WO2025037271A1 - Plasma assay for detecting cns-derived tau peptides - Google Patents

Abstract

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

Description

TITLE

PLASMA ASSAY FOR DETECTING CNS-DERIVED TAU PEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/533,337, filed on 17 August 17 2023, and of U.S. Provisional Application No. 63/545,344, filed on 23 October 2023, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 06 August 2024, is named JAB7215WOPCT1_SL, and is 41,646 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Alzheimer’s Disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment, and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans. More than 5 million people in the United States are living with AD and the number is growing with an aging population, as 10% of people over age 65 have AD and it is the 5th leading cause of death in this population. Worldwide, it is estimated that over 50 million people have AD or related dementia, further demonstrating that AD continues to present as a major public health problem.

[0004] The brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels), and neurofibrillary tangles. Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles of paired helical filaments, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD.

[0005] Neurofibrillary tangles are primarily composed of aggregates of hyperphosphorylated tau protein. The main physiological function of tau is microtubule polymerization and stabilization. The binding of tau to microtubules takes place by ionic interactions between positive charges in the microtubule binding region of tau and negative charges on the microtubule lattice (Butner and Kirschner, 1991). Tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the primary function of tau. Tau that is bound to the axonal microtubule lattice is in a hypophosphorylation state, while aggregated tau in AD is hyper-phosphorylated, providing unique epitopes that are distinct from the physiologically active pool of tau (Iqbal et al., 2010).

[0006] The progression of tauopathy in an AD brain follows distinct spreading patterns. A tauopathy transmission and spreading hypothesis has been described based on the Braak stages of tauopathy progression in the human brain and tauopathy spreading after tau aggregate injections in preclinical tau models (Frost et al., 2009; Clavaguera et al., 2009). It is believed that tauopathy can spread in a prion-like fashion from one brain region to the next. This spreading process would involve an externalization of tau seeds that can be taken up by nearby neurons and induce further tauopathy.

[0007] The National Institute on Aging and Alzheimer’s Association (NIA-AA) Research Framework provides a scheme, termed “AT(N),” for the diagnosis of AD based on measurements that relate to the underlying pathologic processes, beta-amyloid deposition (A), pathologic tau (T), and neurodegeneration (N) (Jack, Jr., et al., 2018). While plasma amyloid-beta and phosphorylated tau (p-tau) have shown promise as reliable biomarkers of A and T, respectively, in the AT(N) framework (Nakamura et al., 2018; Schindler et al., 2019; Palmqvist et al., 2020; Thijssen et al., 2021), a reliable biomarker for N remains elusive. For instance, plasma neurofilament light chain (NFL) has shown that it can effectively identify Alzheimer’s disease compared with controls, but it is unable to distinguish AD from other neurodegenerative diseases (Ashton et al., 2021; Bridel et al., 2019).

[0008] Total-tau (t-tau) has exhibited promise as a biomarker for neurodegeneration. T- tau from CSF has been shown to reliably reflect neurodegeneration in Alzheimer’s disease and not in other neurodegenerative diseases such as Parkinson’s disease, Lewy body dementia, and frontotemporal dementia (Grothe et al., 2021; Sjogren et al., 2001). However, use of CSF in a diagnostic can be difficult, as retrieval of CSF requires patients to undergo invasive lumbar puncture procedures involving physicians inserting a needle into the spinal canal to collect samples of CSF for use in assays; such procedures are uncomfortable and burdensome, and therefore are not desirable for repeating frequently and not suitable for regular monitoring of disease states in patients. And in a plasma assay, t-tau has not demonstrated good diagnostic utility Barthelemy et al., 2020; Frank et al., 2022), as plasma t- tau concentrations do not correlate with CSF t-tau and plasma assays may be detecting tau from tissues outside the central nervous system (CNS) (Barthelemy et al., 2020; Dugger et al., 2016). Thus, while t-tau may have potential as a biomarker of neurodegeneration, there is no current t-tau assay that is practical and reliable.

SUMMARY OF INVENTION

[0009] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0010] In one aspect, provided herein is an assay method of 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau.

[0011] In another aspect, provided herein is an assay method of measuring central nervous system (CNS)-derived total 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau; and (c) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the blood-based sample.

[0012] In some embodiments, the solid phase is a magnetic bead.

[0013] In some embodiments, the blood-based sample is a plasma sample.

[0014] In some embodiments, the methods further comprise obtaining the sample from the subject. In additional embodiments, the methods further comprise washing the captured tau peptides before contacting the captured tau peptides with the detection antibody. The captured tau peptides may be washed, for example, with a stringent buffer.

[0015] In some embodiments, the concentration of the detected tau or t-tau in the bloodbased sample is correlated with concentration of p217+tau in the blood-blood sample. In some embodiments, the concentration of the detected tau or t-tau in the blood-based sample is correlated with concentration of NFL in the blood-blood sample

[0016] In another aspect, provided herein is a method of detecting amyloid status of 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau; and (c) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the blood-based sample. The amyloid status is positive if the amount of CNS- derived t-tau peptides in the blood-based sample is above a predetermined threshold value, and the amyloid status is negative if the amount of CNS-derived t-tau peptides in the bloodbased sample is below a predetermined threshold value.

[0017] In yet another aspect, provided herein is a kit comprising (i) a capture antibody that binds to a tau epitope; and (ii) a detection antibody that binds to an epitope comprising amino acids residues that span the junction of Exon 4 and Exon 5 of central nervous system (CNS)-derived tau.

[0018] In some embodiments, the capture antibody binds to an epitope comprising amino acid residues 7-20 of human tau protein, in which the numbering of the amino acid residues is with reference to the amino acid sequence of SEQ ID NO:1. In certain embodiments, the capture antibody comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively. In some embodiments, the capture antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 12. In preferred embodiments, the capture antibody is hT43. [0019] In some embodiments, the detection antibody binds to an epitope comprising amino acid residues 116-127 of human tau protein, in which the numbering of the amino acid residues is with reference to the amino acid sequence of SEQ ID NO:1. In certain embodiments, the detection antibody comprises HCDR1, HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively. In some embodiments, the detection antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 20. In preferred embodiments, the detection antibody is pT82.

[0020] In other embodiments, the detection antibody binds to an epitope comprising amino acid residues 119-126 of human tau protein, in which the numbering of the amino acid residues is with reference to the amino acid sequence of SEQ ID NO:1. In certain embodiments, the detection antibody comprises (i) HCDR1 comprising an amino acid sequence of SEQ ID NO: 21, HCDR2 comprising an amino acid sequence of SEQ ID NO: 22, HCDR3 comprising an amino acid sequence of SEQ ID NO: 23, LCDR1 comprising an amino acid sequence of SEQ ID NO: 24, LCDR2 comprising an amino acid sequence of SEQ ID NO: 25, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (ii) HCDR1 comprising an amino acid sequence of SEQ ID NO: 27, HCDR2 comprising an amino acid sequence of SEQ ID NO: 28, HCDR3 comprising an amino acid sequence of SEQ ID NO: 29, LCDR1 comprising an amino acid sequence of SEQ ID NO: 24, LCDR2 comprising an amino acid sequence of SEQ ID NO: 25, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (iii) HCDR1 comprising an amino acid sequence of SEQ ID NO: 30, HCDR2 comprising an amino acid sequence of SEQ ID NO: 31, HCDR3 comprising an amino acid sequence of SEQ ID NO: 32, LCDR1 comprising an amino acid sequence of SEQ ID NO: 33, LCDR2 comprising amino acid sequence LVS, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (iv) HCDR1 comprising an amino acid sequence of SEQ ID NO: 35, HCDR2 comprising an amino acid sequence of SEQ ID NO: 36, HCDR3 comprising an amino acid sequence of SEQ ID NO: 23, LCDR1 comprising an amino acid sequence of SEQ ID NO: 24, LCDR2 comprising an amino acid sequence of SEQ ID NO: 25, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (v) HCDR1 comprising an amino acid sequence of SEQ ID NO: 37, HCDR2 comprising an amino acid sequence of SEQ ID NO: 38, HCDR3 comprising an amino acid sequence of SEQ ID NO: 39, LCDR1 comprising an amino acid sequence of SEQ ID NO: 40, LCDR2 comprising an amino acid sequence of SEQ ID NO: 41, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 42. In some embodiments, the detection antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 43, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44. In preferred embodiments, the detection antibody is hT36.

