composed of SEQ ID NO: 33, or LCDR1, comprising amino acid sequence LVS, or LCDR2, and comprising SEQ ID NO: 24, 25, and 26, respectively, or LCDR1, comprising amino acid sequence LVS, or LCDR2, and LCDR3, comprising or composed of SEQ ID NO: 24, 25, and 26, respectively; or amino acid sequences comprising or composed of ... The amino acid sequence NO:26 or LCDR3 composed of the amino acid sequence; or the amino acid sequences of SEQ ID NO:35, 36 and 23 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences; and the amino acid sequences of SEQ ID NO:24, 25 and 26 or LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences; or the amino acid sequences of SEQ ID NO:37, 38 and 39 or HCDR1, HCDR2 and HCDR3 composed of the amino acid sequences; and the amino acid sequences of SEQ ID NO:40, 41 and 42 or LCDR1, LCDR2 and LCDR3 composed of the amino acid sequences. In some embodiments, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 contained within the heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 43; and LCDR1, LCDR2, and LCDR3 contained within the heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain region comprising or consisting of the amino acid sequence of SEQ ID NO: 43 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 44.In a particular embodiment, the antibody or its antigen-binding fragment comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO:45 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO:46.

[0143] An overview of the sequences provided in this article is presented in Table 5.

[0144] Table 5. Sequence Overview .

[0145]

[0146]

[0147] In addition to providing detection antibodies and fragments thereof (the sequences of which are provided in Table 4), this invention also covers variants and equivalents of these detection antibodies and antibody fragments. For example, such variants include humanized, chimeric, optimized, germlined, and / or other forms of detection antibodies having a CDR and / or a variable region having hT36. Similarly, in some embodiments, variants of the sequences disclosed herein containing one or more substitutions, additions, deletions, or other mutations may be used. The amino acid sequences of the heavy chain variable region and / or the light chain variable region, or portions thereof, including the CDR sequence, may be 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to the sequences described herein, and / or contain 1, 2, 3, 4, 5, or more substitutions relative to the sequences described herein, such as conserved substitutions. In some embodiments, the detection antibody according to the invention comprises an amino acid sequence or portion thereof of a heavy chain variable region and / or light chain variable region that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to the amino acid sequence or portion thereof of SEQ ID NO:43 and / or SEQ ID NO:44, and / or comprises 1, 2, 3, 4, 5, or more substitutions relative to that sequence, e.g., conserved substitutions, but contained within a specific CDR sequence found within such heavy chain and / or light chain variable regions, i.e., any mutations (such as substitutions, additions, deletions, etc.) outside the CDR. Such detection antibodies (i.e., heavy chain variable regions and light chain variable regions having a certain percentage of similarity to the heavy chain variable region or light chain variable region, or having one or more substitutions) can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of a nucleic acid molecule encoding the heavy chain variable region and / or variable light chain variable region described herein, followed by testing the binding of the encoded altered antibody molecule to CNS-derived tau.

[0148] In addition to the heavy chain variable region and the light chain variable region, the detection antibody and its fragments (the sequences of which are provided in Table 4) may also include a heavy chain constant region or a fragment thereof. In some embodiments, the heavy chain constant region is a human heavy chain constant region, such as the human IgG constant region, for example, the human IgG1 constant region. Furthermore, the detection antibody may include a light chain constant region or a fragment thereof. In some embodiments, the light chain constant region is a κ constant region or a λ constant region, for example, the human κ constant region or the human λ constant region.

[0149] In another general aspect, the present invention relates to isolated polynucleotides encoding detection antibodies or antigen-binding fragments thereof (the sequences of which are provided in Table 4). Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., by substitution, deletion, insertion, etc.) without changing the amino acid sequence of the protein. Therefore, those skilled in the art will understand that the nucleic acid sequence encoding the detection antibody or antigen-binding fragment of the present invention can be changed without changing the amino acid sequence of the protein. Exemplary isolated polynucleotides are polypeptides encoding HCDR1, HCDR2, and HCDR3 containing or composed of the amino acid sequences shown in Table 4, or polypeptides containing or composed of the amino acid sequences shown in Table 4, or LCDR1, LCDR2, and LCDR3. Other exemplary isolated polynucleotides are polynucleotides encoding the variable regions of the antibodies of the present invention. Other polynucleotides encoding the antibodies of the present invention, given the genetic code degeneracy or codon priority in a given expression system, are also within the scope of the present invention. The isolated nucleic acids of the present invention can be prepared using well-known recombinant or synthetic techniques. DNA encoding monoclonal antibodies can be readily isolated and sequenced using methods known in the art. In the case of hybridoma formation, such cells can be used as a source of such DNA. As an alternative, display techniques associated with the encoded sequences and translation products, such as phage or ribosome display libraries, can be used.

