Assays for detecting MTBR tau

Abstract

Provided herein are methods and assays for detecting MTBR-243 tau peptides in biological samples, including blood-based samples from subjects, involving the use of a capture antibody that binds to a MTBR-tau 243 species, and a detection antibody. In some embodiments, the methods and assays include an immunodepletion step using one or more immunodepletion antibodies.

Description

J&J Docket Number: JAB7242WOPCT1TITLEASSAYS FOR DETECTING MTBR TAUCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 733,269, filed December 12, 2024 and United States Provisional Patent Application No. 63 / 925,819, filed November 26, 2025, the disclosures of each which are incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is being submitted herewith electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 10, 2025, is named JAB7242WOPCTl_SL.xml and is 47,540 bytes in size.TECHNICAL FIELD

[0003] Disclosed herein are compositions and methods for detecting MTBR-243 tau peptides in a sample from a subject.BACKGROUND OF THE INVENTION

[0004] 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.J&J Docket Number: JAB7242WOPCT1

[0005] 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.

[0006] 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).

[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). A recent report showed that the microtubule-binding region (MTBR) of tau containing residue 243 (MTBR-tau 243) exhibits promise as a biomarker specific for AD-related tau pathology and cognitive impairment, as MTBR-tau243 was found to correlate more strongly with tau PET and MMSE scores than other biofluid tau measurements (Horie et al., 2023). Further work in plasma illustrated that a peptide endogenously cleaved at residue 256 but including residue 243 similarly demonstrated a greater association with tau PET and MMSE scores (Horie et al., 2025). There is a need for MTBR-tau 243 biofluid assays that are practical and reliable.SUMMARY OF INVENTION

[0008] 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 intendedJ&J Docket Number: JAB7242WOPCT1 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.

[0009] In one aspect, provided herein is an assay method of detecting MTBR-tau 243 peptides in a sample from a subject, the method comprising: (a) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, to capture tau peptides in the sample; and (b) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

[0010] In another aspect, provided herein is a method of measuring MTBR-243 tau peptides in a sample from a subject, the method comprising: (a) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, to capture tau peptides in the sample; and (b) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1, and (c) detecting the detection antibody to determine an amount of MTBR-243 tau peptides in the sample.

[0011] In some embodiments, the capture antibody is immobilized on a solid phase.

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

[0013] In some embodiments, the method further comprises obtaining the sample from the subject.

[0014] In some embodiments, the sample is a CSF or plasma sample.

[0015] In some embodiments, the method further comprises washing the captured tau peptides before contacting the captured tau peptides with the detection antibody.

[0016] In another aspect, provided herein is a kit comprising: (i) a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein; and (ii) a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.J&J Docket Number: JAB7242WOPCT1

[0017] In some embodiments, the the capture antibody binds to an epitope comprising amino acid residues 249-254 or an epitope comprising amino acid residues 251-254 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.

[0018] In some embodiments, the capture antibody comprises: (i) 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; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively.

[0019] In some embodiments, the capture antibody comprises: (i) 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, or (ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28.

[0020] In some embodiments, the capture antibody is PT15 or PT76.

[0021] In some embodiments, the detection antibody binds to an epitope comprising amino acid residues 151-158 , an epitope comprising amino acid residues 116-127 , or an epitope comprising amino acid residues 215-220 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.

[0022] In some embodiments, the detection antibody comprises (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprisingJ&J Docket Number: JAB7242WOPCT1 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; or (iii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively.

[0023] In some embodiments, the detection antibody comprises (i) 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, or (ii) 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, or (iii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 35, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 36.

[0024] In some embodiments, the detection antibody is pT51, pT82, or pT3.

[0025] In another aspect, provided herein is an antibody or antigen-binding fragment thereof, comprising (i) 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.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain 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.

[0027] In another aspect, provided herein is an antibody or antigen-binding fragment thereof, comprising (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectivelyJ&J Docket Number: JAB7242WOPCT1

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28.

[0029] In another aspect, provided herein is a method of detecting MTBR-243 tau peptides in a sample from a subject, the method comprising: (a) contacting the sample with an immunodepletion antibody, (b) removing from the sample peptides bound by the immunodepletion antibody, thereby producing an immunodepleted sample, (c) contacting the immunodepleted sample with a capture antibody to capture MTBR-243 tau peptides in the sample, wherein the capture antibody binds an epitope comprising amino acid 243 of human tau protein, and (d) contacting the captured MTBR-243 tau peptides with a detection antibody, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NOT.

[0030] In yet another aspect, a method of measuring MTBR-243 tau peptides in a sample from a subject is provided, the method comprising: (a) contacting the sample with an immunodepletion antibody, (b) removing from the sample peptides bound by the immunodepletion antibody, thereby producing an immunodepleted sample, (c) contacting the immunodepleted sample with a capture antibody to capture MTBR-243 tau peptides in the sample, wherein the capture antibody binds an epitope comprising amino acid 243 of human tau protein, contacting the captured MTBR-243 tau peptides with a detection antibody, and (d) detecting the detection antibody to determine an amount of MTBR-243 tau peptides in the sample, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NOT.

[0031] In some embodiments, the immunodepletion antibody binds to an epitope on human tau protein that is upstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody. In some embodiments, the immunodepletion antibody binds to an epitope on human tau protein that is downstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody. In some embodiments, the immunodepletion antibody binds to an epitope that is within amino acids 1 -242 of human tau protein. In some embodiments, the immunodepletion antibody binds to an epitope that is within amino acids 250- 441 of human tau protein. In some embodiments, the capture antibody binds to a tau epitopeJ&J Docket Number: JAB7242WOPCT1 within amino acids 243-254 of human tau protein. In some embodiments, the detection antibody binds to an epitope within amino acids 1-242 of human tau protein.

[0032] In still other embodiments, the capture antibody binds to a tau epitope within amino acids 243-254 of human tau protein, the detection antibody binds to an epitope within amino acids 1-242 of human tau protein, and the immunodepletion antibody binds to an epitope on human tau protein that is upstream of the epitope of the detection antibody.

[0033] In some other embodiments, wherein step (a) comprises contacting the sample with immunodepletion antibodies that bind to different epitopes on human tau. In some embodiments, at least one of the immunodepletion antibodies bind to an epitope on human tau protein that is upstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody, and / or (ii) at least one of the immunodepletion antibodies bind to an epitope on human tau protein that is downstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody.

[0034] In yet some other embodiments, the capture antibody is immobilized on a solid phase. In some embodiments, the solid phase is a magnetic bead.

[0035] In still some other embodiments, the method further comprises obtaining the sample from the subject. In some embodiments, the sample is a CSF or plasma sample. In some embodiments, the method further comprises washing the captured tau peptides before contacting the captured tau peptides with the detection antibody.

[0036] In some embodiments, the immunodepletion antibody or the immunodepletion antibodies is immobilized on a solid phase. In some embodiments, the solid phase is a magnetic bead. In some embodiments, the method further comprises washing the immunodepleted sample before contacting the immunodepleted sample with the capture antibody.

[0037] In another aspect, a kit is provided, the kit comprising: (i) a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein; and (ii) a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, and (iii) an immunodepletion antibody that binds to an epitope within amino acids 1-242 of human tau protein and upstream of the epitope of the detection antibody, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.J&J Docket Number: JAB7242WOPCT1

[0038] In some embodiments, the capture antibody binds to an epitope comprising amino acid residues 249-254 or an epitope comprising amino acid residues 251-254 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.

[0039] In some embodiments, the capture antibody comprises: (i) 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; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively.

[0040] In some embodiments, the capture antibody comprises: (i) 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, or (ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28.

[0041] In some embodiments, the capture antibody is PT15 or PT76.

[0042] In some embodiments, the detection antibody binds to an epitope comprising amino acid residues 151-158 , an epitope comprising amino acid residues 116-127 , or an epitope comprising amino acid residues 215-220 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.