[0021] In yet another aspect, provided herein is an antibody or antigen-binding fragment thereof comprising (i) HCDR1 comprising an amino acid sequence of SEQ ID NO: 21, HCDR2 comprising an amino acid sequence of SEQ ID NO: 22, HCDR3 comprising an amino acid sequence of SEQ ID NO: 23, LCDR1 comprising an amino acid sequence of SEQ ID NO: 24, LCDR2 comprising an amino acid sequence of SEQ ID NO: 25, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (ii) HCDR1 comprising an amino acid sequence of SEQ ID NO: 27, HCDR2 comprising an amino acid sequence of SEQ ID NO: 28, HCDR3 comprising an amino acid sequence of SEQ ID NO: 29, LCDR1 comprising an amino acid sequence of SEQ ID NO: 24, LCDR2 comprising an amino acid sequence of SEQ ID NO: 25, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (iii) HCDR1 comprising an amino acid sequence of SEQ ID NO: 30, HCDR2 comprising an amino acid sequence of SEQ ID NO: 31, HCDR3 comprising an amino acid sequence of SEQ ID NO: 32, LCDR1 comprising an amino acid sequence of SEQ ID NO: 33, LCDR2 comprising amino acid sequence LVS, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (iv) HCDR1 comprising an amino acid sequence of SEQ ID NO: 35, HCDR2 comprising an amino acid sequence of SEQ ID NO: 36, HCDR3 comprising an amino acid sequence of SEQ ID NO: 23, LCDR1 comprising an amino acid sequence of SEQ ID NO: 24, LCDR2 comprising an amino acid sequence of SEQ ID NO: 25, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 26; or (v) HCDR1 comprising an amino acid sequence of SEQ ID NO: 37, HCDR2 comprising an amino acid sequence of SEQ ID NO: 38, HCDR3 comprising an amino acid sequence of SEQ ID NO: 39, LCDR1 comprising an amino acid sequence of SEQ ID NO: 40, LCDR2 comprising an amino acid sequence of SEQ ID NO: 41, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising an amino acid sequence of SEQ ID NO: 43, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44. Also provided herein is an antibody or antigen-binding fragment thereof, comprising (i) HCDR1, HCDR2, and HCDR3 contained within a heavy chain variable region comprising an amino acid sequences of SEQ ID NO: 43, and (ii) LCDR1, LCDR2, and LCDR3 contained within a heavy chain variable region comprising an amino acid sequences of SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 45, and a light chain comprising an amino acid sequence of SEQ ID NO: 46.

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 provides plot of dilution linearity of hT43xpT82 and pT82xhT43 sandwich enzyme-linked immunosorbent assay (ELISA), as described in Example 1.

[0023] FIG. 2 provides plots of standard curve of hT43xpT82 sandwich ELISA using three-step protocol (Panel A), dilution linearity (Panel B), and recovery at different dilutions (Panel C), as described in Example 1.

[0024] FIG. 3 provides a schematic of the structure of Peptide 1 (Panel A) and plots of surface plasmon resonance (SPR) change for antibodies hT36 (Panel B) and pT82 (Panel C) as a measurement of antibody-Peptide 1 binding, as described in Example 2.

[0025] FIG. 4 provides a schematic of the structure of Peptide 2 (Panel A) and plots of surface plasmon resonance (SPR) change for antibodies hT36 (Panel B) and pT82 (Panel C) as a measurement of antibody-Peptide 2 binding, as described in Example 2.

[0026] FIG. 5 provides a schematic of the structure of Peptide 3 (Panel A) and plots of surface plasmon resonance (SPR) change for antibodies hT36 (Panel B) and pT82 (Panel C) as a measurement of antibody-Peptide 3 binding, as described in Example 2.

[0027] FIG. 6 shows plots of p217+tau concentration versus plasma CNS -derived t-tau concentrations in cohort 1 (Panel A) and cohort 2 (Panel B) samples, and plots of NFL concentration versus plasma CNS-derived t-tau concentrations in cohort 1 (Panel C) and cohort 2 (Panel D) samples, as described in Example 3.

[0028] FIG. 7 shows plots of plasma CNS-derived t-tau concentrations in New Therapeutics in Alzheimer’s Disease Longitudinal Cohort study (NT AD) amyloid negative (A-), NT AD amyloid positive (A+), and healthy control (HC) samples in cohort 1 (Panel A) and cohort 2 (Panel B) as described in Example 3. DETAILED DESCRIPTION

[0029] While some of the main embodiments of the present invention are described in the Summary of the Invention, Examples and Claims sections of this patent disclosure, this Detailed Description section provides certain additional description relating to the invention and is intended to be read in conjunction with all other sections of the present patent application.

[0030] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0031] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0032] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.

Definitions

[0033] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0034] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0035] As used herein, the terms “comprising” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude the presence or addition of one or more features, or components, or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting of’. Consequently, the term “consisting of’ can be used in place of the terms “comprising” and “including” to provide for more specific embodiments.

[0036] As used herein, the term “or” is to be interpreted as an inclusive “or” meaning any one or any combination. 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. An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0037] Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase 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).

[0038] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.

Likewise, a disclosed range is a disclosure of each individual value (z.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5- 10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0039] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements is to be understood to refer to every element in the series. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of about 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).

[0040] Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes. [0041] 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 combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The terms “antibody” or “immunoglobulin” are used interchangeably herein.

[0042] A typical antibody comprises at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region and a light chain constant region (CL). The light chain constant region is comprised of one domain, Cl. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can 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 (Clq) of the classical complement system.

[0043] Antibodies can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses (isotypes) thereof e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. There are two classes of mammalian light chains, lambda and kappa.

[0044] The heavy and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with regions that are more conserved, termed framework (FW) regions. The CDRs in each chain are held together in close proximity by the FW regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. Each heavy and light chain variable region is composed of three CDRs: CDR1, CDR2, and CDR3.

[0045] There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Kabat et al., 1991); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs. [0046] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a, according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavy chain FW residue 82.

[0047] The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia & Lesk, 1987). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions 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 regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’ s AbM antibody modeling software.

[0048] IMGT (ImMunoGeneTics) also provides a numbering system for the variable regions, including the CDRs (see, e.g., Lefranc et al., 2003). The IMGT numbering system was based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows for easy comparison of the variable and CDR regions for all species. According to the IMGT numbering schema heavy chain variable region CDR-1 is at positions 26 to 35, heavy chain variable region CDR-2 is at positions 51 to 57, heavy chain variable region CDR-3 is at positions 93 to 102, light chain variable region CDR-1 is at positions 27 to 32, light chain variable region CDR-2 is at positions 50 to 52, and light chain variable region CDR-3 is at positions 89 to 97.

[0049] In addition, the variable regions can be delineated based on “Specificity Determining Residue Usage” (SDRU) (Almagro 2004), where SDR, refers to amino acid residues of an immunoglobulin that are directly involved in antigen contact. This SDRU concept was used to develop the “Contact” method of defining the CDRs, which renamed the SDRs as “contact residues” (MacCallum et al., 1996). [0050] 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 comprising an antigen-determination portion of an antibody; and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0051] A “monoclonal antibody” (mAb) refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term “monoclonal” can apply to both intact and full-length monoclonal antibodies, as well as to antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0052] The term “humanized antibody” refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the CDR are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986; Riechmann et al., 1998; Verhoeyen et al., 1988). In some instances, the Fv FW residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.

[0053] Humanized antibodies can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, humanized antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non- human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanized antibodies can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 5,225,539 and 5,639,641.

[0054] The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. The definition of a human antibody includes intact or full-length antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0055] The term “chimeric antibodies” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[0056] The term “antigen-binding fragment” refers to a portion of an intact antibody comprising the complementarity determining variable regions of the antibody. 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., ScFvs), and multi-specific antibodies formed from antibody fragments.

[0057] “Binding affinity” generally refers to the strength of the sum total 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 indicated otherwise, 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 a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.

[0058] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme- linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BIACORE™ or OCTET® analysis). Direct binding assays as well as competitive binding assay formats can be readily employed (see, e.g., Berzofsky et al., 1984; Kuby, 1992). The measured affinity of a particular antibody- antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD or Kd, K_on, Koff) are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art.