[0150] In another aspect, the present invention relates to a vector comprising isolated polynucleotides encoding the detection antibody or antigen-binding fragment thereof of the present invention. According to this disclosure, any vector known to those skilled in the art, such as plasmids, sticky-terminal plasmids, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements that establish the conventional function of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible, or repressive promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to generate antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention.

[0151] In another aspect, the present invention relates to host cells comprising isolated polynucleotides encoding the detection antibody or antigen-binding fragment thereof of the present invention. In view of this disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the antibody or antigen-binding fragment thereof of the present invention. Such host cells may be eukaryotic cells, bacterial cells, plant cells, or archaea cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines, such as SP2 / 0 (American Center for Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NSO (European Center for Cell Culture Collection (ECACC), Salisbury, Wiltshire, ETC, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is EG266 (ATTC CRL-TIB-196). Other available cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHO-K1 SV (Lonza Biologies), CHO-K1 (ATCC CRL-61, Invitrogen), or DG44.

[0152] In another general aspect, the present invention relates to a method for producing the detection antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing cells containing a polynucleotide encoding the detection antibody or antigen-binding fragment thereof under conditions for producing the detection antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cells or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof may be harvested from the cells and purified according to conventional techniques known in the art.

[0153] Reagent test kit

[0154] In another general aspect, this application relates to a kit comprising (a) a capture antibody that binds to a tau epitope, and (b) a detection antibody that binds to an epitope comprising an amino acid residue spanning the link between exon 4 and exon 5 of CNS-derived tau. The capture antibody and the detection antibody are according to the invention. The kit can be used to detect or measure CNS-derived tau peptides from blood-based samples (e.g., blood, serum, plasma).

[0155] In some implementations, the capture antibody may be pre-bound to a solid phase, such as to a pore in a microtiter dish or magnetic beads.

[0156] In some embodiments, the kit further includes one or more of the components necessary and / or sufficient for performing the assay (including controls), instructions for performing the assay, and any necessary software for analyzing and presenting the results. Those skilled in the art will readily recognize that the disclosed capture antibodies and detection antibodies can be readily incorporated into any of the established kit formats well known in the art.

[0157] The embodiments of this disclosure can be further described and understood with reference to the following non-limiting "Examples," which describe the preparation of certain exemplary sandwich ELISAs using the capture and detection antibodies of the present invention. It will be apparent to those skilled in the art that numerous modifications can be made to the specific descriptions provided in the examples without undue experimentation or departing from the scope of this disclosure.

[0158] Example

[0159] Example 1: Development of Sandwich Measurement

[0160] Following the manufacturer's recommended Homebrew assay development protocol, a sandwich ELISA targeting t-tau was developed on a Simoa HD-X single-molecule array instrument available from Quanterix Corp. (Boston, MA). Monoclonal antibodies hT43 and pT82, along with the commercial antibody hT7, were selected for the assay. Sample diluents (Dilution-1) as described in Triana-Baltzer et al. (2021) were used to screen recombinant full-length tau protein (i.e., SEQ ID NO:1) and human plasma samples. The Simoa analyzer generated an average enzyme count (AEB) output per bead based on these measurements.

[0161] The assay using the pairing of hT43 and pT82 provided maximum sensitivity, yielding a rough lower limit of quantification (LLOQ) of 1 pg / ml (based on the lowest concentration of recombinant tau that generates a signal / noise (S / N) ratio of 2x), utilizing the linear signal from dilutions of several human plasma samples, as well as detectable plasma signals down to dilutions of at least 1:16 (see [link to assay]). Figure 1 (See Tables 6 and 7). In sandwich ELISA, the choice of which antibody to use as the capture antibody and the detection antibody has virtually no impact on the sensitivity of the assay; however, using hT43 as the capture antibody is slightly more sensitive and was chosen as the final pair. The two-step Simoa procedure (without a wash step between capture antibody-sample incubation and contact with the detection antibody) and the three-step Simoa procedure (with a wash step between capture antibody-sample incubation and contact with the detection antibody) were evaluated, and the three-step Simoa procedure provided significantly more signal (data not shown).

