Compounds and Methods Targeting Human TAU

Abstract

The present invention provides compounds and methods targeting human tau, particularly human tau phosphorylated at threonine 217 and tau isoforms expressed only in the CNS, including therapeutic antibodies, pharmaceutical compositions, and diagnostic applications in the field of neurodegenerative diseases such as AD, PSP, and FTD.

Description

[0001] The present invention pertains to the field of medicine. More specifically, the present invention relates to compounds, pharmaceutical compositions, diagnostics, and methods comprising an anti-human tau antibody or fragment thereof. The compounds and methods of the present invention are intended for use in the field of neurodegenerative diseases, particularly tauopathies including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD), including their treatment and related diagnostics.

[0002] Tau is an axonal microtubule-binding protein that is expressed both in the central nervous system (“CNS”) and peripherally and promotes microtubule assembly and stability. Known human tau isoforms are expressed in the CNS and are involved in the abnormal formation and aggregation of neurofibrillary tangles (“NFTs”) within neurons. In neurodegenerative diseases such as AD, the density and neuroanatomical localization of CNS NFTs are correlated with the severity of dementia, the extent of neuronal loss, and overall disease progression. In PSP, the formation of CNS NFTs can also be seen, and their density is also correlated with the severity of neuronal loss.

[0003] AD is a neurodegenerative disease characterized by dementia, causing problems with memory, thinking, and behavior. According to the Alzheimer's Association, an estimated 5.6 million Americans aged 65 or older (i.e., approximately one in ten) have AD, and another 200,000 Americans under 65 have AD. The Alzheimer's Association also states that by 2025, the number of Americans aged 65 and older with AD is expected to increase by more than 26%. This implies substantial healthcare expenditures; in 2019 alone, the direct medical costs associated with AD in the United States were estimated to reach $290 billion, a figure that does not include unpaid care costs. Despite the significant impact of AD on individuals and health, to date, no disease-modifying treatment for AD has been approved, and such treatments remain an unmet medical need.

[0004] In addition, in order to assist in the discovery and / or development of disease-modifying treatments, a reliable and sensitive diagnosis of AD is needed. The approved AD diagnostic application is Amyvid TM . Flourtaucipir is an AD diagnostic application currently under review by the FDA. Amyvid TMBoth [agent name] and flortaucipir are radioactive isotope neuroimaging agents that can be used for the detection and staging of AD and other neurodegenerative diseases. In addition, a diagnostic assay targeting phosphorylated threonine ("hTau-pT181") at residue 181 of human tau (residue numbering based on SEQ ID NO.1) in a patient sample has recently been disclosed. However, the hTau-pT181 diagnostic application lacks the sensitivity required for a diagnostic test, such as identifying different AD stages or patient prognosis in blood, plasma, and cerebrospinal fluid ("CSF") assays. Therefore, there is a need for a diagnosis applicable to blood, plasma, and / or CSF testing that provides a less costly and less invasive diagnostic option, while also being sensitive and reliable. Preferably, such a diagnosis will be able to identify and / or distinguish AD patients (e.g., based on the stage or prognosis of AD). Such a diagnosis also preferably will be able to identify and / or distinguish an effective treatment response. In an embodiment, such a diagnosis also preferably will be able to identify and / or distinguish patients in need of further diagnostic evaluation, e.g., patients for whom neuroimaging (such as flourtaucipir and / or amyvid) is appropriate.

[0005] Accordingly, in one embodiment, the present disclosure provides antibodies and pharmaceutical compositions thereof against human tau phosphorylated at threonine at residue 217 (residue numbering based on SEQ ID NO.1) ("hTau-pT217"), as well as methods and diagnostic applications using such antibodies and pharmaceutical compositions. In addition, according to one embodiment of the present disclosure, antibodies and pharmaceutical compositions thereof against human tau isoforms expressed in the CNS are provided (e.g., recognizing isoforms expressed in the CNS and not recognizing human tau isoforms expressed only outside the CNS).

[0006] According to some embodiments, antibodies that specifically bind to hTau-pT217 are provided. In more specific embodiments, antibodies are provided that bind to an epitope region of human tau that contains phosphorylated threonine at residue 217 of SEQ ID NO.1, wherein such antibodies do not bind human tau if the threonine at residue 217 of SEQ ID NO.1 is not phosphorylated. In more specific embodiments of the present disclosure, such antibodies are provided that comprise a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 has the amino acid sequence SEQ ID NO:13, LCDR2 has the amino acid sequence SEQ ID NO:14, LCDR3 has the amino acid sequence SEQ ID NO:15, HCDR1 has the amino acid sequence SEQ ID NO:10, HCDR2 has the amino acid sequence SEQ ID NO:11, and HCDR3 has the amino acid sequence SEQ ID NO:12. In some embodiments, LCDR1 has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:13, LCDR2 has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:14, LCDR3 has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:15, HCDR1 has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:10, HCDR2 has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:11, and HCDR3 has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:12. In some embodiments of the antibodies provided according to the present disclosure, the LCVR has the amino acid sequence SEQ ID NO:5 and the HCVR has the amino acid sequence SEQ ID NO:3. In some embodiments of the antibodies provided by the present disclosure, the LCVR has the amino acid sequence SEQ ID NO:8 and the HCVR has the amino acid sequence SEQ ID NO:6. In some other embodiments, the LCVR has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:5, and the HCVR has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:3. In other embodiments, the LCVR has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:8, and the HCVR has an amino acid sequence that has at least 95% homology to the amino acid sequence SEQ ID NO:6.

[0007] According to embodiments of the present disclosure, antibodies are provided that specifically bind to human tau isoforms expressed in the CNS (e.g., known human tau isoforms expressed in the CNS), and such antibodies do not bind to human tau isoforms expressed only in regions outside the CNS, including the peripheral nervous system. According to specific embodiments, such antibodies that specifically bind to human tau isoforms expressed in the CNS bind to a human tau epitope region comprising residues 124 (glutamine) and 125 (alanine) of SEQ ID NO.1. In specific embodiments, such antibodies are provided that comprise a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 has the amino acid sequence SEQ ID NO:23, LCDR2 has the amino acid sequence SEQ ID NO:24, LCDR3 has the amino acid sequence SEQ ID NO:25, HCDR1 has the amino acid sequence SEQ ID NO:20, HCDR2 has the amino acid sequence SEQ ID NO:21, and HCDR3 has the amino acid sequence SEQ ID NO:22. In some embodiments, LCDR1 has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:23, LCDR2 has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:24, LCDR3 has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:25, HCDR1 has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:20, HCDR2 has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:21, and HCDR3 has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:22. According to some embodiments of the antibodies provided by the present disclosure, the LCVR has the amino acid sequence SEQ ID NO:17 and the HCVR has the amino acid sequence SEQ ID NO:19. In some other embodiments, the LCVR has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:17, and the HCVR has an amino acid sequence having at least 95% homology to the amino acid sequence SEQ ID NO:19.