[0043] In some embodiments, the detection antibody comprises (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprisingJ&J Docket Number: JAB7242WOPCT1 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; or (iii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively.

[0044] In some embodiments, the detection antibody comprises (i) 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, or (ii) 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, or (iii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 35, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 36.

[0045] In some embodiments, the detection antibody is pT51, pT82, or pT3.

[0046] In some embodiments, the capture antibody is pT15 and the detection antibody is pT3.

[0047] In some embodiments, the immunodepletion antibody or the immunodepletion antibodies comprise one or more antibodies selected from: an antibody that binds to an epitope comprising amino acids 116-127, an antibody that binds to an epitope comprising amino acids 151-158, an antibody that binds to an epitope comprising amino acids 166-182, and an antibody that binds to an epitope comprising amino acids 251-254 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.

[0048] In some embodiments, the immunodepletion antibody or the immunodepletion antibodies comprise one or more antibodies selected from: (i) an antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising aminoJ&J Docket Number: JAB7242WOPCT1 acid sequences of SEQ ID NOs: 40, 41, and 42, respectively; (ii) an antibody comprising 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; (iii) an antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively; and (iv) an antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 45, 46, and 47, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively.

[0049] In some embodiments, the immunodepletion antibody or the immunodepletion antibodies comprise one or more antibodies selected from: (i) an antibody comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28, (ii) an antibody comprising 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, (iii) an antibody comprising 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: 48, and (iv) an antibody comprising 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: 52.

[0050] In some embodiments, the immunodepletion antibody or the immunodepletion antibodies comprises one or more antibodies selected from: pT51, pT82, pT76, and pT89.

[0051] In some embodiments, the immunodepletion antibody or the immunodepletion antibodies binds to an epitope within amino acids 1 -242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1J&J Docket Number: JAB7242WOPCT1

[0052] In some embodiments, the MTBR-243 tau peptide detected or measured has a length of at least 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the MTBR-243 tau peptide detected or measured has a length of or no more than 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, or 50 amino acids. In some embodiments, the MTBR-243 tau peptide detected or measured comprises amino acids 210 to 254 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

[0053] In another aspect, provided herein is an antibody or antigen-binding fragment thereof, comprising (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 45, 46, and 47, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain region comprising an amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 52.BRIEF DESCRIPTION OF THE FIGURES

[0054] FIG. 1 provides epitopes for the antibodies PT15, pT76, pT51, pT82, and pT3 mapped onto the human tau protein sequence (2N4R, SEQ ID NO: 1); wherein the sequence numbering starts from the N-terminus of SEQ ID NO: 1.

[0055] FIGs. 2A-2D provide standard assay curves using indicated antibodies.

[0056] FIGs. 3A-3B provide signal detection results using either pT15 (FIG. 3A) or pT76 (FIG. 3B) as the capture Ab, using AD CSF Pool samples.

[0057] FIGs. 4A-4B provide the dilution linearity results in CSF samples.

[0058] FIGs. 5A-5B provide signal detection results using plasma samples.

[0059] FIGs. 6A-6B provide signal detection results using brain-spiked plasma and AD plasma pool samples.

[0060] FIGs. 7A-7B provide dilution linearity results in brain-spiked plasma samples.J&J Docket Number: JAB7242WOPCT1

[0061] FIG. 8 provides signal detection in samples from healthy controls and AD patients.

[0062] FIG. 9 provides signal detection in plasma samples.

[0063] FIG. 10 provides an illustration of an embodiment of the MTBR-243 tau assay.

[0064] FIG. 11 provides dilution linearity of MTBR+ assay in CSF.

[0065] FIGS. 12A-12C provide a pT217+ and MTBR+ assay comparison in crude CSF from a AD patient population.

[0066] FIG. 13 provides CSF immunodepletion with a single antibody to either the region N-terminal or C-terminal to amino acid 243 (C-terminal).

[0067] FIG. 14 provides higher conservation of MTBR signal in CSF compared to brain using immunodepletion approach developed to specifically target small fragments using 2 immunodepletion antibodies and 4 immunodepletion antibodies.

[0068] FIG. 15 provides fractionation of depleted CSF.

[0069] FIG. 16A provides an illustration of antibodies utilized for immunodepletion and immunoassay. FIGS. 16B-16C provide fractionation of depleted CSF using approach depicted in FIG. 16A

[0070] FIG. 17 provides an outline of a protocol of CSF immunodepletion from healthy controls and AD subjects.

[0071] FIGS. 18A-18B provide bar graphs representing the signal to noise comparison of AD (red) vs. HC (gray) in immunodepleted in crude samples analyzed by either 217+ (FIG.18A) or MTBR+ (FIG. 18B).

[0072] FIGS. 19A-19B provide scatter plots showing the association of MTBR+ with crude 217+ levels in either crude (FIG. 19A) or depleted (FIG. 19B) samples. AD samples are shown in red and HC samples shown in gray.DETAILED DESCRIPTION

[0073] 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 DetailedJ&J Docket Number: JAB7242WOPCT1Description 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.

[0074] 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.

[0075] 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.

[0076] 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

[0077] 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.

[0078] 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.

[0079] 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.J&J Docket Number: JAB7242WOPCT1

[0080] 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.

[0081] 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).

[0082] 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 (i.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.

[0083] 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).

[0084] 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.

[0085] The term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate,J&J Docket Number: JAB7242WOPCT1 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Kabat etal., 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.J&J Docket Number: JAB7242WOPCT1

[0090] 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.

[0091] 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 35 A 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.

[0092] IMGT (ImMunoGeneTics) also provides a numbering system for the variable regions, including the CDRs (see, e.g., Lefranc el 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.

[0093] 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 toJ&J Docket Number: JAB7242WOPCT1 develop the “Contact” method of defining the CDRs, which renamed the SDRs as “contact residues” (MacCallum et al., 1996).

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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, variableJ&J Docket Number: JAB7242WOPCT1 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] “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 bindJ&J Docket Number: JAB7242WOPCT1 antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.

[0102] 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 Ka, Kon, KOff) are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art.

[0103] 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).

[0104] 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.

[0105] 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 otherJ&J Docket Number: JAB7242WOPCT1 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.

[0106] As used herein, when referring to locations within an amino acid sequence of a protein or polypeptide, the term “upstream” refers to amino acid residue(s) N-terminal of a reference point, and the term “downstream” refers to amino acid residue(s) C-terminal of a reference point.

[0107] 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%.

[0108] 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 RNAJ&J Docket Number: JAB7242WOPCT1 characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 microtubulebinding 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.J&J Docket Number: JAB7242WOPCT1

[0113] As used herein, the term “MTBR-243 tau,” “MTBR-tau 243,” “MTBR-tau 243 peptide” or “MTBR-243 tau peptide” refers to tau protein or fragment which contains a microtubule-binding region (MTBR) of tau containing residue 243. The numbering (amino acid residue 243) is with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0114] 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.

[0115] 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.

[0116] 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

[0117] As used herein, “sandwich ELISA” refers to a type of ELIS A 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 antigen-binding molecule is conjugated and facilitates the detection of the antigen.

[0118] 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,J&J Docket Number: JAB7242WOPCT1 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).

[0119] 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.

[0120] 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.

[0121] 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 referenceJ&J Docket Number: JAB7242WOPCT1 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.

[0122] 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

[0123] The present application provides assays and methods for detecting and measuring tau containing residue 243 (MTBR-tau 243) in biological samples, such as cerebrospinal fluid (CSF), blood or plasma.

[0124] The assays and methods of the invention are based in part on the use of capture antibodies that can bind to an epitope within the microtubule-binding region (MTBR) of tau containing residue 243 or downstream of residue 243 (e.g., an epitope within 243-254 of tau), and the use of detection antibodies that can bind to an epitope upstream of residue 243 (e.g., an epitope within residues 1-242).