[0059] As used herein, the term “epitope” refers to a site on an antigen to which an immunoglobulin, antibody, or antigen-binding fragment thereof, specifically binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols, 1996).

[0060] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0061] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids and non-amino acids can interrupt it. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In certain embodiments, the polypeptides can occur as single chains or associated chains.

[0062] An “isolated” polypeptide, antibody, or antigen binding fragment is in a form not found in nature. Isolated polypeptides, antibodies, or antigen binding fragments include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, antibody, or antigen binding fragment that is isolated is substantially pure. When used herein, the term “substantially pure” refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0063] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides,” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0064] As used herein, the term “vector” is a replicon in which another nucleic acid segment can be operably inserted so as to bring about the replication or expression of the segment.

[0065] As used herein, the term “host cell” refers to a cell comprising a nucleic acid molecule of the invention. The “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a “host cell” is a cell transfected with a nucleic acid molecule of the invention. In another embodiment, a “host cell” is a progeny or potential progeny of such a transfected cell. A progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0066] The term “expression” as used herein refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed detection antibody or antigen binding fragment thereof that binds tau can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture, or anchored to the cell membrane.

[0067] As used herein, the term “tau” or “tau protein” or “tau peptide” refers to an abundant central and peripheral nervous system protein having multiple isoforms. In the human central nervous system (CNS), six major tau isoforms ranging in size from 352 to 441 amino acids in length exist due to alternative splicing (Hanger et al., 2009). The isoforms differ from each other by the regulated inclusion of 0-2 N-terminal inserts, and 3 or 4 tandemly arranged microtubule-binding repeats, and are referred to as 0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R. As used herein, the term “control tau” refers to the tau isoform of SEQ ID NO: 1 that is devoid of phosphorylation and other post-translational modifications. As used herein, the term “tau” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full-length wild type tau. The term “tau” also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0068] 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. An example of CNS-derived tau is tau that is present in CSF or tau found in the brain. CNS-derived tau is in contrast to tau that is not in the CNS, for example, that is produced by peripheral nerves or present in other tissues such as the liver, kidney, or heart.

[0069] As used herein, the term “total tau” or “t-tau” refers to a plurality of tau species that can be of any phosphorylation state; for example, the plurality of tau species can include non-phosphorylated tau species, phosphorylated tau species, or both non-phosphorylated tau species and phosphorylated tau species. A measurement of t-tau in a sample refers to measurement of a tau species that is not impacted by phosphorylation state. A “phosphorylated tau species” refers to tau protein phosphorylated in at least one site. The site(s) that are phosphorylated may be any of the sites described in Hanger et al. (2009).

[0070] Unless otherwise indicated, as used herein, the numbering of the amino acid in a tau protein or fragment thereof is with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0071] 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 support. Examples of solid supports include, but are not limited to, microparticles or beads, such as a magnetic beads or paramagnetic beads.

[0072] As used herein, the term “detection antibody” refers to an antibody that binds to an antigen of interest and has a detectable label or is linked to a secondary detection system. Examples of detectable labels 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, a monoclonal antibody that binds to tau protein

[0073] As used herein, “sandwich ELISA” refers to a type of ELISA that involves two antigen-binding molecules, often antibodies, that target different epitopes of the antigen. Typically, one of the antigen-binding molecules is linked to a solid support and is used to “capture” the antigen, i.e., facilitate the immobilization of the antigen. The other antigenbinding molecule is conjugated and facilitates the detection of the antigen.

[0074] As used herein a “tauopathy” encompasses any neurodegenerative disease that involves the pathological aggregation of tau within the brain. In addition to familial and sporadic AD, other exemplary tauopathies are frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, progressive subcortical gliosis, tangle only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy, such as dementia pugulistica (boxing disease) (Morris et al. , 2011).

[0075] As used herein, the term “amyloid status” refers to whether a subject is amyloid positive or amyloid negative. Amyloid status may be determined by methods known in the art, such as by measuring levels of CSF Ap42 or amyloid position emission tomography (PET) scanning; or may be determined by methods of the invention described herein.

[0076] As used herein, “amyloid positive,” “amyloid positivity,” and the like, refers to having cerebral amyloid- [3 accumulation associated with symptoms of Alzheimer’s disease or other amyloid-related conditions. As used herein, “amyloid negative,” “amyloid negativity,” and the like, refers to having no cerebral amyloid- [3 accumulation or in such a low amount that it is not associated with symptoms of Alzheimer’s disease or other amyloid-related conditions.

[0077] As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something present, as well as determining whether it is present or absent.

[0078] As used herein, the term “diagnosis” means detecting a disease or disorder or determining the stage or degree of a disease or disorder, such as a tauopathy. Usually, a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. A diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition, e.g., tau. Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease, i.e., there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease. The term “diagnosis” also encompasses determining the therapeutic effect of a drug therapy, e.g., an anti-tau antibody therapy, or predicting the pattern of response to a drug therapy, e.g., an anti-tau antibody therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical arts for a particular disease or disorder, e.g., Alzheimer’s disease.

[0079] As used herein, the terms “increase” and “decrease” refer to differences in quantity as compared to a control or reference level, such as differences in quantity of a particular biomarker in a sample. For example, the quantity of a particular peptide may be present at an elevated amount or at a decreased amount in samples of patients with a disease compared to a reference level. In one embodiment, an “increase of a level” or “decrease of a level” may be a difference between the level of biomarker present in a sample as 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, an “increase of a level” or “decrease of a level” may be a statistically significant difference between the level of the biomarker present in a sample as compared to a control. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviation, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 standard deviations of the mean of any control or reference group. The reference or control can be, for example, a sample from a healthy individual, or a sample taken from the same individual at an earlier time point, such as a time point prior to administration of a therapeutic or an earlier time point during a therapeutic regimen.

[0080] Other terms are defined elsewhere in this patent disclosure, or else are used in accordance with their usual meaning in the art.

Assays and Methods

[0081] The present application provides assays and methods for detecting and measuring CNS-derived tau peptides in blood-based samples, in particular, plasma. Collection of blood samples is fast and easy to perform and provides a reduced risk of infection or other complications as compared to lumbar puncture used for collection of CSF.

[0082] The assays and methods of the invention are based in part on the use of detection antibodies that can bind to CNS-derived tau peptides and that will not bind to tau peptides that are not CNS-derived, e.g., tau peptides produced by peripheral nerves or from tissues such as the liver, kidney, or heart. The main form of tau in peripheral tissues is distinguishable 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 of CNS-derived tau, Exon 4 is adjacent to Exon 5. Consequently, a detection protein that binds at least in part to amino acids 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.

[0083] Assays and methods of the present application measure CNS-derived t-tau peptides in blood-based samples with sufficient sensitivity, precision and accuracy. Therefore, the present application provides an improved way for measuring and/or monitoring CNS-derived t-tau peptide levels in subjects as compared to CSF-based assays, by minimizing the burden of sample collection on subjects and thereby enabling more frequent assaying and monitoring of changes in CNS-derived t-tau peptide levels, which is particularly desired to monitor and evaluate response to a treatment. The sample used in assays and methods of the present application may be a blood, serum, or plasma sample. Preferably, the sample is a plasma sample. More preferably, the plasma sample has not been immunoprecipitated to concentrate the CNS-derived tau peptides contained therein. In a particular embodiment, the sample is a crude plasma sample.

[0084] The assays and methods of the present application are directed in part to measurement of CNS-derived t-tau peptides in blood-based samples by using a capture antibody which binds to tau peptides in the sample to form capture antibody-tau peptide complexes. The capture antibody is preferably immobilized to a solid phase so that the capture antibody selectively binds to and immobilizes the tau peptides present in the sample to the solid phase. The captured tau peptides, i.e., the capture antibody-tau peptide complexes, are contacted with the detection antibody, which is labeled with a reporter element that allows detection of captured tau species that is CNS-derived. The assays and methods described herein can be used for various diagnostic purposes, e.g., for diagnosing AD, other tauopathies, other diseases characterized by deposits of amyloid-|3, or other amyloidogenic diseases in a subject; monitoring the effectiveness of a treatment; identifying a subject suitable for an anti-tau treatment; pre-screening subjects for PET imaging and/CSF assays for further detection of AD, other tauopathies, other diseases characterized by deposits of amyloid-P, or other amyloidogenic diseases; identification of subjects for enrollment in clinical trials relating to AD, other tauopathies, other diseases characterized by deposits of amyloid-P, or other amyloidogenic diseases; etc.