[0008] According to some embodiments, the antibodies of the present disclosure can be humanized. In some embodiments, the antibodies of the present disclosure comprise IgG4 heavy chains. In some embodiments, the antibodies of the present disclosure comprise κ light chains. According to other embodiments, the present disclosure provides pharmaceutical compositions comprising the antibodies of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0009] According to other embodiments, the present disclosure provides methods for treating neurodegenerative diseases, which include administering to a patient in need an effective amount of an antibody of the present disclosure or a pharmaceutical composition thereof. In some such embodiments, the neurodegenerative disease is a tauopathy. In more specific embodiments, the tauopathy is one of AD, PSP, and FTD.

[0010] According to some embodiments, the present disclosure provides an antibody of the present disclosure or a pharmaceutical composition thereof for treating. In addition, an antibody of the present disclosure or a pharmaceutical composition thereof is provided for treating neurodegenerative diseases. In some such embodiments, the neurodegenerative disease is a tauopathy. In some more specific embodiments, the tauopathy is selected from AD, PSP, and FTD.

[0011] According to some embodiments of the present disclosure, an antibody of the present disclosure or a pharmaceutical composition thereof is provided for preparing a medicament for treating neurodegenerative diseases. In some such embodiments, the neurodegenerative disease is a tauopathy. In more specific embodiments, the tauopathy is selected from AD, PSP, and FTD.

[0012] According to other embodiments of the present disclosure, a method for detecting hTau-pT217 in a patient sample is provided. Such methods include the steps of contacting the patient sample with an antibody of the present disclosure that specifically binds to hTau-pT217 and detecting the signal provided by the contacting step.

[0013] According to an embodiment, a method for detecting human tau isoforms expressed only in the CNS is provided. Such methods include the steps of contacting the patient sample with an antibody of the present disclosure that specifically binds to human tau isoforms expressed in the CNS (i.e., does not bind to human tau isoforms expressed only outside the CNS) and detecting the signal provided by the contacting step.

[0014] According to some embodiments, a method for quantifying hTau-pT217 in a patient sample is provided. Such methods include the steps of contacting the patient sample with an antibody of the present disclosure that specifically binds to hTau-pT217 and detecting the signal provided by the contacting step. In some embodiments, such methods further include contacting a control standard with the antibody and detecting the signal provided by the step of contacting the control standard.

[0015] In some embodiments, the present disclosure provides methods for quantifying hTau-pT217 in a patient sample. Such methods include the steps of contacting the patient sample with an antibody that specifically binds hTau-pT217 of the present disclosure, contacting the patient sample with an antibody that specifically binds a CNS-expressed human tau isoform, wherein the antibodies do not bind overlapping epitopes of the antibodies and one of the antibodies comprises a detectable label; detecting the signal provided by the detectable label when forming a complex comprising the antibody and hTau-pT217; contacting a control standard with the antibody; and detecting the signal provided by the detectable label when forming a complex comprising the antibody and the control standard.

[0016] According to some embodiments of the present disclosure, methods are provided for diagnosing a patient as one or more of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) in need of treatment for a neurodegenerative disease; (iv) being in AD Braak stage I, II, III, IV, V, or VI; or (v) in need of neuroimaging. According to such embodiments, such methods include the step of contacting a patient sample with an antibody that specifically binds hTau-pT217 of the present disclosure and detecting the binding between the antibody and hTau-pT217 in the patient sample. In some such embodiments, the method further includes the step of diagnosing the patient as one of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) in need of treatment for a neurodegenerative disease; (iv) being in AD Braak stage I, II, III, IV, V, or VI; or (v) in need of neuroimaging if the level of hTau-pT217 detected in the patient sample exceeds a reference level.

[0017] In some embodiments of the present disclosure, methods are provided for diagnosing and treating a neurodegenerative disease in a patient. According to such embodiments, the method includes the steps of contacting a patient sample with an antibody that specifically binds hTau-pT217 of the present disclosure; detecting the binding between the antibody and hTau-pT217 in the patient sample; diagnosing a patient having a neurodegenerative disease; and administering a therapeutically effective amount of an anti-human Tau antibody to the diagnosed patient. In some embodiments, the diagnosing step includes diagnosing the patient as having a neurodegenerative disease when the presence of hTau-pT217 in the patient sample exceeds a reference level.

[0018] According to some embodiments of the methods of the present disclosure, such methods further include the step of quantifying hTau-pT217 in a patient sample. In such embodiments, the step of quantifying hTau-pT217 includes quantifying hTau-pT217 in the patient sample relative to a reference standard.

[0019] In some embodiments of the method according to the present disclosure, the patient sample is one of blood, plasma, serum, or CSF.

[0020] In some embodiments of the method according to the present disclosure, the method further comprises the step of contacting the patient sample with an antibody that specifically binds hTau-pT217 and a second antibody that specifically binds a human tau isoform expressed in the CNS. In some such methods, one of the antibody that specifically binds hTau-pT217 or the second antibody comprises a detectable label, and the detecting step comprises detecting a signal provided by the detectable label when a complex comprising the antibody that specifically binds hTau-pT217, the second antibody, and hTau-pT217 is formed. According to some such embodiments, one of the antibody that specifically binds hTau-pT217 and the second antibody is immobilized on a substrate. In some embodiments of the method according to the present disclosure, the step of contacting the patient sample with the antibody and the step of contacting the patient sample with the second antibody occur simultaneously. According to some more specific embodiments, the second antibody comprises an antibody according to the present disclosure that specifically binds a human tau isoform expressed in the CNS as disclosed herein.

[0021] As used herein, an "antibody" is an immunoglobulin molecule that comprises two HCs and two LCs that are interconnected by disulfide bonds. The amino-terminal portion of each LC and HC contains a variable region of about 100-120 amino acids that is primarily responsible for antigen recognition through the CDRs contained therein. The CDRs are interspersed with more conserved regions called framework regions ("FRs"). Each LCVR and HCVR is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3", and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3". The CDRs contain most of the residues that form specific interactions with the antigen. The functional ability of an antibody to bind a specific antigen is largely influenced by the six CDRs. The assignment of amino acids to the CDR domains within the LCVR and HCVR regions of the antibodies of the present invention is based on the well-known Kabat numbering convention (Kabat et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991)) and the Chothia numbering convention (Chothia et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)).

[0022] In some embodiments according to the present disclosure, the LC is classified as κ or λ, and each LC is characterized by a specific constant region known in the art. In some embodiments according to the present disclosure, the HC is classified as γ, μ, α, δ, or ε, and the isotype of the antibody is defined as IgG, IgM, IgA, IgD, or IgE, respectively. In some embodiments, the antibody comprises an IgG HC, which can be further divided into subclasses such as IgG1, IgG2, IgG3, IgG4. The carboxy-terminal portion of each HC defines a constant region that is primarily responsible for effector functions. In a specific embodiment, the antibodies of the present invention have one or more modifications in the constant region of each HC that reduce effector function.

[0023] The antibody of the present invention is a monoclonal antibody. A monoclonal antibody is an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), regardless of the method by which it is produced. For example, monoclonal antibodies can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR grafting), or a combination of such or other techniques known in the art.