[0125] Assays and methods of the present application measure MTBR-tau 243 peptides in samples, including CSF or blood-based samples, with sufficient sensitivity, precision and accuracy. The sample used in assays and methods of the present application may be a fluid sample, such as a CSF, blood, serum, or plasma sample. In some embodiments, the sample is a CSF sample. In some embodiments, the sample is a blood-based sample. Preferably, the sampleJ&J Docket Number: JAB7242WOPCT1 is a plasma sample. In some embodiments, the plasma sample has not been immunoprecipitated to concentrate the MTBR-tau 243 peptides contained therein. In a particular embodiment, the sample is a crude plasma sample.

[0126] In some embodiment, the assays and methods to detect or measure MTBR-tau 243 peptides in samples, including CSF or blood-based samples, include a depletion step to remove larger tau fragments. The depletion step may use one or more immunodepletion antibodies. In some embodiments, the one or more immunodepletion antibodies include an antibody that targets an epitope upstream of the epitope of the capture antibody, an epitope upstream of the epitope of the detection antibody, an epitope downstream of the epitope of the capture antibody, or an epitope downstream of the epitope of the detection antibody.

[0127] The assays and methods of the present application are directed in part to measurement of MTBR-tau 243 peptides in biological 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 MTBR-tau 243 peptides present in the sample to the solid phase. The captured MTBR-tau 243 peptides, i.e., the capture antibody -MTBR-tau 243 peptide complexes, are contacted with the detection antibody, which is labeled with a reporter element that allows detection of captured MTBR-tau 243 species. 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-b, or other amyloidogenic diseases in a subject; monitoring the effectiveness of a treatment; identifying a subject suitable for an anti-tau treatment; prescreening subjects for PET imaging and / CSF assays for further detection of AD, other tauopathies, other diseases characterized by deposits of amyloid-b, or other amyloidogenic diseases; identification of subjects for enrollment in clinical trials relating to AD, other tauopathies, other diseases characterized by deposits of amyloid-b, or other amyloidogenic diseases; etc.

[0128] In some embodiments, the assays and methods of the present application include steps for contacting a biological sample with the capture antibody directed against a tau epitope to bind the capture antibody to MTBR-tau 243 peptides in the sample and form capture antibody- MTBR-tau 243 peptide complexes. The capture antibody- MTBR-tau 243 peptide complexesJ&J Docket Number: JAB7242WOPCT1 may then be contacted with a detection antibody to bind the detection antibody to capture antibody- MTBR-tau 243 peptide complexes that comprise MTBR-243 tau peptides. The detection antibody may then be detected to determine an amount of MTBR-243 tau peptides in the sample.

[0129] In certain embodiments, the capture antibody-MTBR-tau 243 peptide complexes may be washed before being contacted with a detection antibody. For example, the capture antibody- MTBR-tau 243 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 CSF, blood or plasma. In preferred embodiments, the buffer is a stringent buffer.

[0130] As described herein (see BACKGROUND section, infra), the microtubule-binding region (MTBR) of tau containing residue 243 (MTBR-tau 243) was found to exhibit promise as a biomarker specific for AD-related tau pathology and cognitive impairment, as MTBR-tau243 was found to correlate more strongly with tau PET and MMSE scores than other biofluid tau measurements (Horie et al., 2023). The assays and methods of the present application can detect MTBR-tau 243 in biological samples.

[0131] Thus, in one aspect, the assays and methods of the present invention measure MTBR- tau 243 peptides from a biological 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 MTBR-tau 243 peptides measured from the biological 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 MTBR-tau 243 peptide concentration for: differentiating those patients above a level of MTBR-tau 243 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-b (e.g., Ab40 or Ab42), such as in CSF or in plasma; differentiating those patients aboveJ&J Docket Number: JAB7242WOPCT1 a ratio of Ab42 to Ab40, such as in CSF or in plasma, and those below; and differentiating those patients that are cognitively normal and those patients that have dementia.

[0132] 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, cholinesterase inhibitors, N-methyl D-aspartate (NMD A) antagonist, etc.

[0133] 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.

[0134] According to embodiments of the present application, a value related to MTBR-tau 243 peptides in a sample, such as the amount of MTBR-tau 243 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 MTBR-tau 243 peptides is significantly higher than a corresponding baseline amount of MTBR-tau 243 peptides. In some embodiments, it is determined that a subject is suffering from a tauopathy if a ratio related to the MTBR-tau 243 peptides is significantly higher than a corresponding baseline ratio.

[0135] In one aspect, a method of the present invention comprises (i) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, to capture tau peptides in the sample; and (ii) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQJ&J Docket Number: JAB7242WOPCT1ID NO: 1; (iii) detecting the detection antibody to determine an amount of MTBR-tau 243 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 MTBR-tau 243 peptides or a ratio related to the amount of MTBR-tau 243 peptides. Diagnosis can be performed by comparing the amount or concentration of MTBR-tau 243 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 MTBR-tau 243 peptides to a corresponding baseline ratio.

[0136] In another aspect, the effectiveness of a treatment in the subject can be determined by monitoring the amount of the MTBR-tau 243 peptides or a ratio relating to the amount of MTBR-tau 243 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.

[0137] 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).

[0138] Preferably, the tauopathy is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease), FTDP-17, or progressive supranuclear palsy.

[0139] Most preferably, the tauopathy is Alzheimer’s disease (including familial Alzheimer’s disease and sporadic Alzheimer’s disease).J&J Docket Number: JAB7242WOPCT1

[0140] According to another aspect, a method of the present invention comprises (i) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243- 254 of human tau protein, to capture tau peptides in the sample; and (ii) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1, (iii) detecting the detection antibody to determine an amount of MTBR-tau 243 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 MTBR-tau 243 peptides or a ratio related to MTBR-tau 243 peptides.

[0141] According to certain embodiments, it is determined that a subject is suitable for an anti-tau antibody therapy if the amount of MTBR-tau 243 peptides in the biological sample, in particular, plasma sample, or the ratio related to MTBR-tau 243 peptides, is significantly higher than a corresponding baseline value.

[0142] In one aspect, a method of the present invention comprises (i) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, to capture tau peptides in the sample; and (ii) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1, (iii) detecting the detection antibody to determine an amount of MTBR-tau 243 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 MTBR-tau 243 peptides. Diagnosis can be performed by comparing the amount or concentration of MTBR-tau 243 peptides in a sample from the subject to corresponding predetermined threshold levels.

[0143] The present application also relates to measuring MTBR-243 tau peptides that is in complex with antibody in a biological sample, in particular, plasma, as well as free MTBR-243 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, heatinactivation, or other protein disruption techniques. The MTBR-243 tau peptides is separatedJ&J Docket Number: JAB7242WOPCT1 from antibody using rpHPLC, and is measured using methods of the present application, allowing for quantification of antibody-bound MTBR-243 tau peptides.

[0144] 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 the sample with a capture antibody that binds to a tau epitope within amino acids 243- 254 of human tau protein, to capture tau peptides in the sample; and (ii) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1, (iii) detecting the detection antibody to determine an amount of MTBR-tau 243 peptides in the semi-denatured sample. In certain embodiments, the method further comprises obtaining a blood- based sample, in particular, plasma sample, from the subject, and / or obtaining the semi-denatured sample from the blood-based sample containing MTBR-tau 243 peptides.

[0145] The semi-denatured sample may be prepared from the blood-based sample containing MTBR-243 tau peptides by degrading antibodies and / or other blood components that interfere with binding of the capture antibody and / or the detection antibody to MTBR-243 tau peptides or interfere with detection of the detection antibody bound to MTBR-243 tau peptides, without degrading the MTBR-243 tau peptides present in the blood-based sample. In some embodiments, the semi- denatured sample is prepared by heating the blood-based sample at a predetermined temperature 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 MTBR-243 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.

[0146] 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 theJ&J Docket Number: JAB7242WOPCT1 determined amount of MTBR-tau 243 peptides at different time points, wherein a decrease in the amount of a MTBR-tau 243 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 MTBR-tau 243 peptides between an earlier time point and a later time point is indicative of a negative response to treatment.