[0085] In some embodiments, the assays and methods of the present application include steps for contacting a blood-based sample with the capture antibody directed against a tau epitope to bind the capture antibody to tau peptides in the sample and form capture antibody- tau peptide complexes. The capture antibody-tau peptide complexes may then be contacted with a detection antibody to bind the detection antibody to capture antibody-tau peptide complexes that comprise CNS-derived tau peptides. The detection antibody may then be detected to determine an amount of CNS-derived tau peptides in the sample.

[0086] In certain embodiments, the capture antibody-tau peptide complexes may be washed before being contacted with a detection antibody. For example, the capture antibody- tau peptide complexes may be washed with any suitable solution that does not interfere with the assays, such as, for instance, a buffer solution (e.g., phosphate-buffered saline (PBS) solution). In certain embodiments, the buffer is capable of reducing and/or eliminating substantial matrix interference in blood or plasma. In preferred embodiments, the buffer is a stringent buffer.

[0087] As described herein (see BACKGROUND section, infra), historical measurements of plasma t-tau perform poorly as a diagnostic of Alzheimer’ s Disease. In contrast, the assays and methods of the present application can surprisingly rely on t-tau as a diagnostic by measuring t-tau that of CNS-origin, based at least in part on use of a capture antibody that recognizes an amino acid sequence not found in peripheral-origin tau. Therefore, the present application provides improved assays and methods that improve the use of t-tau as a diagnostic.

[0088] Thus, in one aspect, the assays and methods of the present invention measure CNS-derived t-tau peptides from a blood-based sample from a subject and subsequently determines that the subject has or is at risk of developing tauopathy and/or amyloidogenic disease when the amount of CNS-derived t-tau peptides measured from the blood-based sample is above a predetermined threshold value. The predetermined threshold value may be any suitable threshold value for distinguishing those subjects who have or are at risk of developing tauopathy and/or amyloidogenic disease as compared to those subjects who are healthy and not at risk of developing tauopathy and/or amyloidogenic disease. The predetermined threshold value may be determined as a plasma CNS-derived t-tau peptide concentration for: differentiating those patients above a level of tan in the brain or regions of the brain as measured by PET imaging and those below; differentiating those patients above a level of tau (e.g., phosphorylated tau such as pl 81 or p217+tau) in CSF and those below; differentiating those patients above a level of amyloid-[3 e.g., A [340 or A|342), such as in CSF or in plasma; differentiating those patients above a ratio of A|342 to A|340, such as in CSF or in plasma, and those below; and differentiating those patients that are cognitively normal and those patients that have dementia.

[0089] Subjects identified as having or are at risk of developing tauopathy and/or amyloidogenic disease may be directed to obtain further clinical tests, such as, for example, CSF collection and/or PET imaging, to further assess brain pathologies of these subjects. In other embodiments, subjects identified as having or are at risk of developing tauopathy and/or amyloidogenic disease may be administered an active agent for treating cognitive decline or tauopathy and/or amyloidogenic disease, for example, AD. Active agents for treating tauopathy may include anti-tau antibodies, anti-p217+tau antibodies, small interfering RNA (siRNA) against human tau, siRNA against p217+tau, cholinesterase inhibitors, N-methyl D- aspartate (NMD A) antagonist, etc. Active agents for amyloidogenic disease may include anti-amyloid antibodies, beta secretase inhibitors, gamma secretase inhibitors, small interfering RNA (siRNA) against human b-amyloid, cholinesterase inhibitors, N-methyl D- aspartate (NMD A) antagonist, etc.

[0090] In some embodiments, the predetermined threshold value may correspond to a baseline value or a value that is significantly higher than the baseline value. As used herein, “significantly higher” refers to a higher value that is statistically significant, not due to chance alone, which has a p-value of 0.05 or less. “Significantly higher” can be at least about 1%, 2%, 5%, or 10% higher than that found in healthy volunteers, at a p-value of less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc. The baseline value may correspond to a mean level in a population of healthy individuals. The baseline value may also correspond to a mean value of previous levels determined in the same subject.

[0091] According to embodiments of the present application, a value related to 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, it is determined that a subject is suffering from a tauopathy if the amount of CNS-derived t-tau peptides is significantly higher than a corresponding baseline amount of CNS-derived t-tau peptides. In some embodiments, it is determined that a subject is suffering from a tauopathy if a ratio related to the CNS-derived t-tau peptides is significantly higher than a corresponding baseline ratio.

[0092] In one aspect, a method of the present invention comprises (i) contacting a bloodbased sample, preferably a plasma sample, with a capture antibody directed against an epitope of tau to capture tau peptides in the sample, (ii) contacting the captured tau peptides with a detection antibody directed against an epitope comprising amino acids residues at the junction of Exon 4 and Exon 5 of CNS-derived tau, (iii) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the sample, and (iv) determining whether or not the subject suffers from a tauopathy or is at risk of developing a tauopathy based on the amount of the CNS-derived t-tau peptides or a ratio related to 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 the subject to corresponding predetermined threshold levels. Diagnosis can also be performed by comparing a ratio relating to the amount of CNS-derived t-tau peptides to a corresponding baseline ratio.

[0093] In another aspect, the effectiveness of a treatment in the subject can be determined by monitoring the amount of the CNS-derived t-tau peptides or a ratio relating to the amount of CNS-derived t-tau peptides before, during, or after the treatment. A decrease in values relative to baseline signals a positive response to treatment. Values can also increase temporarily in biological fluids as half-life of pathological tau in circulation is increased and/or pathological tau is being cleared from the brain.

[0094] According to some embodiments, the tauopathy includes, but is not limited to, one or more selected from the group consisting of Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick’s disease, progressive subcortical gliosis, tangle only dementia, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia complex, Down syndrome, Gerstmann- Straussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guamanian motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugulistica (boxing disease).

[0095] Preferably, the tauopathy is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease), FTDP-17, or progressive supranuclear palsy.

[0096] Most preferably, the tauopathy is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease).

[0097] According to another aspect, a method of the present invention comprises (i) contacting a blood-based sample, preferably a plasma sample, with a capture antibody directed against a tau epitope to capture tau peptides in the sample, (ii) contacting the captured tau peptides with a detection antibody directed against an epitope comprising amino acids residues at the junction of Exon 4 and Exon 5 of CNS-derived tau, (iii) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the sample, and (iv) determining whether or not the subject is suitable for an anti-tau antibody therapy based on the amount of the CNS-derived t-tau peptides or a ratio related to CNS-derived t-tau peptides.

[0098] According to certain embodiments, it is determined that a subject is suitable for an anti-tau antibody therapy if the amount of CNS-derived t-tau peptides in the blood-based sample, in particular, plasma sample, or the ratio related to CNS-derived t-tau peptides, is significantly higher than a corresponding baseline value.

[0099] In one aspect, a method of the present invention comprises (i) contacting a bloodbased sample, preferably a plasma sample, with a capture antibody directed against an epitope of tau to capture tau peptides in the sample, (ii) contacting the captured tau peptides with a detection antibody directed against an epitope comprising amino acids residues at the junction of Exon 4 and Exon 5 of CNS-derived tau, (iii) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the sample, and (iv) determining whether or not the subject suffers from an amyloidogenic disease or is at risk of developing an amyloidogenic disease based on 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 the subject to corresponding predetermined threshold levels.

[00100] The present application also relates to measuring CNS-derived tau peptides that is in complex with antibody in a blood-based sample, in particular, plasma, as well as free CNS- derived tau peptides in the sample that is not antibody-bound. In certain embodiments, total antibody is captured using affinity techniques, followed by denaturing conditions including chaotrophs, heat-inactivation, or other protein disruption techniques. The CNS-derived tau peptides is separated from antibody using rpHPLC, and is measured using methods of the present application, allowing for quantification of antibody-bound CNS-derived tau peptides.

[00101] According to one aspect, the invention relates to a method of monitoring a tauopathy treatment, such as an anti-tau antibody treatment in a subject, the method comprising: (i) contacting semi-denatured sample containing CNS-derived tau, which was obtained from a blood-based sample from the subject, with a capture antibody directed against tau epitope to capture tau peptides in the semi-denatured sample, (ii) contacting the captured tau peptides with a detection antibody directed against an epitope comprising amino acids residues at the junction of Exon 4 and Exon 5 of CNS-derived tau, (iii) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the semidenatured sample. In certain embodiments, the method further comprises obtaining a bloodbased sample, in particular, plasma sample, from the subject, and/or obtaining the semidenatured sample from the blood-based sample containing CNS-derived tau peptides.