[0024] Methods for producing and purifying antibodies are well known in the art and can be found, for example, in Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Chapters 5-8 and 15, ISBN 0-87969-314-2. For example, mice or rabbits can be immunized with hTau-pT217, and the resulting antibodies can be recovered, purified, and the amino acid sequence determined using conventional methods well known in the art. Similarly, a phage library can be screened, whereby thousands of Fab fragments are screened for interaction with hTau-pT217, and the resulting interactions can be recovered, purified, and the amino acid sequence determined using conventional methods well known in the art, whereby an initial lead antibody can be constructed. Embodiments of the antibodies of the present disclosure include antibodies engineered to contain one or more human framework regions surrounding the CDRs derived from non-human antibodies. For example, human framework germline sequences can be obtained from ImMunoGeneTics (INGT) via its website http: / / imgt.cines.fr or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351.

[0025] In a specific embodiment of the present invention, the antibody or the nucleic acid encoding the antibody is provided in isolated form. As used herein, the term "isolated" refers to a protein, peptide, or nucleic acid that is free or substantially free of other macromolecular species found in a cellular environment.

[0026] The antibodies provided by the present disclosure can be used to treat patients. More specifically, embodiments of the antibodies of the present disclosure can be used to treat neurodegenerative diseases or conditions, including tauopathies, including AD, PSP, and FTD. Although the antibodies of the present invention can be used to treat AD, PSP, and FTD, such antibodies can also be used to treat other neurodegenerative diseases, especially diseases involving tau pathology such as NFT formation. The terms "treatment" and / or "treating" and / or "treat" used interchangeably herein are intended to refer to all processes in which there can be a slowing, interruption, arrest, control, cessation, or reversal of the progression of the diseases described herein, but this does not necessarily mean that all disease symptoms are completely eliminated. Treatment includes administering the antibodies of the present invention to treat human diseases or conditions that would benefit from a reduction in the spread of at least one of tau aggregation formation, NFT formation, and neuronal loss, and includes: (a) inhibiting the further progression of the disease, i.e., preventing its development; and (b) alleviating the disease, i.e., causing the disease or condition to regress or alleviating its symptoms or complications.

[0027] The terms "patient", "subject", and "individual" used interchangeably herein refer to a human being. In certain embodiments, the patient is further characterized by a disease, condition, or disorder (such as a neurodegenerative disease) that would benefit from a reduction in the spread of at least one of tau aggregation formation, neurofibrillary tangle formation, and neuronal loss. In another embodiment, the patient is further characterized as being at risk of developing a neurodegenerative disease, disorder, or condition that would benefit from a reduction in the spread of at least one of tau aggregation formation, NFT formation, and neuronal loss.

[0028] The term "specifically binds to hTau-pT217" as used herein refers to the interaction of an antibody with a human tau epitope region containing phosphorylated threonine at residue 217 of SEQ ID NO.1. This binding is dependent on the phosphorylation of threonine at residue 217 of SEQ ID NO.1. It should be understood that there are known human tau variants or isoforms, such as those resulting from splicing variants. It should also be understood that such known variants can result in a change in the residue numbering of some amino acid residues of SEQ ID NO.1, including phosphorylated threonine, provided in the human tau sequence shown herein with reference to SEQ ID NO.1.

[0029] As used herein, the term "specifically binds to human tau isoforms expressed in the CNS" refers to the interaction of the antibodies of the present disclosure with an epitope region common to or present on the human tau isoforms expressed in the CNS, which epitope region is not present on human tau isoforms expressed only outside the CNS. Antibodies that specifically bind to human tau isoforms expressed in the CNS do not bind to human tau isoforms expressed only outside the CNS (such as isoforms expressed only in other regions of the body, such as the peripheral nervous system). According to some embodiments, an antibody that specifically binds to human tau isoforms expressed in the CNS binds to or recognizes an epitope region of the human tau isoforms expressed in the CNS, which epitope region comprises glutamine at residue 124 (Q124) and alanine at residue 125 (A125), with residue numbering referring to SEQ ID NO.1. It should be understood that there are known human tau variants or isoforms, such as those resulting from splicing variants, and such variants may result in a change in the residue numbering of some amino acid residues with reference to SEQ ID NO.1, including the glutamine and alanine provided herein with reference to the human tau sequence shown in SEQ ID NO.1.

[0030] As used herein, the term "epitope region" refers to a discrete three-dimensional site of an antigen that is recognized in whole or in part by an antibody of the present invention. The amino acids of the epitope region provide the chemically active surface groups of human tau and form the specific three-dimensional structure of human tau, and may provide specific charge characteristics. The difference between conformational epitopes and non-conformational / linear epitopes is that in the presence of a denaturing solvent, binding to the conformational epitope region is lost, while binding to the linear epitope region is not lost.

[0031] The antibodies of the present invention can be incorporated into pharmaceutical compositions, which can be prepared by methods well known in the art and contain the antibodies of the present invention and one or more pharmaceutically acceptable carriers and / or diluents (e.g., Remington, The Science and Practice of Pharmacy, 22nd edition, edited by Loyd V., Pharmaceutical Press, 2012, which provides a general overview of formulation techniques well known to practitioners). Carriers suitable for use in pharmaceutical compositions include any material that retains the activity of the molecule when combined with the antibodies of the present invention and is non-reactive with the patient's immune system. The pharmaceutical compositions containing the antibodies of the present invention can be administered to a patient at risk of or exhibiting a disease or condition described herein by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). The pharmaceutical compositions of the present invention contain an "effective" or "therapeutically effective" amount (used interchangeably herein) of the antibodies of the present invention. An effective amount refers to the amount necessary to achieve the desired therapeutic effect (dose, time, and mode of administration). The effective amount of the antibody can vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the ability of the antibody to elicit the desired response in the subject. An effective amount is also the amount at which the therapeutic benefit of the antibodies of the present invention exceeds any toxic or harmful effects.

[0032] The percent homology referred to in the context of two or more amino acid sequences herein means that two or more sequences have a specified percentage of identical amino acid residues when compared and aligned using a sequence comparison algorithm (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection to obtain maximum correspondence. Depending on the application, percent homology may exist in a region of the sequences being compared, such as a functional domain, or, in the full length of the two sequences being compared. As an example, the percent homology of a sequence can be compared to a reference sequence. For example, when using a sequence comparison algorithm, a test sequence and a reference sequence can be input into a computer (subsequence coordinates and sequence algorithm program parameters can be further specified as needed). Then, the sequence comparison algorithm calculates the percent sequence identity or homology of the test sequence relative to the reference sequence according to the specified program parameters. Exemplary sequence alignment and / or homology algorithms can be obtained by: Smith & Waterman, Adv. Appl. Math. 2:482 (1981); Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); GAP, BESTFIT, FASTA, and TFASTA (in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally see Ausubel et al., below). An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0033] As used herein, a patient “sample” refers to a human sample. Non-limiting sources of samples for use in the present invention include blood, plasma, serum, and CSF. In addition, a sample can also refer to lymph fluid, biopsy aspirates, ascites, body fluid extracts, solid tissues, external portions of the skin, respiratory tract, nasal cavity, intestine, and urogenital tract, tears, saliva, milk, tumors, organs, cell cultures, and / or cell culture components.