[0147] In certain embodiments, the method of monitoring a treatment may further comprise determining values of antibody-free MTBR-243 tau peptides and antibody-bound MTBR-243 tau peptides. The effectiveness of the treatment may be determined by comparing the determined values of antibody-free MTBR-243 tau peptides and antibody-bound MTBR-243 tau peptides at different time points, wherein a decrease in the amount of values of MTBR-243 tau peptides at a later time point relative to a previous time point, or an increase in values of antibody-bound MTBR-243 tau peptides at a later time point relative to an earlier time point, and therefore an increase in the ratio of the antibody-bound MTBR-243 tau peptides to the antibody-free MTBR- 243 tau peptides at a later time point relative to an earlier time point, signals a positive response to treatment.

[0148] 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.

[0149] In some embodiments, the concentration of the detected tau or MTBR-tau 243 in the sample is correlated with a concentration of p217+tau in the blood-blood sample.

[0150] 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 populationJ&J Docket Number: JAB7242WOPCT1 of healthy individuals. The baseline value may also correspond to a mean value of previous levels determined in the same subject.

[0151] 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.

[0152] According to certain embodiment of the present invention, the amount of MTBR-tau 243 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 MTBR-tau 243 peptides in a biological sample, for example a bloodbased sample (specifically a plasma sample).

[0153] 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-b (Ab), 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.

[0154] In some embodiments, the assays and methods of the present invention provide a bead-based assay for measuring MTBR-tau 243 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 non-ionic surfactant is Triton X- 100. The sample diluent may also comprise tris(hydroxymethyl)aminomethane (Tris). InJ&J Docket Number: JAB7242WOPCT1 particular embodiments, the sample diluent may further include other suitable components, such as NaCl, ethylenediaminetetraacetic acid (EDTA), heterophilic blocker, and / or Bovine Serum Albumin.

[0155] In some embodiments of the present invention, MTBR-tau 243 peptide measurements obtained from biological samples, for example blood-based samples are further analyzed in a computing device to detect and / or predict tauopathy in a subject. In particular, the MTBR-tau 243 peptide measurements obtained from biological samples are analyzed by a computing device in combination with data corresponding to measurements obtained for other biomarkers that are also detectable from biological 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 biological 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.

[0156] In some embodiments, a computing device obtains the biological peptide measurements detected by the assay to generate tau data corresponding to the amount of biological peptides. The tau data may represent the amount of biological 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. The computing device analyzes the tau data and the biomarker data using a machine learning module to determine or predict whether the subject suffers fromJ&J Docket Number: JAB7242WOPCT1 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.

[0157] 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.

[0158] 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, Detection, and Immunodepletion Antibodies

[0159] 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 249 and 254 of human tau protein, or between aminoJ&J Docket Number: JAB7242WOPCT1 acid residues 251 and 254 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 249-254 of human tau protein. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the capture antibody binds to an epitope comprising amino acid residues 251-254 of human tau protein. In some embodiments, the epitope comprises the amino acid sequence of SEQ ID NO: 4 (see Table 1).Table 1. Amino acid sequences of human tau protein and epitopes thereof.J&J Docket Number: JAB7242WOPCT1

[0160] In some embodiments, exemplary capture antibodies of the present invention may comprise the CDRs of antibody pT15, 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 pT15, 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 some embodiments, the capture antibody is pT15.Table 2. Amino acid sequences of pT15 CDRs and heavy and light chain variable regions.J&J Docket Number: JAB7242WOPCT1

[0161] In some embodiments, exemplary capture antibodies or immunodepletion antibodies of the present invention may comprise the CDRs of antibody pT76, 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: 25, 26, and 27, respectively; and / or the heavy chain variable region and light chain variable region of antibody pT76, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 28 (see Table 3). In some embodiments, the capture antibody is pT76. In some embodiments, the immunodepletion antibody is pT76.Table 3. Amino acid sequences of pT76 CDRs and heavy and light chain variable regions.J&J Docket Number: JAB7242WOPCT1In some embodiments, the detection antibody or immunodepletion antibody for use in the assays and methods of the present invention binds to an epitope within amino acids 1-242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

[0162] In some embodiments, the detection antibody or immunodepletion 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.

[0163] In some embodiments, the detection antibody or immunodepletion 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 or immunodepletion 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 4).J&J Docket Number: JAB7242WOPCT1Table 4. Amino acid sequences of pT82 CDRs and heavy and light chain variable regions.

[0164] In some embodiments, the exemplary detection antibody or immunodepletion antibody of the present invention may comprise the CDRs of antibody pT51, i.e., 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 or immunodepletion antibodies of the present invention may comprise the heavy chain variable region and light chain variable region of antibody pT51, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 43, and a light chainJ&J Docket Number: JAB7242WOPCT1 variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 44 (seeTable 5)Table 5. Amino acid sequences of pT51 CDRs (Kabat) and heavy and light chain variable regions.

[0165] An aspect of the present invention is also directed to an antibody or antigen binding fragment thereof that can bind to tau 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. In some embodiments, the antibody or antigen binding fragment thereof comprises HCDR1 , HCDR2, and HCDR3 contained within a heavy chain variable region comprising orJ&J Docket Number: JAB7242WOPCT1 consisting of an amino acid sequences of SEQ ID NO: 27; and LCDR1, LCDR2, and LCDR3 contained within a heavy chain variable region comprising or consisting of an amino acid sequences of SEQ ID NO: 28.

[0166] In some embodiments, the exemplary detection antibody of the present invention may comprise the CDRs of antibody pT3, i.e., HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively. Antibody pT3 has been previously described, for example, in U.S. Patent No. 9,371,376 which is incorporated herein by reference in its entirety. Exemplary detection antibodies of the present invention may comprise the heavy chain variable region and light chain variable region of antibody pT3, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 35, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 36 (see Table 6).Table 6. Amino acid sequences of pT3 CDRs (Kabat) and heavy and light chain variable regions.J&J Docket Number: JAB7242WOPCT1

[0167] An aspect of the present invention is also directed to an antibody or antigen binding fragment thereof that can bind to tau 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: 45, 46, and 47, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 49, 50 and 51, 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: 48; and LCDR1, LCDR2, and LCDR3 contained within a heavy chain variable region comprising or consisting of an amino acid sequences of SEQ ID NO: 52.In some embodiments, the exemplary immunodepletion antibody of the present invention may comprise the CDRs of antibody pT89, i.e., HCDR1, HCDR2, and HCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 45, 46, and 47, respectively, and LCDR1, LCDR2, and LCDR3 comprising or consisting of amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively. Exemplary immunodepletion antibodies of the present invention may comprise the heavy chain variable region and light chain variable region of antibody pT89, i.e., a heavy chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising or consisting of an amino acid sequence of SEQ ID NO: 52 (see Table 7).Table 7. Amino acid sequences of pT89 CDRs and heavy and light chain variable regions.J&J Docket Number: JAB7242WOPCT1

[0168] In addition to providing the capture antibodies, detection antibodies, immunodepletion antibodies and fragments thereof, whose sequences are provided in Tables 2-7, the present invention also encompasses variants and equivalents of these antibodies and antibody fragments. For example, such variants include humanized, chimeric, optimized, germlined, and / or other versions of any of the antibodies having the CDRs and / or variable regions of pTl 5 or pT76. 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 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: 11 and / or SEQ ID NO: 12, and / or comprise 1, 2, 3, 4, 5 or more substitutions, e.g., conservative substitutions, relative to that sequence, butJ&J Docket Number: JAB7242WOPCT1 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. . In some embodiments an 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: 24 and / or SEQ ID NO: 28, 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 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 MTBR-243 tau.

[0169] The antibodies and fragments thereof, whose sequences are provided in Table 2-6, 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 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.

[0170] In another general aspect, the present invention relates to an isolated polynucleotide encoding a antibody or antigen-binding fragment thereof whose sequences are provided in Tables 2-7. 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 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, andJ&J Docket Number: JAB7242WOPCT1HCDR3 comprising or consisting of the amino acid sequences shown in Tables 2-7, or polypeptides comprising LCDR1, LCDR2, and LCDR3 comprising or consisting of the amino acid sequences shown in Tables 2-7. 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.