[00102] The semi-denatured sample may be prepared from the blood-based sample containing CNS-derived tau peptides by degrading antibodies and/or other blood components that interfere with binding of the capture antibody and/or the detection antibody to CNS- derived tau peptides or interfere with detection of the detection antibody bound to CNS- derived tau peptides, without degrading the CNS-derived tau peptides present in the bloodbased sample. In some embodiments, the semi-denatured sample is prepared by heating the blood-based sample at a predetermined temperature that denatures antibodies for a predetermined amount of time. The predetermined temperature may be from 75 °C to 100 °C, from 80 °C to 90 °C, or 85 °C. The predetermined amount of time may be 0.1 to 30 minutes, 1 to 15 minutes, 2 to 10 minutes, 3 to 9 minutes, or 7 minutes. Following heat denaturing, the sample may optionally be cooled to a temperature that is suitably stable for the CNS-derived tau peptides (e.g., at or below 4 °C), to stop further degradation of proteins within the semi-denatured sample. In certain embodiments, the semi-denatured sample is prepared by heating the blood-based sample to 85 °C for 7 minutes and subsequently cooled in a 4 °C ice bath for 10 minutes. [00103] In certain embodiments, the method of monitoring a treatment is performed at one or more time points before treatment is administered, during treatment, after treatment, or a combination thereof. The effectiveness of the treatment may be determined by comparing the determined amount of CNS-derived t-tau peptides at different time points, wherein a decrease in the amount of a CNS-derived t-tau peptides between an earlier time point and a later time point is indicative of a positive response to treatment, and an increase in the amount of a CNS-derived t-tau peptides between an earlier time point and a later time point is indicative of a negative response to treatment.

[00104] In certain embodiments, the method of monitoring a treatment may further comprise determining values of antibody-free CNS-derived tau peptides and antibody-bound CNS-derived tau peptides. The effectiveness of the treatment may be determined by comparing the determined values of antibody-free CNS-derived tau peptides and antibodybound CNS-derived tau peptides at different time points, wherein a decrease in the amount of values of CNS-derived tau peptides at a later time point relative to a previous time point, or an increase in values of antibody-bound CNS-derived tau peptides at a later time point relative to an earlier time point, and therefore an increase in the ratio of the antibody-bound CNS-derived tau peptides to the antibody-free CNS-derived tau peptides at a later time point relative to an earlier time point, signals a positive response to treatment.

[00105] The indication of whether a response is positive or negative to treatment may be used for numerous different purposes including use as a decision tool to determine if the dose level or dosing interval of the treatment should be increased or decreased to ensure attainment or maintenance of efficacious or safe drug levels; use as an aid in the initiation of anti-tau drug therapy by providing evidence of the attainment of minimum pharmacokinetic (pK) levels; and use as an indication that a patient should be excluded from or included in a clinical trial and as an aid in the subsequent monitoring of adherence to clinical trial medication requirements.

[00106] In some embodiments, the concentration of the detected tau or t-tau in the sample is correlated with a concentration of p217+tau in the blood-blood sample. In other embodiments, the concentration of the detected tau or t-tau in the blood-based sample is correlated with a concentration of NFL in the sample.

[00107] According to one aspect, the invention relates to a method of detecting amyloid status of a subject. The method comprises 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau; and detecting the detection antibody to determine an 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 value, the amyloid status of the subject is positive. If the amount of CNS-derived t-tau peptides in the bloodbased sample is below a predetermined threshold value, the amyloid status is negative.

[00108] The predetermined threshold value may be any suitable threshold value for distinguishing those subjects who are amyloid positive as compared to those subjects who are amyloid negative. The predetermined threshold value may be determined as a plasma CNS- derived t-tau peptide concentration for: differentiating those patients above a level of amyloid in the brain or regions of the brain as measured by PET imaging and those below; differentiating those patients above a level of amyloid-[3 (e.g., A [340 or A|342), such as in CSF or in plasma; differentiating those patients above a ratio of A|342 to A|340, such as in CSF or in plasma, and those below; and differentiating those patients that are cognitively normal and those patients that have dementia.

[00109] In some embodiments, the predetermined threshold value may correspond to a value of plasma CNS-derived t-tau peptide concentration or an average value of plasma CNS- derived t-tau peptide concentration in subjects who were determined to be amyloid positive or amyloid negative via other diagnostic methods, for example, through measurement of A [342 in CSF, or ratio of A[342 to A|340 in CSF, or amyloid PET scanning, or a combination thereof.

[00110] In some embodiments, the predetermined threshold value may correspond to a baseline value or a value that is significantly higher than the baseline value. As used herein, “significantly higher” refers to a higher value that is statistically significant, not due to chance alone, which has a p-value of 0.05 or less. “Significantly higher” can be at least about 1%, 2%, 5%, or 10% higher than that found in healthy volunteers, at a p-value of less than 0.05, 0.04, 0.03, 0.01, 0.005, 0.001, etc. The baseline value may correspond to a mean level in a population of healthy individuals. The baseline value may also correspond to a mean value of previous levels determined in the same subject. [0001] In one aspect, the method of detecting amyloid status of a subject may be included or involved in an in vivo method, for example, a method of treating a subject with an amyloid-related condition, or a method for preventing, ameliorating, treating and/or decreasing amyloid- [3 deposition in an amyloid-related condition. The in vivo method may comprise administering a treatment to the subject, wherein the subject was determined to have the amyloid-related condition by the method of detecting amyloid status of a subject as described herein; if the amyloid status is positive, then the subject is determined to have an amyloid-related condition. Alternatively, the in vivo method may comprise detecting amyloid status of a subject, and if the amyloid status is positive, then administering a treatment to the subject. An amyloid-related condition may be characterized by the formation of plaques containing beta-amyloid protein. Such conditions include, but are not limited to Alzheimer’s disease, dementia associated with Trisomy 21 (Down's Syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy or hereditary cerebral hemorrhage with amyloidosis of the Dutch-type (HCHWA-D). Treatments of amyloid-related conditions are known in the art. In certain embodiments, the treatment may comprise an antibody or antigen binding fragment thereof that targets amyloid-|3, for examples, an antibody or antigen binding fragment thereof described in International Application Nos. PCT/EP2020/058395 or PCT/IB2017/056831, each of which is incorporated herein by reference.

[0002] In some embodiments, the method of detecting amyloid status of a subject may be included or involved in a method of diagnosing Alzheimer’s Disease, for example, as part of the AT(N) framework for diagnosing Alzheimer’s Disease.

[00111] According to some embodiments, the capture antibody of methods of the present invention is first bound to a solid support (e.g., microtiter dish, magnetic beads, etc.) before contacting with a sample. The detection antibody can contain or be attached to any detectable label (e.g., fluorescent molecule, biotin, etc.), which is directly detectable or detectable via a secondary reaction (e.g., reaction with streptavidin). Alternatively, a second reagent containing the detectable label can be used, where the second reagent has binding specificity for the primary antibody. In a particular embodiment, the detection antibody is biotinylated.

[00112] According to certain embodiment of the present invention, the amount of CNS- derived t-tau peptides measured in methods of the present application can be determined using any suitable techniques known in the art, including enzyme-linked immunosorbent assay (ELISA) and single molecule array platform. According to particular aspects, methods of the present application use a high sensitivity array platform, such as Quanterix Simoa or MSD S-plex, to measure the amount of CNS-derived t-tau peptides in a blood-based sample (specifically a plasma sample).

[00113] According to some embodiments, the assays and methods may further comprise measuring other biomarkers related to or indicative of tauopathy. Such biomarkers include, but are not limited to, amyloid-|3 (A|3), phosphorylated (p)-taul81, p217+tau, p231+tau, and NFL. Exemplified assays and methods for measuring such biomarkers are disclosed in PCT Publication Nos. WO/2018/083628, WO 2019/171258, and WO 2022/013286, which are incorporated herein by reference.