[0162] Table 6. Calibration curves for hT43xpT82 (based on LLOQ=0.009 and AEB=approximately 1.25 pg / L for S / N>2 and CV<20%). ml） .

[0163]

[0164] Table 7. Calibration curves for pT82xhT43 (based on LLOQ=0.011 and AEB=approximately 1.25 pg / L for S / N>2 and CV<20%). ml） .

[0165]

[0166] Sandwich ELISA conditions were further developed using hT43 and pT82 antibodies and calibration peptides containing hT43 epitopes (amino acid residues 7-20) and pT82 epitopes (amino acid residues 116-127) separated by PEG4 linkers. Assays were tested under various conditions: (i) either antibody as the capture / detection antibody; (ii) two-step and three-step protocols; and (iii) sample diluent-1 with Quanterix's proprietary diluent, according to the manufacturer's instructions. Success was determined based on the maximum sensitivity of the calibration peptide, the maximum sensitivity of plasma samples, the dilution linearity of plasma, and the spiked recovery of the calibrator in plasma.

[0167] The assay conditions yielded the most sensitive assay while maintaining good dilution linearity and spiked recovery. The assay exhibited a dynamic range of 0.4 pg / ml to 300 pg / ml, with 97% of plasma samples measured within the linear range (see [reference]). Figure 2 Figure A). LOD was determined to be 28 fg / ml, and LLOG was calculated to be 123 fg / ml. Dilution linearity was shown from 1:8 to 1:32 dilutions, and was preferably achieved with the smallest 1:8 dilution (while maintaining the sample signal within the linear range of the assay) (see Figure A). Figure 2 Figure B). Furthermore, after spiked samples to calibration peptide concentrations of 4 pg / ml, 80 pg / ml, and 1600 pg / ml, the recoveries of the calibration peptides in the samples were measured to be between 85% and 93% (average) (see Figure B). Figure 2 (Figure C). Finally, the measured in-test precision was calculated to be 13% ± 21.2% (mean ± SD).

[0168] Example 2: Detection of CNS-derived Tau

[0169] As described herein (see the Assays and Methods section below), CNS-derived tau RNA in the brain and spinal cord has been reported to be spliced ​​to remove exon 4A, while peripherally derived tau mRNA does not exhibit this splicing event. Therefore, centrally derived tau will have a seamless junction from exons 4–5, while peripherally derived tau does not. The last amino acid of exon 4 corresponds to amino acid 124, and the first amino acid of exon 5 corresponds to amino acid 125. The exon 4a sequence is not considered in this numbering scheme because most literature describes CNS-derived tau, thus ignoring exon 4A. The antibody pT82 recognizes an epitope corresponding to amino acid residues 116–127 of tau and is therefore specific for CNS-derived tau. The antibody hT36 has also been found to recognize this same epitope.

[0170] To confirm the hypothesis that the hT43xpT82 assay only recognizes CNS-derived tau species, a series of peptides corresponding to the exon 4-4a junction (peptide 1; see [link]). Figure 3 Figure A), exon 4-5 junction (peptide 2; see Figure A). Figure 4 Figure A) and the exon 4A-5 junction (peptide 3; see Figure A) Figure 5 Figure A). As mentioned above, peptide 2 will correspond to a sequence found only in CNS-derived tau, while peptides 1 and 3 will correspond to sequences found only in peripheral-derived tau. pT82 and hT36 antibodies (as a backup of pT82) were immobilized on a Biacore gold chip, and changes in surface plasmon resonance (SPR) were measured after increasing concentrations of peptides 1, 2, or 3 flowed across the chip.

[0171] Neither antibody showed activity at peptide 1 (see [link to antibody]). Figure 3 Figures B and C) or peptide 3 (see ... Figure 5 The SPR change (a measure of antibody / peptide binding) in the cases shown in Figures B and C does indeed show substantial binding to peptide 2 (see Figures B and C). Figure 4 (Figures B and C). These results provide evidence that pT82 and hT36 recognize sequences found in CNS-derived tau but not in peripherally derived tau. pT82 exhibits a slightly tighter binding, confirming its suitability as an ideal antibody for Simoa assays.