[0171] In another aspect, the present invention relates to a vector comprising an isolated polynucleotide encoding a 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.

[0172] In another aspect, the present invention relates to a host cell comprising an isolated polynucleotide encoding a 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 suchJ&J Docket Number: JAB7242WOPCT1 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.

[0173] In another general aspect, the invention relates to a method of producing a antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof under conditions to produce a 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.KitsIn another general aspect, the present application relates to a kit comprising (a) a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, and (b) a detection antibody that binds to an epitope within amino acids 1-242 of human tau protein. The capture antibody and detection antibody are in accordance with the present invention. The kit can be used to detect or measure MTBR-243 tau peptides from biological samples (e.g., CSF, blood, serum, plasma).

[0174] 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.

[0175] 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.

[0176] 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 certainJ&J Docket Number: JAB7242WOPCT1 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.EXAMPLESExample 1: Generation of antibodies

[0177] Antibodies (Abs) PT15, pT76, pT51, and pT89 were generated using standard methods. The amino acid sequences for these antibodies are shown in Tables 2, 3, 5, and 7.

[0178] Amino acid sequences for pT82 antibody are shown in Table 4. Amino acid sequences for pT3 antibody are shown in Table 6.

[0179] Epitopes for the antibodies PT15, pT76, pT51, pT82, pT89, and pT3 are shown mapped onto the human tau protein sequence (SEQ ID NO: 1) in FIG 1; sequence numbering starts from the N-terminus of SEQ ID NO: 1.Example 2. Capture antibody and detection antibody paring.

[0180] Next, capture antibody / detection antibody paring was characterized, with the goal to improve assay sensitivity.

[0181] FIG 2A shows the average enzyme per bead (AEB), or raw signal, from the Single Molecule Assay (SiMoA) assay derived from a pT15 bead capture and either pT51 (circles) or pT82 (squares) detection antibody. FIG 2B shows signal-to-noise values derived from the assays in FIG 2A for the pT15 capture with pT51 detect assay (circles) and pT15 capture with pT82 detect assay (squares). FIG 2C shows AEB or raw SiMoA assay derived from a pT76 bead capture and either pT51 (circles) or pT82 (squares) detection antibody. FIG 2D shows signal-to- noise derived from the assays in FIG 2C for the pT76 capture with pT51 detect assay (circles) and pT76 capture with pT82 detect assay (squares).

[0182] Materials and Methods. pT15 capture beads were prepared according to Quanterix manufacturer’s instructions using the in-house pT15 antibody conjugated at 0.3 mg / mL. pT51 and pT82 biotinylated detection antibodies were prepared according to Quanterix manufacturer’s instructions and run in the assay at a concentration of 0.9 g / mL. Assays were run as 3 -stepJ&J Docket Number: JAB7242WOPCT1SiMoA assays on the Quanterix HD-X instrument. Calibrant protein was recombinant 2N4R tau (Sigma, SAE0076) resuspended in PBS and diluted in blocking buffer to the appropriate concentration. AEB is reported directly from the HD-X instrument. AEB results are plotted as the average of duplicate wells. Signal-to-noise is calculated as the average AEB signal from replicated wells of the sample divided by the average background AEB.

[0183] Results. pT15 capture paired with either pT51 or pT82 detection made a high sensitivity assay that was responsive to recombinant tau 2N4R concentration levels. pT76 capture similarly paired with pT51 and pT76 detection but the assays were less sensitive requiring a higher concentration of recombinant tau to generate a response.Example 3. Signal detection in in AD CSF samples.

[0184] Next, pT15 capture paired with either pT51 or pT82 detection antibodies were tested in samples of cerebrospinal fluid (CSF) from Alzheimer’s Disease (AD) patients.

[0185] FIG 3A shows results of pT15 capture Ab based assays in various dilutions of AD CSF samples. pT15 capture Ab paired with pT51 detection Ab is shown in circles. pT15 capture paired with pT82 detect is in squares. Results are plotted as signal-to-noise ratio. FIG3B shows results of pT76 capture based assays in various dilutions of AD CSF samples. pT76 capture Ab paired with pT76 detection Ab is shown in circles. pT76 capture paired with pT82 detect is shown in squares. Results are plotted as signal-to-noise ratio.

[0186] Materials and Methods . Utilizing the reagents and assay combinations described in Example 2, samples of pooled CSF from AD patients were analyzed on the Quanterix HD-X. CSF was diluted by a factor of 2, 4, 8 or 16 in blocking buffer for pTl 5 analyses and a factor of 4 or 8 for pT76 analyses. AEB is reported directly from the HD-X instrument. Signal-to-noise is calculated as the average AEB signal from replicated wells of the sample divided by the average background AEB.

[0187] Results. pT15 capture paired with either pT51 or pT82 detection showed a concentration dependent response to CSF from AD patients. pT76 capture paired with either pT51 or pT82 detection showed limited response in CSF samples at dilution factor of 4 which was numerically smaller at a dilution factor of 8.J&J Docket Number: JAB7242WOPCT1Example 4. Signal detection in CSF samples of varying dilutions.

[0188] Next, the dilution linearity for select capture / detection Ab pairs was evaluated.

[0189] FIG 4 shows the results of dilution linearity test in CSF samples. One pool of CSF is shown in circles, and one pool is shown in squares. Dotted lines represent the threshold for acceptable dilution linearity and are placed at 80% and 120% of the 1 to 4 dilution condition concentration. FIG 4A shows results of pT15 capture paired with pT51. FIG 4B shows results of pT15 capture paired with pT82 detect.

[0190] Materials and Methods. Samples of pooled CSF from AD patients were analyzed using the reagents and assay combinations described in Example 2 on the Quanterix HD-X. CSF was diluted by a factor of 2, 4, 8 or 16 in blocking buffer. The samples and raw values for AD CSF 1 were the same as those represented in Example 3. AD CSF 2 was an additional pool of CSF from AD patients. The results were presented as a % of the 1 to 4 dilution condition, which was chosen as the optimal dilution, to demonstrate dilution linearity. Values were calculated using the dilution factor corrected concentration calculated for each sample based on a recombinant tau 441 standard curve fit on the Quanterix HD-x software. The calculated concentration reflected the average of 2 replicate wells.

[0191] Results. The pT15 capture with pT51 detection assay demonstrated acceptable dilution linearity for CSF derived from AD patients at a 1 to 4 and 1 to 8 dilution conditions. The pT15 capture with pT82 detection assay demonstrated acceptable dilution linearity at all dilution conditions between a 1 to 2 and 1 to 16 dilution.Example 5. Signal detection in plasma samples.

[0192] Next, the select capture / detection Ab pairs were tested using plasma samples.

[0193] FIGs 5A-5B demonstrate results of human plasma analysis, including both healthy control samples and AD patient sample, as indicated. FIG. 5A shows the bar graph summarizing the results of pT15 capture with pT51 detection Ab analysis of human plasma samples. Results were plotted as signal-to-noise and each bar represents the reported value for one sample. FIG. 5B shows the bar graph summarizing the results of pT15 capture with pT82 detection Ab analysis of human plasma samples. Results were plotted as signal-to-noise and each bar represents the reported value for one sample.J&J Docket Number: JAB7242WOPCT1

[0194] Materials and Methods . Utilizing the reagents and assay combinations described inExample 2, individual patient plasma samples were analyzed on the Quanterix HD-X. All plasma samples were diluted 1 to 2 in blocking buffer. Healthy control samples 1-3 are from individual volunteers. All other plasma samples are unique pools either from healthy control or AD patients. Signal-to-noise ratios were calculated as the average AEB signal from replicated wells of the sample divided by the average background AEB.

[0195] Results. pT15 paired with either pT51 or pT82 gave low signal in plasma samples. The signal varied by sample and was near background levels for some samples.Example 6. Signal detection in brain-spiked plasma samples and AD plasma pools.