[0034] The present disclosure also relates to methods for clinical diagnosis, prognosis or treatment of subjects by medical professionals using the methods disclosed herein. The methods described herein can be performed, for example, by an individual, a health professional, or a third party (e.g., a service provider that interprets information from a subject). As explained herein, a medical professional can initiate or modify treatment after receiving information about the diagnostic methods disclosed herein. For example, a medical professional can recommend treatment, change treatment, or perform additional diagnostic assessments (e.g., neuroimaging).

[0035] The anti-tau antibodies of the present disclosure that specifically bind to hTau-pT217 can be used to isolate, detect and / or quantify hTau-pT217 by techniques such as affinity chromatography, immunoprecipitation, immunohistochemistry or ELISA-based assays, etc. Such assays can be used to detect and / or evaluate the abundance and / or pattern of hTau-pT217 expression for diagnostic, prognostic or therapeutic purposes, to monitor, for example, the levels of the polypeptide in serum, plasma, blood or CSF, as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen.

[0036] The anti-tau antibodies of the present disclosure that specifically bind to human tau isoforms expressed in the CNS can be used to separate and / or detect human tau isoforms expressed in the CNS (excluding human tau isoforms expressed only outside the CNS) by techniques such as affinity chromatography, immunoprecipitation, immunohistochemistry, or ELISA-based assays. Such assays can be used to detect and / or evaluate the abundance and / or pattern of human tau expression isoforms expressed in the CNS for diagnostic, prognostic, or therapeutic purposes to monitor, for example, levels of polypeptides in serum, plasma, blood, or CSF as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. As understood in the art, the antibodies of the present invention can be coupled to a detectable substance or label to facilitate its detection. Examples of detectable substances or labels include a variety of enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, chemiluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichloroazoxide fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, ruthenium and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3H. The antibodies of the invention can also be used in pharmacogenomic analysis. Such embodiments can be used to identify subjects who may benefit from a particular or modified treatment modality and / or to monitor the efficacy of a current treatment regimen.

[0037] The level or measurement of hTau-pT217 provided by the assays of the invention can be an absolute value (e.g., concentration within a biological sample) or a relative value (e.g., concentration compared to a reference). As used herein, hTau-pT217 in a patient sample is said to be "increased" if the method used to detect hTau-pT217 indicates that the level or concentration of hTau-pT217 in the patient sample is higher than a reference value. Conversely, hTau-pT217 in a patient sample is said to be "decreased" if the level or concentration of hTau-pT217 in the patient sample is lower than a reference value or, for example, the hTau-pT217 value measured in a previous patient sample.

[0038] As used herein, "reference value" refers to a known or approximate concentration of hTau-pT217 associated with a particular condition. The concentration level in the reference value can be an absolute or relative amount of hTau-pT217, a range of amounts, or a minimum, average, and / or median amount. The reference value can also serve as a baseline for hTau-pT217 for comparison with patient samples.

[0039] As used herein, "control standard" refers to a sample that can be used to compare the results obtained from patient samples in the methods of the invention. The control standard can be cells, blood, plasma, CSF, tissue, or a known protein concentration added to a medium. The concentration level in the control standard can be an absolute or relative amount of hTau-pT217, a range of amounts, or a minimum, average, and / or median amount. The control standard can also serve as a baseline for hTau-pT217 for comparison with patient samples. The control standard can include concentration values from the same patient or a known normal reference for hTau-pT217. Additionally, in some embodiments, the control standard can represent hTau-pT217 concentration in the form of a standard curve.

[0040] As used herein, the term "capture antibody" refers to an antibody that binds hTau-pT217. In such embodiments, the capture antibody is capable of binding and capturing hTau-pT217 in a patient sample under appropriate conditions, e.g., specifically binding hTau-pT217 (e.g., not binding human Tau if the threonine at residue 217 of SEQ ID NO.1 is not phosphorylated), such that the capture antibody-hTau-pT217 complex can be separated from the remainder of the sample. In some embodiments, the capture antibody can be an antibody that specifically binds human tau isoforms expressed in the CNS (e.g., which may include hTau phosphorylated at threonine at residue 217), and an antibody that specifically binds hTau-pT217 is used as the "second (or detection) antibody". In some embodiments, the capture antibody is immobilized. In some embodiments, the detection antibody is labeled with a detectable tag. In some embodiments, the capture antibody is immobilized in a "sandwich" immunoassay, and the capture antibody or the first antibody specifically binds to an epitope region of human tau that includes phosphorylated threonine at residue 217 of SEQ ID NO.1. In such sandwich immunoassays, a "detection (or second) antibody" is also used. According to some embodiments, the detection or second antibody can specifically bind to the capture antibody and can be labeled with a detectable tag. In some embodiments, the second antibody specifically binds to the detection of hTau-pT217 that has been bound or captured by the capture antibody or the first antibody. In such embodiments, the detection antibody binds hTau-pT217 in a second epitope region that does not overlap with the first or capture antibody, and can be labeled with a detectable tag. In some such embodiments, the second antibody is an antibody of the invention that specifically binds human tau isoforms expressed in the CNS.

[0041] As used herein, a "detectable tag" is a moiety, composition, or technique that can be used to detect the formation of a complex between an antibody of the invention that specifically binds hTau-pT217 and hTau-pT217. According to some embodiments, the detectable tag can be conjugated directly or indirectly to the antibody (capture or detection, as appropriate). Exemplary embodiments of detectable tags include biotin; radioisotopes; fluorophores or other fluorescent moieties; and enzyme moieties.

[0042] The terms “diagnosis” or “diagnosing,” which may be used interchangeably herein, refer to methods by which one of ordinary skill in the art can estimate and / or determine the probability (“likelihood”) that a patient has a given disease or condition. In the context of the present invention, “diagnosing” a patient includes using the assay results of the present invention to identify or diagnose a neurological disease, such as AD, PSP, or FTD, and to identify a patient, for example, in whom a neurological disease or condition is present or occurring or who is in need of treatment, or the effectiveness of treatment of a patient for a neurological disease. According to the present invention, a diagnosis can be achieved based on a combination of other clinical metrics understood by a medical professional. In some embodiments of the present invention, the diagnostic application of the present invention can be used to diagnose a patient in AD Braak stage I, II, III, IV, V, or VI. The AD Braak staging is as known in the art and is described, for example, in Braak et al., (2006) Acta Neuropathol 112(4):389-404. Example