[0172] Example 3: Detection of amyloid protein positivity in Alzheimer's disease

[0173] The clinical performance of the Simoa CNS-derived t-tau assay, using hT43 as the capture antibody and pT82 as the detection antibody, was evaluated in the detection of amyloid positivity. Two cohorts were evaluated, involving healthy control (HC) samples and samples from NTAD:

[0174] Cohort 1 = 58 subjects: 8 HC (0% A+) and 49 NTAD (61% A+).

[0175] Cohort 2 = 87 subjects: 12 HC (0% A+) and 75 NTAD (80% A+).

[0176] (“A+” = Amyloid positive). HC samples are all amyloid negative as determined by Meso Scale Discovery (MSD) assay of Aβ42 / 40 in CSF. NTAD samples are 73% amyloid positive as determined by CSF Aβ42 / 40 ratio or amyloid PET scan.

[0177] The results showed that the plasma concentration of CNS-derived t-tau was correlated with the p217+tau concentration in both cohort 1 and cohort 2 (see [references]). Figure 6 Figures A and B), and correlated with NFL concentrations in both Cohort 1 and Cohort 2 (see Figures A and B, respectively). Figure 6 Figures C and D (see also Table 8). Furthermore, in both cohort 1 and cohort 2, plasma concentrations of CNS-derived t-tau were elevated in amyloid-positive subjects compared to those who were amyloid-negative (see Figures C and D respectively). Figure 7 (See Figures A and B) (see also Table 9).

[0178] Table 8. Association between CNS-derived t-tau and plasma 217+tau and NFL concentrations as measured by Simoa. sex .

[0179]

[0180] Table 9. CNS-derived t-tau in subjects with positive amyloid and those with negative amyloid (A-) average concentration .

[0181]

[0182] * * * * *

[0183] This invention is not limited to the specific embodiments described herein. In fact, various modifications of the invention will become apparent to those skilled in the art from the foregoing description and drawings, in addition to those described. Such modifications are intended to fall within the scope of the appended claims.

[0184] All references cited in this paper are incorporated herein by reference in full for all purposes, to the extent that each individual publication, patent, or patent application is specifically and individually represented in full and incorporated herein by reference for all purposes.

[0185] References

[0186] Al-Lazikani B et al., Standard conformations for the canonical structures of immunoglobulins. Journal of Molecular Biology, 1997, 273(4): 927-948.

[0187] Almagro JC, Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires. Journal of Molecular Recognition, 2004, 17(2): 132-143.

[0188] Ashton NJ et al., A multicentre validation study of the diagnostic value of plasma neurofilament light, Nature Communications, 2021, 12(1): 3400.

[0189] Barthélemy NR et al., Blood plasma phosphorylated-tau isoforms track CNS change in Alzheimer's disease, Journal of Experimental Medicine, 2020, 217(11): e20200861.

[0190] Berzofsky JA et al., Antibody-Antigen Interactions. In Fundamental Immunology, Paul WE ed., Raven Press: New York, 1984.

[0191] Bridel C et al., Diagnostic value of cerebrospinal fluid neurofilament light protein in neurology: a systematic review and meta-analysis, JAMA Neurology, 2019, 76(9): 1035-1048。

[0192] Butner KA and Kirschner MW, Tau protein binds to microtubules through a flexible array of distributed weak sites, Journal of Cell Biology, 1991, 115(3): 717-730。

[0193] Chothia C & Lesk AM, Canonical structures for the hypervariable regions of immunoglobulins. Journal of Molecular Biology, 1987, 196(4): 901-917。

[0194] Clavaguera F et al., Transmission and spreading of tauopathy in transgenic mouse brain, Nature Cell Biology, 2009, 11(7): 909-913。

[0195] Couchie D et al., Primary structure of high molecular weight tau present in the peripheral nervous system, Proceedings of the National Academy of Sciences of the U.S.A, 1992, 89(10): 4378-4381。

[0196] Dugger BN et al., The presence of select tau species in human peripheral tissues and their relation to Alzheimer’s disease, Journal of Alzheimer’s Disease, 2016, 51(2): 345-356.

[0197] Epitope Mapping Protocols, 1996, edited by Morris GE, 66.