[0196] Next, the select capture / detection Ab pairs were tested using brain-spiked plasma samples and AD plasma pools.

[0197] FIGs. 6A-6B demonstrate the results of crude and brain-spiked plasma. AD plasma pool samples are shown as circles and squares. The brain-spiked plasma sample is shown in triangles. Results were plotted as signal-to-noise for both 1 to 2 and 1 to 4 dilution conditions. FIG. 6A shows the pTl 5 capture Ab with pT51 detection Ab analysis. FIG. 6B shows the pTl 5 capture with pT82 detection Ab analysis.

[0198] Materials and Methods. Samples of pooled plasma from AD patients were analyzed using the reagents and assay combinations described in Example 2 on the Quanterix HD-X. An additional plasma sample was spiked with 5000 fg / mL of human AD patient brain lysate. This lysate is known to be enriched for pathological tau common to AD relative to biofluid samples and thus serves as a positive control and is termed “plasma with AD brain spike”. All three samples were diluted 1 to 2 and 1 to 4 in blocking buffer. Signal-to-noise is calculated as the average AEB signal from replicated wells of the sample divided by the average background AEB.

[0199] Results. pT15 paired with either pT51 or pT82 gave a low signal in plasma samples. The signal was substantially higher in brain-spiked samples confirming that a signal was detectable in plasma and increasing the presumed concentration of tau generates a greater response. These results demonstrated a large dynamic range of the assay in plasma.J&J Docket Number: JAB7242WOPCT1Example 7. Signal detection in brain-spiked plasma samples of varying dilutions.

[0200] Next, the select capture / detection Ab pairs were tested using brain-spiked plasma samples of varying dilutions.

[0201] FIGs 7A-7B demonstrate results of dilution linearity test in plasma sample spiked with AD patient brain lysate. Dotted lines represent the threshold for acceptable dilution linearity and are placed at 80% and 120% of the 1 to 4 dilution condition concentration. FIG. 7A shows the results of pT15 capture Ab paired with pT51 detection Ab. FIGs 7B shows the results of pT15 capture Ab paired with pT82 detection Ab.

[0202] Materials and Methods. A plasma sample was spiked with 5000 fg / mL of human AD patient brain lysate and diluted by a factor of 2, 4, 8 or 16 in blocking buffer for analysis. Assays were run with the reagents and methods outlined in Example 2. The results were presented as a % of the 1 to 4 dilution condition, which was chosen as the optimal dilution, to demonstrate dilution linearity. Values were calculated using the dilution factor corrected concentration calculated for each sample based on a recombinant tau 441 standard curve fit on the Quanterix HD-x software. The calculated concentration reflects the average of 2 replicate wells.

[0203] Results. Both assays demonstrated a range of dilution linearity when plasma samples were spiked with AD patient derived brain lysate. pT15 capture paired with pT51 detection Ab met the criteria for dilution linearity from a factor of 2 to a factor of 8. pTl 5 capture paired with pT82 detection Ab met the criteria for dilution linearity from a factor of 4 to a factor of 16. While crude plasma was too dilute to demonstrate a range of linearity, when the matrix was spiked to increase signal, linearity can be demonstrated. These results suggested there was not a matrix effect on dilution linearity.Example 8. Signal detection in CSF samples from AD patients and healthy controls.

[0204] Next, the select capture / detection Ab pairs were tested using CSF samples from AD patients and healthy controls.

[0205] FIG 8 shows the bar graph representing the results from a comparison of a pT15 capture Ab with pT3 detection Ab assay in CSF samples from healthy control (HC) and AD patients. Bar graphs show the mean and standard deviation of each group; **** pO.OOOlJ&J Docket Number: JAB7242WOPCT1

[0206] Materials and Methods. 10 individual AD patients and 10 individual healthy volunteer CSF were selected from the commercial suppliers. Samples diluted 1 to 4 in blocking buffer. Samples were analyzed with a pTl 5 capture antibody and pT3 detection antibody. The capture reagents were the same as in prior examples. The pT3 detection antibody was made according to Quanterix manufacturer’s instructions and ran in the assay at a concentration of 0.9 g / mL. Assays were run as 3-step SiMoA assays on the Quanterix HD-X instrument. Results were plotted as signal-to-noise ratio, which was calculated as the average AEB signal from replicated wells of the sample divided by the average background AEB. A significant difference between the two groups was calculated using a two-tailed Mann-Whitney test.

[0207] Results. We detected a significant increase in signal from a pT15 capture and pT3 detect assay in AD patient CSF compared to healthy control CSF.Example 9. Signal detection in plasma samples from AD patients of varying dilutions.

[0208] FIG 9 shows the the signal-to-noise ratio identified in AD plasma samples with a pT15 capture and pT3 detection assay. One plasma pool is shown in circles and one is in suqares.

[0209] Materials and Methods. Samples of pooled plasma from AD patients were analyzed using the reagents and assay combinations described in example 8 on the Quanterix HD-X. Samples were diluted by a factor of 2, 4, 8 or 16 in blocking buffer. Results were plotted as signal-to-noise ratio, which is calculated as the average AEB signal from replicated wells of the sample divided by the average background AEB.

[0210] Results. The signal obtained was insufficient to demonstrate dilution linearity in AD plasma utilizing an assay that detected a significant difference in AD CSF compared to healthy control. The lowest dilution feasible on the instrument, 1 to 2, gave a signal that is approximately 50% above background.Example 10. MTBR+ Immunoassay

[0211] FIG. 10 is an illustration of an embodiment of the MTBR-243 tau assay, which uses pT15 as capture antibody and pT3 as detection antibody (hereinafter referred to as MTBR+ Immunoassay or MTBR+ assay). Domains of tau are denoted by different shades of gray:J&J Docket Number: JAB7242WOPCT1Projection Domain, PD; Proline Rich Region, PRR; Microtubule Binding Region, MTBR; and C-terminal region, C-term. General antibody binding regions are illustrated for the two antibodies, pT15 and pT3, denoting the minimum required fragment length for detection by this assay as roughly amino acid 210 to 254.

[0212] FIG. 11 shows dilution linearity of MTBR+ assay in CSF. Two AD patient pool samples, including moderate to severe disease stages, were analyzed for dilution linearity. Dilution factors of 4 to 8 fell within the acceptable criteria of 20% variation from baseline, which was defined as the 1 :2 dilution.Example 11. pT217+ tau assay and MTBR+ Immunoassay

[0213] The pT217+ tau assay using pT3 as capture antibody and hT43 as detection antibody is described in published patent applications WO2019 / 171258 and WO2022 / 013286.

[0214] FIGS. 12A-12C show a pT217+ and MTBR+ assay comparison in crude CSF from AD patient population with elevated levels of pT217+ tau. FIGS. 12Aand 12B show signal-to- noise plotted as a bar graph for healthy control (HC, N=10, grey) and AD (N=10, red) subjects. FIG. 12A shows pT217+ assay results. Mann- Whitey demonstrates a significant group difference. FIG. 12B shows MTBR+ assay results. Mann- Whitey demonstrates a significant group difference. FIG. 12C shows correlation between 217+ and MTBR+ assay results, Spearman correlation demonstrates a significant positive correlation between the two assays. These show that the MTBR+ assay, which is an assay that spans from phosphorylated 217 to early N-terminal region of MTBR, can reliably differentiate advanced AD from HC but is highly correlated with a longer fragment assay of pT217+ tau.Example 12. Optimization of immunodepletion approach to specifically target small fragments

[0215] As shown in FIG. 13, CSF was immunodepleted with a single antibody to either the region N-terminal or C-terminal to amino acid 243. Brain lysate depletion was included as a positive control. On average 45.8% of signal was depleted by a single N-terminal antibody. An average of 4.2% signal increase was observed following C-terminal immunodepletion. N- terminal depletion removes -50% of signal from brain and CSF. C-terminal depletion removes 50% of signal from brain but has little to no effect on CSF. These results indicate most tauJ&J Docket Number: JAB7242WOPCT1 fragments are already cleaved between 254-303 in CSF but N-terminal depletion can remove larger peptides.