[0043] Anti-hTau-pT217 antibody

[0044] The anti-hTau-pT217 antibodies of the present disclosure, or antibodies that specifically bind hTau-pT217, are produced by the hybridoma method (e.g., as first described by Kohler et al., Nature, 256:495 (1975)). Briefly, as an example, rabbits are immunized with a peptide that contains phosphorylated threonine and four or more amino acids N-terminal and C-terminal to such threonine, as shown in SEQ ID NO.1 (SEQ ID NO.26 provides an example of a peptide that can be used for immunization). Lymphocytes capable of producing antibodies that bind hTau-pT217 are isolated and fused with a myeloma cell line using a suitable fusing agent to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)). The hybridomas are inoculated and cultured in a suitable medium (preferably containing one or more substances that inhibit the survival of unfused myeloma cells). Then, the binding specificity of the monoclonal antibodies produced by the hybridomas is determined using hTau-pT217 and recombinant tau that is not phosphorylated at threonine residue 217 (numbering based on reference to SEQ ID NO.1) by an in vitro binding assay (e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)). Antibodies that specifically bind hTau-pT217 are identified (e.g., do not bind human tau that is not phosphorylated at residue 217, numbering based on SEQ ID NO.1). Preferably, the hybridomas are subcloned by a limiting dilution procedure and cultured by standard methods, including as ascites tumors in animals (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by the hybridomas (and / or subclones) are purified according to conventional procedures, such as affinity chromatography (e.g., protein A or protein G-agarose gel) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0045] The cDNA encoding the antibody of the present invention is sequenced using conventional procedures. The exemplary rabbit anti-hTau-pT217 antibody (“mAb A”), which is produced substantially according to the procedures described herein, comprises the heavy chain of SEQ ID NO.2 and the light chain of SEQ ID NO.4. The complementarity determining regions (CDRs) or variable regions of the sequenced antibody can be used to generate chimeric or humanized antibodies, and / or to generate other mammalian IgG forms. For example, the clone can be converted to a murine IgG chimeric antibody, such as an exemplary rabbit variable region, murine IgG constant region chimeric anti-hTau-pT217 antibody (“mAb B”) having the heavy chain variable region of SEQ ID NO.6 and the heavy chain of SEQ ID NO.7 and the light chain variable region of SEQ ID NO.8 and the light chain of SEQ ID NO.9. The binding specificity can then be re-evaluated. The cDNA sequences encoding the heavy and light chains can be cloned and engineered into a GS (glutamine synthetase) expression vector. Then, the engineered immunoglobulin expression vector can be stably transfected into CHO cells. Those skilled in the art will understand that mammalian expression of the antibody will result in glycosylation, typically at N-glycosylation sites that are highly conserved in the Fc region. It can be verified whether the stable clone expresses an antibody that specifically binds hTau-pT217. The positive clones can be amplified into serum-free medium for antibody production in a bioreactor. The medium into which the antibody has been secreted can be purified by conventional techniques. For example, the medium can be conveniently applied to a Protein A or G Sepharose FF column that has been equilibrated with a compatible buffer, such as phosphate buffered saline. The chromatography column is washed to remove non-specific binding components. For example, the bound antibody is eluted by a pH gradient and the antibody fractions are detected, such as by SDS-PAGE, and then pooled. The antibody can be concentrated and / or sterile filtered using common techniques. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, for example, frozen at -70 °C, or it can be lyophilized.

[0046] Antibodies specific for hTau isoforms expressed only in the CNS

[0047] Antibodies that specifically bind to CNS-expressed human tau isoforms of the present disclosure can be generated by hybridoma methods (e.g., as first described by Kohler et al., Nature, 256:495 (1975)). Briefly, as an example, a non-human mammal (e.g., a mouse or a rabbit) can be immunized with a human tau protein (e.g., hTau given in SEQ ID NO.1) or its peptide containing glutamine at residue 124 and alanine at residue 125 (numbered as shown in SEQ ID NO.1). Lymphocytes capable of producing antibodies that specifically bind to CNS-expressed human tau isoforms can be isolated and fused with a myeloma cell line using a suitable fusogen to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)). The hybridomas can be inoculated and cultured in a suitable medium (preferably containing one or more substances that inhibit the survival of unfused myeloma cells). Then, the binding specificity of the monoclonal antibodies produced by the hybridomas is determined by in vitro binding assays (e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)) against both CNS-expressed human tau isoforms (e.g., a peptide having the sequence of SEQ ID NO.1) and / or peripherally-expressed human tau isoforms (e.g., a peptide having the sequence given in SEQ ID NO.27, which does not contain glutamine at residue 124 adjacent to alanine at residue 125 as shown in SEQ ID NO.1). Antibodies that specifically bind to CNS-expressed human tau isoforms (e.g., do not bind peripherally-expressed human tau) can be identified. Preferably, the hybridomas can be subcloned by limiting dilution procedures and cultured by standard methods, including as ascites tumors in animals (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by the hybridomas (and / or subclones) are purified by conventional procedures, such as affinity chromatography (e.g., protein A or protein G-agarose gel) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc.

[0048] The cDNA encoding the antibody of the present invention was sequenced using a conventional process. An exemplary murine antibody ("mAb C") that specifically binds to CNS-expressed human tau isoforms of the present disclosure, generated substantially according to the process described herein, comprises a heavy chain of SEQ ID NO. 16 and a light chain of SEQ ID NO. 18. The complementarity-determining regions (CDRs) or variable regions of the sequenced antibody can be used to generate chimeric or humanized antibodies and / or to generate other mammalian IgG forms, and to express their components in host cells such as CHO cells.

[0049] Binding kinetics and affinity

[0050] Using an Octet available from ForteBio The binding of the antibodies of the present disclosure that specifically bind hTau-pT217 to the recombinant hTau-pT217 protein having the amino acid sequence SEQ ID NO: 1 was measured by a biolayer interferometry (BLI) assay measured using an instrument (run at 25 °C using HBS-EP+ running buffer (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% surfactant P20)).

[0051] Unless otherwise noted, all reagents and materials were from ForteBio (Freemont, CA). A Protein A biosensor was used to immobilize the antibody of interest for analysis. An exemplary antibody sample of the present invention (mAb A) was prepared at 5 μg / mL by dilution into running buffer. The recombinant hTau-pT217 protein was prepared at concentrations of 300, 100, 33.3, 11.1, 3.7, 1.24, 0.4115, and 0 (blank) nM by dilution into running buffer. Each analysis included: (1) capturing the antibody sample on the biosensor for 240 seconds; (2) establishing a baseline by incubating the antibody-loaded biosensor with running buffer for 60 seconds; (3) incubating the antibody-loaded biosensor with serially diluted recombinant hTau-pT217 protein for 300 seconds to monitor the binding phase; (4) returning the biosensor to running buffer to monitor the dissociation phase.

[0052] Binding data was processed using standard dual-reference processing and fit to a 1:1 binding model using Data Analysis v9.0 evaluation software to determine the association rate (kon, M -1 s -1 units) and the dissociation rate (koff, s -1 units). The equilibrium dissociation constant (K D ) was calculated from the relationship K D = koff / kon, in molar units. The results are provided in Table 1.

[0053] Table 1: BLI binding data with recombinant hTau-pT217.

[0054]

[0055] *K D Results are considered relative as they are not normalized for the effect of affinity.