[0198] Frank B et al., Plasma p-tau181 shows stronger network association to Alzheimer's disease dementia than neurofilament light and total tau, Alzheimer's & Dementia, 2022, 18(8): 1523-1536.

[0199] Frost B et al., Propagation of tau misfolding from the outside to the inside of a cell, Journal of Biological Chemistry, 2009, 284(19): 12845-12852.

[0200] Georgieff IS et al., High molecular weight tau: preferential localization in the peripheral nervous system, Journal of Cell Science, 1991, 100(pt.1): 55-60.

[0201] Gonzalez-Ortiz F et al., Brain-derived tau: a novel blood-based biomarker for Alzheimer's disease-type neurodegeneration, Brain, 2023, 146(3): 1152-1165.

[0202] Grothe MJ et al., Associations of fully automated CSF and novel plasmabiomarkers with Alzheimer disease neuropathology at autopsy, Neurology, 2021,97(12): e1229-e1242。

[0203] Hanger DP et al., Tau phosphorylation: the therapeutic challenge forneurodegenerative disease, Trends in Molecular Medicine, 2009, 15(3): 112-119。

[0204] Iqbal K et al., Tau in Alzheimer disease and related tauopathies,Current Alzheimer Research, 2010, 7(8): 656-664。

[0205] Jack, Jr. CR et al., NIA-AA research framework: toward a biologicaldefinition of Alzheimer’s disease, Alzheimer's & Dementia, 2018, 14(4): 535-562。

[0206] Jones PT et al., Replacing the complementarity-determining regions in ahuman antibody with those from a mouse.Nature, 1986, 321(6069): 522-525。

[0207] Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th ed.Public Health Service, National Institutes of Health, Bethesda, Md., 1991。

[0208] Kuby. Immunology, W. H. Freeman and Company: New York, 1992。

[0209] Lefranc M-P et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental and Comparative Immunology, 2003, 27(1): 55-77。

[0210] Morris M et al., The many face of tau, Neuron, 2011, 70(3): 410-416。

[0211] Nakamura A et al., High performance plasma amyloid-β biomarkers for Alzheimer's disease, Nature, 2018, 554(7691): 249-254。

[0212] Palmqvist S et al., Discriminative accuracy of plasma phospho-tau217 for Alzheimer disease vs other neurodegenerative disorders, JAMA, 2020, 324(8): 772-781。

[0213] Riechmann L et al., Reshaping human antibodies for therapy. Nature, 1988, 332(6162): 323-327。

[0214] Schindler S et al., High-precision plasma β-amyloid 42 / 40 predicts current and future brain amyloidosis, Neurology, 2019, 93(17): e1647-e1659。

[0215] Sjögren M et al., Both total and phosphorylated tau are increased in Alzheimer's disease, Journal of Neurology, Neurosugery, and Psychiatry, 2001, 70(5): 624-630.

[0216] Thijssen EH et al., Plasma phosphorylated tau 217 and phosphorylated tau 181 as biomarkers in Alzheimer's disease and frontotemporal lobe degeneration: a retrospective diagnostic performance study, Lancet. Neurology, 2021, 20(9): 739-752.

[0217] Triana-Baltzer G et al., Development and validation of a high-sensitivity assay for measuring p217+tau in plasma, Alzheimer's & Dementia (Amsterdam, Netherlands), 2021, 13 (1): e12204.

[0218] Verhoeyen M et al., Reshaping human antibodies: grafting an antilysozyme activity. Science, 1988, 239(4847): 1534-1536.

Claims

1. A method for detecting central nervous system (CNS)-derived tau peptides in a blood-based sample from a subject, the method comprising: (a) Contact the blood-based sample with a capture antibody that binds to the tau epitope to capture the tau peptide in the blood-based sample; and (b) Contact the captured tau peptide with a detection antibody that binds to an epitope containing an amino acid residue that spans the junction of exon 4 and exon 5 of CNS-derived tau.

2. A method for measuring total central nervous system (CNS)-derived tau (t-tau) peptides in a blood-based sample from a subject, the method comprising: (a) Contact the blood-based sample with a capture antibody that binds to the tau epitope to capture the tau peptide in the blood-based sample; (b) Contacting the captured tau peptide with a detection antibody that binds to an epitope containing an amino acid residue at the junction of exon 4 and exon 5 across CNS-derived tau; and (c) Detect the detection antibody to determine the amount of CNS-derived t-tau peptide in the blood-based sample.