[0216] FIG. 14 shows that the immunodepletion approach developed to specifically target small fragments still gets a signal in AD CSF. The schematic at the top of FIG. 14 shows the epitopes on human tau targeted by the capture antibody, detection antibody, and immunodepletion antibodies. Depletion is greater in brain where tau is expected to be less fragmented. Signal is diminished between 2 antibody depletion (using immunodepletion antibodies pT89 and pT76) and 4 antibody depletion (using immunodepletion antibodies pT51, pT82, pT89, and pT76).

[0217] FIG. 15 shows that fractionation of depleted CSF demonstrates a significant diminution of the major peak. Although signal is significantly reduced by immunodepletion, the major peak is still present. The minor peak around fraction 5 is diffuse but present in similar levels in immunodepleted fractions; thus it represents a larger share of the overall signal and may represent smaller fragments, thus increasing the specificity of the assay for smaller tau peptides.

[0218] FIGS. 16A-16C shows that fractionation of depleted CSF demonstrates a distinct pattern of signal between the full-length pT217+ assay and MTBR-specific pT217+ assay following immunodepletion. FIG. 16A illustrates the antibodies utilized for immunodepletion and immunoassay. CSF was first immunodepleted with a mixture of 4 antibodies pT51, pT76, pT89, and pT82; 2 targeting the PD(PD #1 and PD #2, which are pT82 and pT51, respectively) , one targeting the PRR (pT89), and one targeting the MTBR in a portion C-terminal to the MTBR+ capture antibody (pT76). The remaining tau proteoforms following immunodepletion were detected by either the pT217+ or MTBR+ assay. Both assays rely on the pT217+ epitope but the pT217+ assay relies on an antibody in the N-terminal portion of the PD and the MTBR+ relies on an antibody in the N-terminal portion of the MTBR.

[0219] In FIG. 16B, CSF was immunodepleted according to the strategy outlined in FIG. 16A and then fractionated via HPLC on a Cl 8 column. Crude CSF was fractionated by the same protocol. Replicate samples were analyzed by the pT217+ (black and gray) or MTBR+ assay (red and blue). Earlier fractions represent peptides eluted at a lower precent acetonitrile. In FIG. 16C, the immunodepleted samples presented in FIG. 16B were analyzed as a signal in immunodepleted fractions relative to signal in crude of the same fraction. Values >100% represent fractions where immunodepleted signal is greater than crude. Note in early fractionsJ&J Docket Number: JAB7242WOPCT1(likely smaller fragments) MTBR+ assay is higher for the immunodepleted fraction, this is the opposite of the conventional pT217+ assay which is lower in the immunodepleted fraction relative to crude. pT217+ is even lower in crude than the immunodeplted MTBR+ which aligns with this assay measuring a small fragment that is missed by the conventional 217+ assay.Example 13. Effect of immunodepletion on MTBR+ assay’s differentiation of Alzheimer’s disease

[0220] FIGS. 17-19 show the effect of immunodepletion on MTBR+ assay’s differentiation of Alzheimer’s disease. In FIGS. 17-19, CSF was immunodepleted according to the protocol outlined in FIG. 17 from HC (N=8) and AD (N=10) subjects.. Both crude and immunodepleted samples were analyzed by MTBR+ (duplicate) and pT217+ assays (singlet). FIGS. 18A-18B show bar graph representing the signal to noise comparison of AD (red) vs. HC (gray) in immunodepleted in crude samples analyzed by either pT217+ (FIG. 18A) or MTBR+ (FIG.18B).

[0221] In FIGS. 18A-18D, pT217+ and MTBR is near signal-to-noise in most depleted samples. Due to limited volume ran samples as 1 :3 rather than 1:2. Depletion is largest in higher baseline MTBR signals, likely reflects that most samples are below S / N to begin with. If analysis is separated by a depletion efficiency >75%, there is a trend toward increase in AD vs. HC in the high depletion group. All HC have a depletion efficiency <75%.

[0222] FIGS. 19A-19B show scatter plots showing the association of MTBR+ with crude pT217+ levels in either crude (FIG. 19A) or depleted (FIG. 19B) samples. AD samples are shown in red and HC samples shown in gray. The immunodepleted samples only give signal more than 20% above background in the MTBR+ assay as opposed to indication of correlation in crude samples.

[0223] 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.

[0224] 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 patentJ&J Docket Number: JAB7242WOPCT1 application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.J&J Docket Number: JAB7242WOPCT1REFERENCESAl-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 multi centre 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.J&J Docket Number: JAB7242WOPCT1Chothia C & Lesk AM, Canonical structures for the hypervariable regions of immunoglobulins.Journal of Molecular Biology, 1987, 196(4): 901-917.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 isease, Journal of Alzheimer s’ Disease, 2016, 51(2): 345-356.Epitope Mapping Protocols, 1996, Morris GE, ed., 66.Frank B, et al. , Plasma p-taul 81 shows stronger network association to Alzheimer's disease dementia than neurofilament light and total tau, Alzheimer's & Dementia, 2022, 18(8): 1523- 1536.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 tau: 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.J&J Docket Number: JAB7242WOPCT1Hanger DP, et al., Tau phosphorylation: the therapeutic challenge for neurodegenerative disease, Trends in Molecular Medicine, 2009, 15(3): 112-119.Horie K, et al., CSF MTBR-tau243 is a specific biomarker of tau tangle pathology in Alzheimer's disease, Nature Medicine, 2023, 29: 1954-1963Horie K, Salvado G, Koppisetti RK, et al. Plasma MTBR-tau243 biomarker identifies tau tangle pathology in Alzheimer's disease. Nat Med. 2025;31(6):2044-2053Iqbal 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.J&J Docket Number: JAB7242WOPCT1Lefranc 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-0 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 0-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.J&J Docket Number: JAB7242WOPCT1Triana-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. J&J Docket Number: JAB7242WOPCT1CLAIMS1. A method of detecting MTBR-243 tau peptides in a sample from a subject, the method comprising:(a) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, to capture tau peptides in the sample; and(b) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1 -242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

2. A method of measuring MTBR-243 tau peptides in a sample from a subject, the method comprising:(a) contacting the sample with a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein, to capture tau peptides in the sample; and(b) contacting the captured tau peptides with a detection antibody that binds to an epitope within amino acids 1 -242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1, and(c) detecting the detection antibody to determine an amount of MTBR-243 tau peptides in the 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 sample is a CSF or 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. A kit comprising:J&J Docket Number: JAB7242WOPCT1(i) a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein; and(ii) a detection antibody that binds to an epitope within amino acids 1 -242 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

9. The method or kit of any one of claims 1-8, wherein the capture antibody binds to an epitope comprising amino acid residues 249-254 or an epitope comprising amino acid residues 251-254 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.

10. The method or kit of claim 9, wherein the capture antibody comprises: (i) 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; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively;.

11. The method or kit of claim 10, wherein the capture antibody comprises: (i) 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, or (ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28.

12. The method or kit of claim 11, wherein the capture antibody is PT15 or PT76.

13. The method or kit of any one of claims 1-12, wherein the detection antibody binds to an epitope comprising amino acid residues 151-158 , an epitope comprising amino acid residues 116-127 , or an epitope comprising amino acid residues 215-220 of human tau protein,J&J Docket Number: JAB7242WOPCT1 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 detection antibody comprises (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively; or (ii) 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; or (iii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively.

15. The method or kit of claim 14, wherein the detection antibody comprises (i) 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, or (ii) 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, or (iii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 35, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 36.

16. The method or kit of claim 15, wherein the detection antibody is pT51, pT82, or pT3.

17. An antibody or antigen-binding fragment thereof, comprising (i) 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, respectivelyJ&J Docket Number: JAB7242WOPCT118. The antibody or antigen-binding fragment thereof of claim 17, comprising a heavy chain 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.