[00114] In some embodiments, the assays and methods of the present invention provide a bead-based assay for measuring CNS-derived t-tau peptides in blood-based samples, for example, assay and methods in which the capture antibody is bound to magnetic beads before contacting with a blood-based sample. In some embodiments, the assays and methods involve the use of a sample diluent that comprises a nonionic surfactant. In certain embodiments, the nonionic surfactant includes a hydrophilic polyethylene oxide chain and/or an aromatic hydrocarbon lipophilic or hydrophobic group. In certain embodiments, the nonionic surfactant is Triton X-100. The sample diluent may also comprise tris(hydroxymethyl)aminomethane (Tris). In particular embodiments, the sample diluent may further include other suitable components, such as NaCl, ethylenediaminetetraacetic acid (EDTA), heterophilic blocker, and/or Bovine Serum Albumin.

[00115] In some embodiments of the present invention, CNS-derived t-tau peptide measurements obtained from blood-based samples are further analyzed in a computing device to detect and/or predict tauopathy in a subject. In particular, the CNS-derived t-tau peptide measurements obtained from blood-based samples are analyzed by a computing device in combination with data corresponding to measurements obtained for other biomarkers that are also detectable from blood-based samples to provide further improved detection and/or prediction of tauopathy in the subject. The improved ability to detect and/or predict tauopathy, specifically AD, using biomarker(s) that can be adequately measured from bloodbased samples can be used for various diagnostic purposes, e.g., for diagnosing AD or other tauopathies in a subject, monitoring the effectiveness of a treatment, identifying a subject suitable for an anti-tau treatment, pre-screening subjects for PET imaging and/or CSF assays for further detection of AD or other tauopathies, identification of subjects for enrollment in clinical trials relating to AD or other tauopathies, etc.

[00116] In some embodiments, a computing device obtains the CNS-derived t-tau peptide measurements detected by the assay to generate tau data corresponding to the amount of CNS-derived t-tau peptides. The tau data may represent the amount of CNS-derived t-tau peptides detected by the assay. Alternatively, the tau data may represent a binary status (yes/no) indicating whether the amount of is above a predetermined threshold value. The assay is sufficiently sensitive, as discussed above, such that the predetermined threshold value is above a LLOQ of the assay method. The computing device may also obtain medical data of the subject, such as, for example, demographic information (e.g., age, sex), medical history, Electronic Medical Records (EMR), pharmacy data corresponding to the patient’s medication records, etc. In particular, the computer device may obtain biomarker data corresponding to measurement or binary status for at least one biomarker detected from the patient. The biomarker may be any suitable biomarker for tauopathy. Preferably, the biomarker is detectable from blood-based samples, in particular, plasma samples, of a subject. For example, the biomarker may be selected from a group consisting of amyloid- [3 (A|3), p-taul81, p217+tau, p231+tau, NFL, adiponectin, leptin, and other inflammatory or metabolic markers. More specifically, the biomarker is selected from NFL, adiponectin and leptin. The computing device analyzes the tau data and the biomarker data using a machine learning module to determine or predict whether the subject suffers from tauopathy or is at risk of developing tauopathy. The machine learning module is trained using a set of reference data. The machine learning module compares the tau data and the biomarker data to a set of reference data to determine or predict whether the subject has tauopathy or is at risk of developing tauopathy. The set of reference data includes tau data and biomarker data, along with data corresponding to brain pathology of tauopathy (e.g., stage of disease, amount of tau detected in CSF, PET measurements of tau in brain tissue, etc.), for a reference group of patients.

[00117] The machine learning module may be a supervised and/or unsupervised machine learning module. The machine learning module may be a machine learning classifier, for identifying dataset as correlating to one of two categories. The machine learning module may include support vector machine, random forest, logistic regression, gradient boosting module, or ensemble modules thereof. In some embodiments the machine learning module is an ensemble module comprising at least one of support vector machine, random forest, logistic regression, and/or gradient boosting module.

[00118] Those skilled in the art will understand that the exemplary computer-implemented embodiments described herein may be implemented in any number of manners, including as a separate software module, as a combination of hardware and software, etc. For example, the exemplary methods may be embodiment in one or more programs stored in a non- transitory storage medium and containing lines of code that, when compiled, may be executed by one or more processor cores or a separate processor. A system according to one embodiment comprises a plurality of processor cores and a set of instructions executing on the plurality of processor cores to perform the exemplary methods discussed above. The processor cores or separate processor may be incorporated in or may communicate with any suitable electronic device, for example, on board processing arrangements within the device or processing arrangements external to the device, e.g., a mobile computing device, a smart phone, a computing tablet, a computing device, etc. , that may be in communications with at least a portion of the device.

Capture and Detection Antibodies

[00119] The capture antibody for use 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 of human tau protein, or between amino acid residues 150 and 250 of human tau protein, in which the numbering of the amino acid is with reference to the amino acid sequence of SEQ ID NO:1 (see Table 1). In certain embodiments, the capture antibody binds to an epitope comprising amino acid residues 7-20 of human tau protein. In preferred embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 2 (see Table 1).

Table 1. Amino acid sequences of human tau protein and epitopes thereof.

[00120] Exemplary capture antibodies of the present invention may comprise the CDRs of antibody hT43, i.e., HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; and/or the heavy chain variable region and light chain variable region of antibody hT43, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 12 (see Table 2). In preferred embodiments, the capture antibody is hT43.

Table 2. Amino acid sequences of hT43 CD Rs and heavy and light chain variable regions.

[00121] The detection antibody for use in the assays and methods of the present invention binds to an epitope comprising amino acid residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau. The “junction of Exon 4 and Exon 5” refers to the point in CNS- derived tau in which Exon 4 and Exon 5 meet. Thus, amino acid residues that “span the junction of Exon 4 and Exon 5” comprise, in the N-terminal-to-C-terminal direction, at least the last amino acid residue in Exon 4 and the first amino acid residue of Exon 5.

[00122] In some embodiments, the detection antibody capture antibody is a monoclonal antibody that binds to an epitope that (a) comprises amino acid residue(s) 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) comprises amino acid residue(s) 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, of SEQ ID NO: 1.

[00123] In some embodiments, the detection antibody binds to an epitope comprising amino acid residues 116-127 of human tau protein. In certain 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 CDRs of antibody pT82, i.e., HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively; and/or the heavy chain variable region and light chain variable region of antibody pT82, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 20 (see Table 3). . Table 3. Amino acid sequences of pT82 CDRs and heavy and light chain variable regions.

[00124] In some embodiments, the detection antibody binds to an epitope comprising amino acid residues 119-126 of human tau protein. In preferred embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 4 (see Table 1). Exemplary detection antibodies of the present invention may comprise the CDRs of antibody hT36, i.e., HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 21,

22, and 23, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively, and LCDR1 comprising or consisting of an amino acid sequence of SEQ ID NO: 33, LCDR2 comprising or consisting of amino acid sequence LVS, and LCDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 26; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 35, 36, and

23, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively. Exemplary detection antibodies of the present invention may comprise the heavy chain variable region and light chain variable region of antibody hT36, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 43, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 44; and/or the heavy chain and light chain of antibody hT36, i.e., a heavy chain comprising or consisting of amino acid sequence of SEQ ID NO: 45 and a light chain comprising or consisting of amino acid sequence of SEQ ID NO: 46 (see Table 4).

Table 4. Amino acid sequences of hT36 CD Rs, heavy and light chain variable regions, and heavy and light chains.

[00125] An aspect of the present invention is also directed to an antibody or antigen binding fragment thereof that can bind to CDR-derived tan peptides. In some embodiments, the antibody or antigen binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 30, 31, and 32, respectively, and LCDR1 comprising or consisting of an amino acid sequence of SEQ ID NO: 33, LCDR2 comprising or consisting of amino acid sequence LVS, and LCDR3 comprising or consisting of an amino acid sequence of SEQ ID NO: 26; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 35, 36, and 23, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 24, 25, and 26, respectively; or HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively. In some embodiments, the antibody or antigen binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 contained within a heavy chain variable region comprising or consisting of an amino acid sequences of SEQ ID NO: 43; and LCDR1, LCDR2, and LCDR3 contained within a heavy chain variable region comprising or consisting of an amino acid sequences of SEQ ID NO: 44. In certain embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain region comprising or consisting of an amino acid sequence of SEQ ID NO: 43, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 44. In particular embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of an amino acid sequence of SEQ ID NO: 45, and a light chain comprising or consisting of an amino acid sequence of SEQ ID NO: 46.