[0056] Binding specificity to hTau-pT217

[0057] Using an Octet available from ForteBio The specificity of exemplary antibodies (mAb A and mAb B) that specifically bind hTau-pT217 of the present invention was determined using a BLI assay measured with an instrument (operated at 25 °C using HBS-EP+ running buffer (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% surfactant P20)). The N-terminal biotinylated peptide of SEQ ID NO. 26 with or without phosphorylated threonine at residue 7 was immobilized on a streptavidin biosensor (ForteBio). The peptide was incubated with IgG of mAb A and mAb B diluted to 5 μg / mL in the running buffer for 300 seconds and then dissociated for 300 seconds. Binding data were determined using Data Analysis v9.0 evaluation software. The binding signal (nm) of each peptide at the end of dissociation is provided in Table 2.

[0058] Table 2: BLI binding data of mAb A and mAb B with recombinant hTau with and without pT217

[0059] Exemplary antibody Phosphorylated threonine (nm units) Non-phosphorylated threonine (nm units) mAb A 4.4178 -0.0024 mAb B 4.7715 0.3389

[0060] hTau-pT217 immunoassay

[0061] An immunoassay for measuring hTau-pT217 in plasma is designed to measure disease-related differences in AD patients. As an example, the immunoassay of the present disclosure is performed on a streptavidin small spot plate using the Meso Scale Discovery (MSD) platform. Monoclonal antibody A or monoclonal antibody B is used as a capture antibody and biotinylated. A SULFO-TAG secondary antibody such as monoclonal antibody C (an antibody of the present disclosure that specifically binds to human tau isoforms expressed in the CNS) is used as a detection antibody. The antibodies are conjugated with Sulfo-NHS-Biotin (Thermo Scientific, catalog number: 21329) or MSD GOLD SULFO-TAG NHS-Ester (MSD, catalog number: R91AO-1) according to the manufacturer's protocol. The assay is calibrated using recombinant tau (4R2N, NCBI-tau-v2) protein phosphorylated in vitro in a reaction with glycogen synthase kinase-3 and characterized by mass spectrometry. Samples are thawed on wet ice, vortexed briefly, and plasma is diluted 1:4 in sample buffer: for monoclonal antibody A (phosphate buffered saline (PBS), 0.5% bovine serum albumin (BSA), 0.5% Tween 20, 5 mM EDTA, 5 mM EGTA); for monoclonal antibody 2 (50 mM HEPES, 300 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 1% MSD blocker A, 2% PEG), heterophilic blocker 1 (Scantibodies Inc, catalog number: 3KC533) is added to a concentration of 200 μg / mL. A calibration diluent is made by mixing sample buffer 50 / 50 with Knock-Out Serum Replacement (Gibco, 10828-010).

[0062] Block the MSD streptavidin (MSD, L45SA)-coated plate for 1 hour at room temperature by shaking with 200 μL of PBS containing 3% bovine serum albumin on a shaker at 650 rpm. Wash the plate three times with 200 μL of wash buffer (PBS + 0.05% Tween 20). For the hTau-pT217 plate, add 25 μL of biotinylated capture antibody (mAb A) at 0.464 μg / mL (diluted in DPBS + 0.1% BSA + 0.05% Tween 20, for mAb 2, add 2% PEG), and incubate with shaking at 650 rpm on a shaker for 1 hour at room temperature. Wash the plate three times again with 200 μL of wash buffer. Add 50 μL of diluted calibrator or sample to the plate, and incubate with shaking at 650 rpm on a shaker for 2 hours at room temperature. Then wash the plate three times with 200 μL of wash buffer. For the hTau-pT217 plate, add 25 μL of SULFO-tagged detection antibody (mAb C) at 0.25 μg / mL (diluted in MSD diluent 35, for mAb 2, add 2% PEG), and incubate with shaking at 650 rpm on a shaker for 1 hour at room temperature. Wash the plate three times for the last time with 200 μL of wash buffer. Add 150 μL of surfactant-containing 2X MSD read plate buffer T (MSD, R92TC) to each plate, and read the plate on an MSD SQ120 within 10 minutes after adding the read plate buffer. The results are calculated by the MSD software using 4PL, 1 / y 2 weight of the standard curve for interpolation using the following equation: Y = b 1 + ((b 2 – b 1 ) / (1 + (x / b 3 )) b4 ).

[0063] The hTau-pT217 immunoassay as a prognostic assay for neuroimaging

[0064] Evaluate the levels of hTau-pT217 and hTau-pT181 in the blood of subjects recruited for AD clinical trials. The hTau-pT217 immunoassay described herein was used to evaluate hTau-pT217 in the plasma of AD subjects from two studies (Study 1: N = 42; Study 2: N = 185). In addition, the hTau-pT181 immunoassay previously described in the art was used to measure hTau-pT181 in the same patients. All patients underwent tau positron emission tomography (PET) measured by flortaucipir neuroimaging. Samples were collected during two unique clinical trials, including at baseline, and stored at -80 °C for future biomarker studies. Flortaucipir SUVR was determined in the target neocortical region according to the reference signal in the white matter. The correlation between flortaucipir SUVR and plasma pTau (pT181 and pT217, respectively) was evaluated using Spearman rank correlation. Receiver operating characteristic (ROC) curve analysis used a logistic regression model incorporating age and gender as covariates, with a flortaucipir positive cutoff SUVR > 1.1. pTau was evaluated by quartiles, and the baseline pTau predicting future cognitive decline was evaluated using a mixed effects model.

[0065] As shown in Table 3, in both studies, the hTau-pT217 immunoassay showed a statistically significantly higher correlation with Flortaucipir PET (p-value < 0.05).

[0066] Table 3. Correlation of hTau-pT217 and hTau-pT181 immunoassays with Flortaucipir PET

[0067] <![CDATA hTau-pT217 immunoassay > <![CDATA hTau-pT181 immunoassay > Study 1 0.783 0.332 Study 2 0.463 0.308

[0068] In both studies, the area under the ROC for flortaucipir positive showed a higher value for hTau-pT217 than for hTau-pT181 (Study 1: 0.88 vs. 0.79; Study 2: 0.83 vs. 0.81). In addition, the quartile mixed effects model for hTau-pT271 showed a significant increase in cognitive decline. As demonstrated by the results of this example, the hTau-pT217 immunoassay of the present disclosure is capable of identifying subjects suitable for neuroimaging and at risk of cognitive decline due to neurodegenerative diseases, and shows a significant improvement compared to the hTau-pT181 immunoassay.

[0069] hTau-pT217 immunoassay as a diagnostic marker for AD and disease progression

[0070] The level of hTau-pT217 in CSF was evaluated using the described immunoassay and compared with the results of the hTau-pT181 immunoassay. Briefly, CSF samples from non-impaired elderly (CU, n = 65), patients with mild cognitive impairment due to AD (MCI-AD, n = 29), AD dementia (n = 43), and other neurodegenerative diseases (n = 57) from the Swedish BioFINDER study were evaluated using hTau-pT217 and hTau-p181 immunoassays. 184 participants underwent 18 F-Flortaucipir positron emission tomography (PET). Uptake of 18 F-Flortaucipir was quantified in a priori defined regions related to tau pathology in AD (including tau Braak I-II, III-IV, and V-VI stages and worse).