19. An antibody or antigen-binding fragment thereof, comprising (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively20. The antibody or antigen-binding fragment thereof of claim 19, comprising a heavy chain region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28.

21. A method of detecting MTBR-243 tau peptides in a sample from a subject, the method comprising:(a) contacting the sample with an immunodepletion antibody,(b) removing from the sample peptides bound by the immunodepletion antibody, thereby producing an immunodepleted sample,(c) contacting the immunodepleted sample with a capture antibody to capture MTBR-243 tau peptides in the sample, wherein the capture antibody binds an epitope comprising amino acid 243 of human tau protein, and(d) contacting the captured MTBR-243 tau peptides with a detection antibody, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

22. A method of measuring MTBR-243 tau peptides in a sample from a subject, the method comprising:(a) contacting the sample with an immunodepletion antibody,(b) removing from the sample peptides bound by the immunodepletion antibody, thereby producing an immunodepleted sample,(c) contacting the immunodepleted sample with a capture antibody to capture MTBR-243 tau peptides in the sample, wherein the capture antibody binds an epitope comprising amino acid 243 of human tau protein,J&J Docket Number: JAB7242WOPCT1(d) contacting the captured MTBR-243 tau peptides with a detection antibody, and(e) detecting the detection antibody to determine an amount of MTBR-243 tau peptides in the sample, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

23. The method of claim 21 or 22, wherein the immunodepletion antibody binds to an epitope on human tau protein that is upstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody.

24. The method of claim 21 or 22, wherein the immunodepletion antibody binds to an epitope on human tau protein that is downstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody.

25. The method of claim 23, wherein the immunodepletion antibody binds to an epitope that is within amino acids 1-242 of human tau protein.

26. The method of claim 24, wherein the immunodepletion antibody binds to an epitope that is within amino acids 250-441 of human tau protein.

27. The method of any one claims 21-26, wherein the capture antibody binds to a tau epitope within amino acids 243-254 of human tau protein.

28. The method of any one of claim 21-27, wherein the detection antibody binds to an epitope within amino acids 1-242 of human tau protein.

29. The method of any one of claims 21-28, wherein the capture antibody binds to a tau epitope within amino acids 243-254 of human tau protein, the detection antibody binds to an epitope within amino acids 1-242 of human tau protein, and the immunodepletion antibody binds to an epitope on human tau protein that is upstream of the epitope of the detection antibody.

30. The method of any one of claims 21-29, wherein step (a) comprises contacting the sample with immunodepletion antibodies that bind to different epitopes on human tau.

31. The method of claim 30, wherein (i) at least one of the immunodepletion antibodies bind to an epitope on human tau protein that is upstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody, (ii) at least one of the immunodepletion antibodies bind to an epitope on human tau protein that is downstream of the epitope bound by the capture antibody and / or the epitope bound by the detection antibody, (iii) at least one of the immunodepletion antibodies bind to an epitope withinJ&J Docket Number: JAB7242WOPCT1 amino acids 1 -242 on human tau protein, (iv) each of the immunodepletion antibodies binds to an epitope that is within amino acids 1-242 on human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO:1.

32. The method of any one of claims 21-31, wherein the capture antibody is immobilized on a solid phase.

33. The method of claim 32, wherein the solid phase is a magnetic bead.

34. The method of any one of claims 21-33, further comprising obtaining the sample from the subject.

35. The method of any one of claims 21-34, wherein the sample is a CSF or plasma sample.

36. The method of any one of claims 21-35, further comprising washing the captured tau peptides before contacting the captured tau peptides with the detection antibody.

37. The method of any one of claims 21-36, wherein the immunodepletion antibody or the immunodepletion antibodies is immobilized on a solid phase.

38. The method of claim 37, wherein the solid phase is a magnetic bead.

39. The method of any one of claims 21-38, further comprising washing the immunodepleted sample before contacting the immunodepleted sample with the capture antibody.

40. A kit comprising:(i) a capture antibody that binds to a tau epitope within amino acids 243-254 of human tau protein; and(ii) a detection antibody that binds to an epitope within amino acids 1 -242 of human tau protein,(iii) an immunodepletion antibody that binds to an epitope within amino acids 1-242 of human tau protein and upstream of the epitope of the detection antibody, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

41. The method or kit of any one of claims 21-40, wherein the capture antibody binds to an epitope comprising amino acid residues 249-254 or an epitope comprising amino acidJ&J Docket Number: JAB7242WOPCT1 residues 251-254 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.

42. The method or kit of claim 41, wherein the capture antibody comprises: (i) 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; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively;.

43. The method or kit of claim 42, wherein the capture antibody comprises: (i) 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, or (ii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28.

44. The method or kit of claim 43, wherein the capture antibody is PT15 or PT76.

45. The method or kit of any one of claims 21-44, wherein the detection antibody binds to an epitope comprising amino acid residues 151-158 , an epitope comprising amino acid residues 116-127 , or an epitope comprising amino acid residues 215-220 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.

46. The method or kit of claim 45, wherein the detection antibody comprises (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 37, 38, and 39, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively; or (ii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, andJ&J Docket Number: JAB7242WOPCT1HCDR3 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; or (iii) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 29, 30, and 31, respectively, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 32, 33, and 34, respectively.

47. The method or kit of claim 46, wherein the detection antibody comprises (i) 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, or (ii) 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, or (iii) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 35, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 36.

48. The method or kit of claim 47, wherein the detection antibody is pT51, pT82, or pT3.

49. The method of kit of claim 48, wherein the capture antibody is pT15 and the detection antibody is pT3.

50. The method or kit of any one of claims 21-50, wherein the immunodepletion antibody or the immunodepletion antibodies comprise one or more antibodies selected from: an antibody that binds to an epitope comprising amino acids 116-127, an antibody that binds to an epitope comprising amino acids 151-158, an antibody that binds to an epitope comprising amino acids 166-182, and an antibody that binds to an epitope comprising amino acids 251-254 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.

51. The method or kit of claim 50, wherein the immunodepletion antibody or the immunodepletion antibodies comprise one or more antibodies selected from:(i) an antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences ofJ&J Docket Number: JAB7242WOPCT1SEQ ID NOs: 37, 38, and 39, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 40, 41, and 42, respectively;(ii) an antibody comprising 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;(iii) an antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively; and(iv) an antibody comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 45, 46, and 47, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively.

52. The method or kit of claim 51 , wherein the immunodepletion antibody or the immunodepletion antibodies comprise one or more antibodies selected from:(i) an antibody comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 28,(ii) an antibody comprising 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,(iii) an antibody comprising 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: 48, andJ&J Docket Number: JAB7242WOPCT1(iv) an antibody comprising 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: 52.

53. The method or kit of claim 52, wherein the immunodepletion antibody or the immunodepletion antibodies comprises one or more antibodies selected from: pT51, pT82, pT76, and pT89.

54. The method or kit of any one of claims 1-53, wherein the MTBR-243 tau peptide detected or measured has a length of at least 40 amino acids, 45 amino acids, or 50 amino acids.

55. The method or kit of claim 54, wherein the MTBR-243 tau peptide detected or measured has a length of or no more than 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, or 50 amino acids.

56. The method or kit of claim 55, wherein the MTBR-243 tau peptide detected or measured comprises amino acids 210 to 254 of human tau protein, wherein the numbering of the amino acids is with reference to the amino acid sequence of SEQ ID NO: 1.

57. An antibody or antigen-binding fragment thereof, comprising (i) heavy chain complementarity determining regions (HCDRs) HCDR1 , HCDR2, and HCDR3 comprising amino acid sequences of SEQ ID NOs: 45, 46, and 47, respectively, and light chain complementarity determining regions (LCDRs) LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences of SEQ ID NOs: 49, 50, and 51, respectively.

58. The antibody or antigen-binding fragment thereof of claim 57, comprising a heavy chain region comprising an amino acid sequence of SEQ ID NO: 48, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 52.

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