[00126] A summary of the sequences provided herein are presented in Table 5.

Table 5. Sequence summary.

[00127] In addition to providing the detection antibodies, and fragments thereof, whose sequences are provided in Table 4, the present invention also encompasses variants and equivalents of these detection antibodies and antibody fragments. For example, such variants include humanized, chimeric, optimized, germlined, and/or other versions of any of the detection antibodies having the CDRs and/or variable regions of hT36. Likewise, in some embodiments variants of the sequences disclosed herein that comprise one or more substitutions, additions, deletions, or other mutations may be used. A heavy chain variable region and/or light chain variable region amino acid sequence or portion thereof, including a CDR sequence, can be, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to a sequence set forth herein, and/or comprise 1, 2, 3, 4, 5 or more substitutions, e.g., conservative substitutions, relative to a sequence set forth herein. In some embodiments a detection antibody according to the present invention comprises a heavy chain variable region and/or light chain variable region amino acid sequence, or portion thereof, that is 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to that of SEQ ID NO: 43 and/or SEQ ID NO: 44, and/or comprise 1, 2, 3, 4, 5 or more substitutions, e.g., conservative substitutions, relative to that sequence, but comprises the specific CDR sequences found within such heavy chain and/or light chain variable regions — i.e., any mutations (such as substitutions, additions, deletions, etc.) are outside of the CDRs. Such detection antibodies, i.e., having heavy chain and light chain variable regions with a certain percent similarity to a 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 nucleic acid molecules encoding heavy chain and/or variable light chain variable regions described herein, followed by testing of the encoded altered antibody molecule for binding to CNS-derived tau.

[00128] The detection antibodies and fragments thereof, whose sequences are provided in Table 4, can include, in addition to a heavy chain variable region and a light chain variable region, a heavy chain constant region or fragment thereof. In certain embodiments the heavy chain constant region is a human heavy chain constant region, e.g., a human IgG constant region, e.g., a human IgGl constant region. In addition, the detection antibody can include a light chain constant region or fragment thereof. In certain embodiments the light chain constant region is a kappa constant region or a lambda constant region, e.g., a human kappa constant region or a human lambda constant region.

[00129] In another general aspect, the present invention relates to an isolated polynucleotide encoding a detection antibody or antigen-binding fragment thereof whose sequences are provided in Table 4. It will be appreciated by those skilled in the art that the coding sequence of a protein can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding detection antibodies or antigen-binding fragments thereof of the invention can be altered without changing the amino acid sequences of the proteins. Exemplary isolated polynucleotides are polynucleotides encoding polypeptides comprising HCDR1 , HCDR2, and HCDR3 comprising or consisting of the amino acid sequences shown in Table 4, or polypeptides comprising LCDR1, LCDR2, and LCDR3 comprising or consisting of the amino acid sequences shown in Table 4. Other exemplary isolated polynucleotides are polynucleotides encoding antibody variable regions of the invention. Other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the antibodies of the invention are also within the scope of the invention. The isolated nucleic acids of the present invention can be made using well known recombinant or synthetic techniques. DNA encoding the monoclonal antibodies is readily isolated and sequenced using methods known in the art. Where a hybridoma is produced, such cells can serve as a source of such DNA.

Alternatively, display techniques wherein the coding sequence and the translation product are linked, such as phage or ribosomal display libraries, can be used. [00130] In another aspect, the present invention relates to a vector comprising an isolated polynucleotide encoding a detection antibody or antigen-binding fragment thereof of the invention. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an antibody or antigen-binding fragment thereof in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the invention.

[00131] In another aspect, the present invention relates to a host cell comprising an isolated polynucleotide encoding a detection antibody or antigen-binding fragment thereof of the invention. Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of antibodies or antigen-binding fragments thereof of the invention. Such host cells can be eukaryotic cells, bacterial cells, plant cells or archaeal cells. Exemplary eukaryotic cells can be of mammalian, insect, avian or other animal origins. Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NSO (European Collection of Cell Cultures (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 useful 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.

[00132] In another general aspect, the invention relates to a method of producing a detection antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a polynucleotide encoding the detection antibody or antigen-binding fragment thereof under conditions to produce a detection antibody or antigen-binding fragment thereof of the invention, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). Expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art.

Kits

[00133] In another general aspect, the present 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau. The capture antibody and detection antibody are in accordance with the present invention. The kit can be used to detect or measure CNS-derived tau peptides from blood-based samples (e.g., blood, serum, plasma).

[00134] In some embodiments, the capture antibody may be pre-bound to a solid phase, such as to the wells of a microtiter dish or to magnetic beads.

[00135] In some embodiments, the kit further comprises one or more of the components necessary and/or sufficient to perform a detection assay, including controls, directions for performing assays, and any necessary software for analysis and presentation of results. One skilled in the art will readily recognize that the disclosed capture and detection antibodies can be readily incorporated into any of the established kit formats that are well known in the art.

[00136] Embodiments of the present disclosure can be further described and understood by reference to the following non-limiting “Examples,” which describe in the preparation of certain exemplary sandwich ELISAs using the capture and detection antibodies of the invention. It will be apparent to those skilled in the art that many modifications to the specific description provided in the Examples can be practiced without undue experimentation and without departing from the scope of the present disclosure.

EXAMPLES

Example 1: Development of Sandwich Assays

[00137] Sandwich ELISAs to t-tau were developed on the Single molecule array (Simoa) HD-X instrument available from Quanterix Corp. (Boston, MA) per manufacturer- recommended Homebrew Assay development protocols. Monoclonal antibodies hT43 and pT82 as well as commercial antibody hT7 were selected for the assay. Screening was performed on recombinant full length tau protein (z.e., SEQ ID NO: 1) as well as human plasma samples, using sample diluent as described in Triana-Baltzer et al. (2021) (Diluent-1). The Simoa analyzer generates an output for an average number of enzymes per bead (AEB) based on these measurements.

[00138] Assays pairing hT43 and pT82 provided the greatest sensitivity, yielding rough lower limit of quantification (LLOQ) of 1 pg/ml (based on lowest concentration of recombinant tau that generated Signal/Noise (S/N) of 2x), dilution linear signal with several human plasma samples, and detectable plasma signal until at least 1:16 dilution (see FIG. 1, Tables 6 and 7). Selection of which antibody served as the capture antibody versus the detection antibody in the sandwich ELISAs did not substantially impact the sensitivity of the assays, however use of hT43 as the capture antibody was slightly more sensitive and was selected as the final pair. Two-step Simoa program (no wash step between capture antibodysample incubation and contact with detection antibody) and three-step Simoa program (with a wash step between capture antibody-sample incubation and contact with detection antibody) were evaluated, and the three-step Simoa program provided substantially more signal (data not shown).

Table 6. Calibration curve of hT43xpT82 (LLOQ based on S/N > 2 and CV < 20% = AEB of 0.009 = -1.25 pg/ml).

Table 7. Calibration curve of pT82xhT43 (LLOQ based on S/N > 2 and CV < 20% = AEB of 0.011 = ~1.25 pg/ml).

[00139] The sandwich ELISA conditions were further developed, using the hT43 and pT82 antibodes and a calibrant peptide comprising the hT43 epitope (amino acid residues 7- 20) and the pT82 epitope (amino acid residues 116-127) seperated by a PEG4 linker. The assay was tested under various conditions: (i) either antibody as the capture/detection antibody; (ii) two-step versus three-step protocol; and (iii) the sample Diluent- 1 vs Quanterix homebrew diluent, according to manufacturer’s instructions. Success was determined based on maximal sensitivity with calibrant peptide, plasma samples, dilution linearity with plasma, and spike recovery of calibrant in plasma.

[00140] The assay conditions yielded the most sensitive assay that still preserved good dilution linearity and spike recovery. The assay exhibited a dynamic range of 0.4 to 300 pg/ml, with 97% of the plasma samples measuring in the linear range (see FIG. 2, Panel A). LOD was detemined to be 28 fg/ml and LLOG was calculated as 123 fg/ml. Dilution linearity was shown from 1:8 to 1:32 dilution, and was achieved best with minimal 1:8 dilution (while preserving sample signal in assay linear range) (see FIG. 2, Panel B). Further, after spiking the samples with calibrant peptide at concentrations of 4 pg/ml, 80 pg/ml, and 1600 pg/ml, the assay showed a spike recovery of the calibrant peptide in the samples within 85% to 93% (average) (see FIG. 2, Panel C). Finally, intra-test precision of the assay was calculated as 13 ± 21.2 % (average ± SD).