[0071] In CU patients, both the hTau-pT217 immunoassay and the hTau-pT181 immunoassay were correlated with 18 F-Flortaucipir in Braak I-II stage; in AD patients, both the hTau-pT217 immunoassay and the hTau-pT181 immunoassay were correlated in Braak III-IV and V-VI stage regions; in MCI patients, hTau-pT217 was correlated with 18 F-Flortaucipir regions in Braak I-II, III-IV, and V-VI stages, while hTau-pT181 was only correlated in Braak I-II stage regions. Importantly, in all three diagnostic groups (CU, MCI, and AD) and all regions (Braak I-II, III-IV, and V-VI stages), the correlation between regional 18 F-Flortaucipir and the hTau-pT217 immunoassay was statistically significantly better than the correlation between the hTau-pT181 immunoassay and 18 F-Flortaucipir (p < 0.001 - 0.016).

[0072] Compared with the hTau-pT181 immunoassay, the hTau-pT217 immunoassay showed consistently statistically significantly higher correlation coefficients with 18 F-Flortaucipir in all regions (all p < 0.001): 0.698 - 0.752 for hTau-pT217 versus 0.572 - 0.706 for hTau-pT181. In addition, the hTau-pT217 immunoassay proved to be pathological in all regions18 F-Flortaucipir status was a significantly more accurate predictor statistically (p<0.001) (hTau-pT217: AUC 0.890 - 0.929; hTau-pT181 immunoassay: AUC 0.859 - 0.904). In addition, compared with the hTau-pT181 immunoassay, the hTau-pT217 immunoassay showed a statistically significant improvement in differentiating AD from non-AD neurodegenerative diseases (hTau-pT217: AUC 0.943; hTau-pT181: AUC 0.914) (p = 0.026). These results suggest that the hTau-pT217 immunoassay is associated with 18 F-Flortaucipir neuroimaging, can distinguish AD from other neurological diseases and stages, and shows significant improvement compared with the hTau-pT181 immunoassay.

[0073] The hTau-pT217 immunoassay is associated with Tau PET SUVr

[0074] Tau PET SUVr has been shown in the literature to be associated with Tau pathology. In a study measuring hTau-pT217 in plasma, serum, and CSF of patients with mild AD, the hTau-pT217 immunoassay disclosed herein was associated with Tau PET SUVr. Briefly, 190 subjects had the hTau-pT217 immunoassay in plasma at baseline. Among these 190 subjects, 185 patients had the Tau PET SUVr assay. The data were analyzed using the spearman test, and a significant correlation with spearman ρ = 0.49 and uncorrected p value <0.001 was observed. In addition, 187 subjects had hTau-pT217 measured in serum at baseline examination. Among these 187 subjects, 182 had the Tau PET SUVr assay. The data were analyzed using the spearman test, and a significant correlation with spearman ρ = 0.41 and uncorrected p value <0.001 was observed. In addition, 86 subjects had hTau-pT217 measured in CSF at baseline examination. Among these 86 subjects, 29 had the Tau PET SUVr assay. The data were analyzed using the spearman test, and a significant correlation with spearman ρ = 0.70 and uncorrected p value <0.001 was observed. The results support using the pTau217 level measured in CSF, plasma, or serum to identify Tau pathology.

[0075] The hTau-pT217 immunoassay is associated with the cognitive status of mild AD

[0076] The mean values of hTau-pT217 in plasma, serum, and CSF of mild AD subjects were calculated according to the above immunoassay. The results in each matrix are provided in Table 4.

[0077] Table 4. Mean values of hTau-pT217 in plasma, serum, and CSF of mild AD subjects

[0078] Body fluid Mean Standard deviation N Plasma 14.2 6.2 190 Serum 13.4 6.2 187 CSF 684.9 531.1 86

[0079] The hTau-pT217 immunoassay disclosed herein is correlated with the cognitive status (based on the Mini-Mental State Examination, MMSE) of patients with mild AD and the change in MMSE from baseline relative to placebo. hTau-pT217 was evaluated by immunoassay in the plasma, serum, and CSF of patients with mild AD. Briefly, for each matrix (plasma, serum, and CSF), the Spearman test was used to evaluate the significance of subjects with hTau-pT217 measurement results (as described herein) at baseline and MMSE assessment results and assessment results of change from baseline. The hTau-pT217 immunoassay showed a statistically significant correlation with cognitive status and change from baseline in plasma and serum (the number of CSF samples was too small to reach statistical significance, but the data showed a correlation, and it is expected that with an increase in the number of samples, such as the assessment of serum and plasma, CSF will have a statistically significant correlation with MMSE and change in MMSE from baseline). The results are provided in Table 5.

[0080] Table 5. Correlation of hTau-pT217 immunoassay results in plasma, serum, and cerebrospinal fluid of subjects diagnosed with mild AD with MMSE and change in MMSE from baseline

[0081]

[0082]

[0083] *Not considered statistically significant due to the small sample size of patients evaluated for change in MMSE from baseline.

[0084] These results demonstrate the cross-sectional correlation of hTau-pT217 with the cognitive measurement MMSE and demonstrate the utility of hTau-pT217 in determining the risk of future cognitive decline.

[0085] The hTau-pT217 immunoassay is correlated with amyloid status

[0086] The hTau-pT217 immunoassay disclosed herein is associated with amyloid status. Briefly, plasma samples from four different groups of patients with known AD and amyloid status (based on PET neuroimaging) were evaluated for hTau-pT217 correlation: (i) unaffected elderly amyloid-positive (CU-A+); (ii) unaffected elderly amyloid-negative (CU-A-); (iii) elderly amyloid-positive AD (AD-A+); and (iv) clinically unaffected young (CUY-A-). Samples from each group were analyzed using the hTau-pT217 immunoassay described herein. The results are provided in Table 6.

[0087] Table 6. hTau-pT217 is associated with AD and amyloid status

[0088] Group Mean Standard deviation N Alzheimer's disease - amyloid positive 11.4 6.9 14 Clinically unaffected elderly - amyloid positive 6.7 1.9 14 Clinically unaffected elderly - amyloid negative 3.6 1.4 16 Clinically unaffected young - amyloid negative 3.6 1.3 10

[0089] By using the student t-test to evaluate the results provided in Table 6 for different groups, the evaluation of the hTau-pT217 immunoassay for identifying amyloid-positive subjects was determined. The results are provided in Table 7.

[0090] Table 7. Student t-test of the mean difference in plasma hTau-pT217 associated with AD and amyloid status

[0091]

[0092]

[0093] As shown in Tables 6 and 7, the mean value of the AD-A+ group was 11.4 pg / mL, which was 3.6 times higher than the mean value of the age-matched CU-A- group, resulting in an uncorrected p-value of 4.60E-06. Receiver operating characteristic (ROC) curve analysis was also used to evaluate the sensitivity and specificity of hTau-pT217 in discriminating amyloid-positive subjects. The area under the ROC curve was 0.94, as Figure 1 shown.

[0094] The data provided herein demonstrate that the disclosed hTau-pT217 assay is capable of discriminating amyloid-positive subjects; diagnosing AD, and determining the cognitive status of subjects related to AD; identifying subjects at risk of AD and / or in the earliest stage of AD; and diagnosing AD progression. The data also demonstrate that the disclosed hTau-pT217 assay is related to neuroimaging, functions in serum, plasma, and CSF matrices, and is superior to the known hTau-pT181 assay.