3. The method according to claim 1 or 2, wherein the capture antibody is immobilized on a solid phase.

4. The method according to claim 3, wherein the solid phase is a magnetic bead.

5. The method according to any one of claims 1 to 4, the method further comprising obtaining the sample from the subject.

6. The method according to any one of claims 1 to 5, wherein the blood-based sample is a plasma sample.

7. The method according to any one of claims 1 to 6, the method further comprising washing the captured tau peptide before contacting the captured tau peptide with the detection antibody.

8. The method of claim 7, wherein the captured tau peptide is washed with a harsh buffer solution.

9. The method according to any one of claims 1 to 8, wherein the concentration of tau or t-tau detected in the blood-based sample is associated with the concentration of p217+tau in the blood-blood sample.

10. The method according to any one of claims 1 to 8, wherein the concentration of tau or t-tau detected in the blood-based sample is correlated with the concentration of neurofilament light chains in the blood-blood sample.

11. A method for detecting the amyloid status of a subject, the method comprising: (a) Contact a blood-based sample from the subject with a capture antibody that binds to a tau epitope to capture tau peptides in the blood-based sample; (b) Contacting the captured tau peptide with a detection antibody that binds to an epitope containing an amino acid residue at the junction of exon 4 and exon 5 across CNS-derived tau; and (c) Detect the detection antibody to determine the amount of CNS-derived t-tau peptide in the blood-based sample. If the amount of CNS-derived t-tau peptide in the blood-based sample is higher than a predetermined threshold, then the amyloid status is positive, and If the amount of CNS-derived t-tau peptide in the blood-based sample is below a predetermined threshold, the amyloid status is negative.

12. A kit comprising: (i) Capture antibodies that bind to tau epitopes; and (ii) Detection antibodies that bind to epitopes containing amino acid residues at the junction of exon 4 and exon 5, which are tau originating from the central nervous system (CNS).

13. The method or kit according to any one of claims 1 to 12, wherein the capture antibody binds to an epitope comprising amino acid residues 7-20 of human tau protein. The amino acid residues are numbered according to the amino acid sequence of SEQ ID NO:

1.

14. The method or kit of claim 13, wherein the capture antibody comprises heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2 and HCDR3 containing the amino acid sequences of SEQ ID NO: 5, 6 and 7, respectively, and light chain complementarity-determining regions (LCDR) LCDR1, LCDR2 and LCDR3 containing the amino acid sequences of SEQ ID NO: 8, 9 and 10, respectively.

15. The method or kit of claim 14, wherein the capture antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

12.

16. The method or kit of claim 15, wherein the capture antibody is hT43.

17. The method or kit according to any one of claims 1 to 16, wherein the detection antibody binds to an epitope comprising amino acid residues 116-127 of human tau protein. The amino acid residues are numbered according to the amino acid sequence of SEQ ID NO:

1.

18. The method or kit of claim 17, wherein the detection antibody comprises heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2 and HCDR3 containing the amino acid sequences of SEQ ID NO: 13, 14 and 15, respectively, and light chain complementarity-determining regions (LCDR) LCDR1, LCDR2 and LCDR3 containing the amino acid sequences of SEQ ID NO: 16, 17 and 18, respectively.

19. The method or kit of claim 18, wherein the detection antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

20.

20. The method or kit of claim 19, wherein the detection antibody is pT82.

21. The method or kit according to any one of claims 1 to 16, wherein the detection antibody binds to an epitope comprising amino acid residues 119-126 of human tau protein. The amino acid residues are numbered according to the amino acid sequence of SEQ ID NO:

1.