Example 2: Detection of CNS-Derived Tan

[00141] As described herein (see Assays and Methods section, infra), it has been reported that CNS-derived tau mRNA is spliced in the brain and spinal cord to remove Exon 4A, while tau mRNA of peripheral origin does not have this splice event. Thus, tau of central origin would have a seamless junction from Exon 4-5, while tau of peripheral origin would not. The final amino acid of Exon 4 corresponds to amino acid 124 and the first amino acid of Exon 5 corresponds to amino acid 125. Exon 4a sequence is not considered in this numbering scheme, as most literature describes tau of CNS origin, thus ignores Exon 4A. Antibody pT82 recognizes an epitope corresponding to amino acid residues 116-127 of tau, and thus is specific for CNS-derived tau. Antibody hT36 is also found to recognize this same epitope.

[00142] To confirm the hypothesis that the hT43xpT82 assay recognizes only tau species of CNS origin, a series of peptides were synthesized that correspond to the Exon 4-4a junction (Peptide 1; see FIG. 3, Panel A), Exon 4-5 junction (Peptide 2; see FIG. 4, Panel A), and Exon 4A-5 junction (Peptide 3; see FIG. 5, Panel A). As noted above Peptide 2 would correspond to a sequence only found in CNS-derived tau, while Peptide 1 and Peptide 3 would correspond to sequences only found in peripheral-derived tau. The pT82 antibody and the hT36 antibody (as a backup for pT82) were immobilized on Biacore gold chip and surface plasmon resonance (SPR) change was measured after flowing increasing concentrations of Peptide 1, 2, or 3 over the chip.

[00143] Both antibodies did not show SPR change (a measure of antibody/peptide binding) with Peptide 1 (see FIG. 3, Panels B and C) or Peptide 3 (see FIG. 5, Panels B and C), but did show substantial binding to Peptide 2 (see FIG. 4, Panels B and C). These results provide evidence that pT82 and hT36 recognize a sequence found in CNS-derived tau, but not that found in peripheral-origin tau. pT82 had slightly tighter binding, confirming it as the ideal antibody to use in the Simoa assay.

Example 3: Detection of Amyloid Positivity in Alzheimer’s Disease

[00144] The Simoa CNS-derived t-tau assay using hT43 as the capture antibody and pT82 as the detection antibody was assessed for its clinical performance in detecting amyloid positivity. The assessments involved two cohorts of healthy control (HC) samples and samples from NTAD, as follows:

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

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

(“A+” = amyloid positivity). The HC samples were all amyloid negative as determined in CSF by Meso Scale Discovery (MSD) assay for AP42/40. The NTAD samples were 73% amyloid positive as determined by CSF A [>42/40 ratio or amyloid PET scanning. [00145] The results indicate that the plasma concentration of CNS -derived t-tau correlated with p217+tau concentrations in both cohorts 1 and 2 (see FIG. 6, Panels A and B, respectively), and with NFL concentrations in both cohorts 1 and 2 (see FIG. 6, Panels C and D, respectively) (see also Table 8). Further, the plasma concentration of CNS-derived t- tau were elevated in subjects who were amyloid positive versus negative subjects in both cohorts 1 and 2 (see FIG. 7, Panels A and B, respectively) (see also Table 9).

Table 8. Correlation between CNS-derived t-tau measured by the Simoa assay and plasma 217+tau and NFL concentrations.

Table 9. Average concentrations of CNS-derived t-tau in subjects with amyloid positivity and subjects with amyloid negativity (A-).

[00146] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying Figures. Such modifications are intended to fall within the scope of the appended claims.

[00147] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. REFERENCES

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

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.

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

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

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

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.

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.

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

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

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.

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.

Epitope Mapping Protocols, 1996, Morris GE, ed., 66.

Frank B, et al., Plasma p-taul81 shows stronger network association to Alzheimer's disease dementia than neurofilament light and total tau, Alzheimer's & Dementia, 2022, 18(8): 1523- 1536.

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

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

Grothe MJ, et al. , Associations of fully automated CSF and novel plasma biomarkers with Alzheimer disease neuropathology at autopsy, Neurology, 2021, 97(12): el229-el242.

Hanger DP, et al., Tau phosphorylation: the therapeutic challenge for neurodegenerative disease, Trends in Molecular Medicine , 2009, 15(3): 112-119.

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

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

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

Kabat EA, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

Kuby. Immunology, W. H. Freeman and Company: New York, 1992.

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.

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

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

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

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

Schindler S, et al., High-precision plasma P-amyloid 42/40 predicts current and future brain amyloidosis, Neurology, 2019, 93(17): el647-el659.

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

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): el2204.

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

Claims

1. An assay method of detecting central nervous system (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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau.

2. An assay method of measuring central nervous system (CNS)-derived total 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau; and

(c) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the blood-based sample.

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

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

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

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

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

8. The method of claim 7, wherein the captured tau peptides are washed with a stringent buffer.

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

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

11. A method of detecting amyloid status of 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 amino acids residues that span the junction of Exon 4 and Exon 5 of CNS-derived tau; and

(c) detecting the detection antibody to determine an amount of CNS-derived t-tau peptides in the blood-based sample, wherein the amyloid status is positive if the amount of CNS-derived t-tau peptides in the blood-based sample is above a predetermined threshold value, and wherein the amyloid status is negative if the amount of CNS-derived t-tau peptides in the blood-based sample is below a predetermined threshold value.

12. A kit comprising:

(i) a capture antibody that binds to a tau epitope; and

(ii) a detection antibody that binds to an epitope comprising amino acids residues that span the junction of Exon 4 and Exon 5 of central nervous system (CNS)-derived tau.

13. The method or kit of any one of claims 1-12, wherein the capture antibody binds to an epitope comprising amino acid residues 7-20 of human tau protein, wherein the numbering of the amino acid residues is with reference 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 (HCDRs) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively.

15. The method or kit of claim 14, wherein the capture antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising an 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 of any one of claims 1-16, wherein the detection antibody binds to an epitope comprising amino acid residues 116-127 of human tau protein, wherein the numbering of the amino acid residues is with reference 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 (HCDRs) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 16, 17, and 18, respectively.

19. The method or kit of claim 18, wherein the detection antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising an 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 of any one of claims 1-16, wherein the detection antibody binds to an epitope comprising amino acid residues 119-126 of human tau protein, wherein the numbering of the amino acid residues is with reference 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 (HCDRs) HCDR1, HCDR2, and HCDR3 and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein:

(i) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 22, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 23, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(ii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 28, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 29, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(iii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 30, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 31, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 32, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 33, the LCDR2 comprises amino acid sequence LVS, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(iv) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 35, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 36, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 23, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(v) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 37, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 38, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 39, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 40, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 41, and the LCDR3 comprises an 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 comprising an amino acid sequence of SEQ ID NO: 43, and a light chain variable region comprising an 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 antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein:

(i) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 22, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 23, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(ii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 28, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 29, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(iii) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 30, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 31, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 32, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 33, the LCDR2 comprises amino acid sequence LVS, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(iv) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 35, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 36, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 23, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 26; or

(v) the HCDR1 comprises an amino acid sequence of SEQ ID NO: 37, the HCDR2 comprises an amino acid sequence of SEQ ID NO: 38, the HCDR3 comprises an amino acid sequence of SEQ ID NO: 39, the LCDR1 comprises an amino acid sequence of SEQ ID NO: 40, the LCDR2 comprises an amino acid sequence of SEQ ID NO: 41, and the LCDR3 comprises an amino acid sequence of SEQ ID NO: 42.

26. The antibody or antigen-binding fragment thereof of claim 25, comprising a heavy chain region comprising an amino acid sequence of SEQ ID NO: 43, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 44.

27. An antibody or antigen-binding fragment thereof, comprising

(i) heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 contained within a heavy chain variable region comprising an amino acid sequences of SEQ ID NO: 43, and

(ii) light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 contained within a heavy chain variable region comprising an amino acid sequences of SEQ ID NO: 44.

28. The antibody or antigen-binding fragment thereof of any one of claims 25-27, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 45, and a light chain comprising an amino acid sequence of SEQ ID NO: 46.