22. The method or kit of claim 21, wherein the detection antibody comprises heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2, and HCDR3 and light chain complementarity-determining regions (LCDR) LCDR1, LCDR2, and LCDR3, wherein: (i) HCDR1 contains the amino acid sequence of SEQ ID NO: 21, HCDR2 contains the amino acid sequence of SEQ ID NO: 22, HCDR3 contains the amino acid sequence of SEQ ID NO: 23, LCDR1 contains the amino acid sequence of SEQ ID NO: 24, LCDR2 contains the amino acid sequence of SEQ ID NO: 25, and LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (ii) HCDR1 contains the amino acid sequence of SEQ ID NO: 27, HCDR2 contains the amino acid sequence of SEQ ID NO: 28, HCDR3 contains the amino acid sequence of SEQ ID NO: 29, LCDR1 contains the amino acid sequence of SEQ ID NO: 24, LCDR2 contains the amino acid sequence of SEQ ID NO: 25, and LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (iii) HCDR1 contains the amino acid sequence of SEQ ID NO: 30, HCDR2 contains the amino acid sequence of SEQ ID NO: 31, HCDR3 contains the amino acid sequence of SEQ ID NO: 32, LCDR1 contains the amino acid sequence of SEQ ID NO: 33, LCDR2 contains the amino acid sequence LVS, and LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (iv) The HCDR1 contains the amino acid sequence of SEQ ID NO: 35, the HCDR2 contains the amino acid sequence of SEQ ID NO: 36, the HCDR3 contains the amino acid sequence of SEQ ID NO: 23, the LCDR1 contains the amino acid sequence of SEQ ID NO: 24, the LCDR2 contains the amino acid sequence of SEQ ID NO: 25, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (v) The HCDR1 contains the amino acid sequence of SEQ ID NO: 37, the HCDR2 contains the amino acid sequence of SEQ ID NO: 38, the HCDR3 contains the amino acid sequence of SEQ ID NO: 39, the LCDR1 contains the amino acid sequence of SEQ ID NO: 40, the LCDR2 contains the amino acid sequence of SEQ ID NO: 41, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

42.

23. The method or kit of claim 22, wherein the detection antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 43 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

44.

24. The method or kit of claim 23, wherein the detection antibody is hT36.

25. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising heavy chain complementarity-determining regions (HCDR) HCDR1, HCDR2 and HCDR3 and light chain complementarity-determining regions (LCDR) LCDR1, LCDR2 and LCDR3, wherein: (i) HCDR1 contains the amino acid sequence of SEQ ID NO: 21, HCDR2 contains the amino acid sequence of SEQ ID NO: 22, HCDR3 contains the amino acid sequence of SEQ ID NO: 23, LCDR1 contains the amino acid sequence of SEQ ID NO: 24, LCDR2 contains the amino acid sequence of SEQ ID NO: 25, and LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (ii) HCDR1 contains the amino acid sequence of SEQ ID NO: 27, HCDR2 contains the amino acid sequence of SEQ ID NO: 28, HCDR3 contains the amino acid sequence of SEQ ID NO: 29, LCDR1 contains the amino acid sequence of SEQ ID NO: 24, LCDR2 contains the amino acid sequence of SEQ ID NO: 25, and LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (iii) HCDR1 contains the amino acid sequence of SEQ ID NO: 30, HCDR2 contains the amino acid sequence of SEQ ID NO: 31, HCDR3 contains the amino acid sequence of SEQ ID NO: 32, LCDR1 contains the amino acid sequence of SEQ ID NO: 33, LCDR2 contains the amino acid sequence LVS, and LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (iv) The HCDR1 contains the amino acid sequence of SEQ ID NO: 35, the HCDR2 contains the amino acid sequence of SEQ ID NO: 36, the HCDR3 contains the amino acid sequence of SEQ ID NO: 23, the LCDR1 contains the amino acid sequence of SEQ ID NO: 24, the LCDR2 contains the amino acid sequence of SEQ ID NO: 25, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 26; or (v) The HCDR1 contains the amino acid sequence of SEQ ID NO: 37, the HCDR2 contains the amino acid sequence of SEQ ID NO: 38, the HCDR3 contains the amino acid sequence of SEQ ID NO: 39, the LCDR1 contains the amino acid sequence of SEQ ID NO: 40, the LCDR2 contains the amino acid sequence of SEQ ID NO: 41, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

42.

26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain region containing the amino acid sequence of SEQ ID NO: 43 and a light chain variable region containing the amino acid sequence of SEQ ID NO:

44.

27. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising... (i) The heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 contained within the heavy chain variable region containing the amino acid sequence of SEQ ID NO:43, and (ii) Light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained within the heavy chain variable region containing the amino acid sequence of SEQ ID NO:

44.

28. The antibody or antigen-binding fragment thereof according to any one of claims 25 to 27, wherein the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 45 and a light chain containing the amino acid sequence of SEQ ID NO: 46.

Images