[0095] Exemplary embodiments of the present disclosure

[0096] 1. An antibody that specifically binds to human tau phosphorylated at threonine at residue 217 of SEQ ID NO.1 (“hTau-pT217”).

[0097] 2. An antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,

[0098] wherein LCDR1 has the amino acid sequence SEQ ID NO:13, LCDR2 has the amino acid sequence SEQ ID NO:14, LCDR3 has the amino acid sequence SEQ ID NO:15, HCDR1 has the amino acid sequence SEQ ID NO:10, HCDR2 has the amino acid sequence SEQ ID NO:11, and HCDR3 has the amino acid sequence SEQ ID NO:12.

[0099] 3. An antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,

[0100] wherein LCDR1 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:13, LCDR2 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:14, LCDR3 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:15, HCDR1 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:10, HCDR2 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:11, and HCDR3 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:12.

[0101] 4. The antibody of embodiment 2 or 3, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) selected from:

[0102] a. An LCVR having the amino acid sequence SEQ ID NO:5 and an HCVR having the amino acid sequence SEQ ID NO:3; and

[0103] b. An LCVR having the amino acid sequence SEQ ID NO:8 and an HCVR having the amino acid sequence SEQ ID NO:6.

[0104] 5. The antibody of embodiment 2 or 3, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) selected from:

[0105] a. An LCVR having an amino acid sequence with at least 95% homology to the amino acid sequence SEQ ID NO:5, and an HCVR having an amino acid sequence with at least 95% homology to the amino acid sequence SEQ ID NO:3; and

[0106] b. An LCVR having an amino acid sequence with at least 95% homology to the amino acid sequence SEQ ID NO:8, and an HCVR having an amino acid sequence with at least 95% homology to the amino acid sequence SEQ ID NO:6.

[0107] 6. The antibody according to any one of embodiments 1-5, wherein the antibody is humanized.

[0108] 7. The antibody according to any one of embodiments 1-6, wherein the antibody comprises an IgG4 heavy chain.

[0109] 8. The antibody according to any one of embodiments 1-7, wherein the antibody comprises a κ light chain.

[0110] 9. A pharmaceutical composition comprising the antibody according to any one of embodiments 1-8 and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0111] 10. An antibody that specifically binds to human tau isoforms expressed in the CNS.

[0112] 11. The antibody of embodiment 10, wherein the antibody binds to a human tau epitope region comprising glutamine at residue 124 and alanine at residue 125 of SEQ ID NO.1.

[0113] 12. An antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementary determining regions (CDRs) LCDR1, LCDR2 and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2 and HCDR3,

[0114] wherein LCDR1 has the amino acid sequence SEQ ID NO:23, LCDR2 has the amino acid sequence SEQ ID NO:24, LCDR3 has the amino acid sequence SEQ ID NO:25, HCDR1 has the amino acid sequence SEQ ID NO:20, HCDR2 has the amino acid sequence SEQ ID NO:21, and HCDR3 has the amino acid sequence SEQ ID NO:22.

[0115] 13. An antibody comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,

[0116] wherein LCDR1 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:23, LCDR2 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:24, LCDR3 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:25, HCDR1 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:20, HCDR2 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:21, and HCDR3 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:22.

[0117] 14. The antibody of embodiment 12 or 13, comprising a light chain variable region (LCVR) having the amino acid sequence SEQ ID NO:17 and a heavy chain variable region (HCVR) having the amino acid sequence SEQ ID NO:19.

[0118] 15. The antibody of embodiment 12 or 13, comprising a light chain variable region (LCVR) having an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:17, and a heavy chain variable region (HCVR) having an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO:19.

[0119] 16. The antibody according to any one of embodiments 10 - 15, wherein the antibody is humanized.

[0120] 17. The antibody according to any one of embodiments 10 - 16, wherein the antibody comprises an IgG4 heavy chain.

[0121] 18. The antibody according to any one of embodiments 10-17, wherein the antibody comprises a κ light chain.

[0122] 19. A pharmaceutical composition comprising the antibody according to any one of embodiments 10-18 and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0123] 20. A method for treating a neurodegenerative disease, comprising administering to a patient in need thereof an effective amount of the antibody according to any one of embodiments 1-19 or a pharmaceutical composition thereof.

[0124] 21. The method of embodiment 20, wherein the neurodegenerative disease is a tauopathy.

[0125] 22. The method of embodiment 21, wherein the tauopathy is one of AD, PSP, and FTD.

[0126] 23. The antibody according to any one of embodiments 1-19 or a pharmaceutical composition thereof, for use in therapy.

[0127] 24. The antibody according to any one of embodiments 1-19 or a pharmaceutical composition thereof, for use in treating a neurodegenerative disease.

[0128] 25. The antibody or a pharmaceutical composition thereof according to embodiment 24, wherein the neurodegenerative disease is a tauopathy.

[0129] 26. The antibody or a pharmaceutical composition thereof according to embodiment 25, wherein the tauopathy is selected from AD, PSP, and FTD.

[0130] 27. The antibody according to any one of embodiments 1-19 or a pharmaceutical composition thereof, for use in the preparation of a medicament for treating a neurodegenerative disease.

[0131] 28. The antibody or a pharmaceutical composition thereof according to embodiment 27, wherein the neurodegenerative disease is a tauopathy.

[0132] 29. The antibody or a pharmaceutical composition thereof according to embodiment 28, wherein the tauopathy is selected from AD, PSP, and FTD.

[0133] 30. A method for detecting hTau-pT217 in a patient sample, comprising the steps of:

[0134] Contacting the patient sample with the antibody according to any one of embodiments 1-8; and detecting a signal provided by the contacting step.

[0135] 31. A method for quantifying hTau-pT217 in a patient sample, comprising the steps of:

[0136] Contact a patient sample with an antibody as described in any one of embodiments 1-8; and detect the signal provided by the contacting step.

[0137] 32. The method of embodiment 31, further comprising the steps of: contacting a control standard with the antibody; and detecting the signal provided by the step of contacting the control standard.

[0138] 33. A method for quantifying hTau-pT217 in a patient sample, comprising the steps of: contacting a patient sample with an antibody of embodiments 1-8; contacting the patient sample with a second antibody, wherein the second antibody is an antibody of embodiments 10-18, and one of the antibody of embodiments 1-8 and the second antibody comprises a detectable label; detecting the signal provided by the detectable label when forming a complex comprising the antibody of embodiments 1-8, the second antibody, and hTau-pT217; contacting a control standard with the antibody; contacting the control standard with the second antibody, and one of the antibody of embodiments 1-8 and the second antibody comprises a detectable label; detecting the signal provided by the detectable label when forming a complex comprising the antibody of embodiments 1-8, the second antibody, and the control standard.

[0139] 34. A method for diagnosing a patient as one or more of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) in need of treatment for a neurodegenerative disease; or (iv) in need of neuroimaging, comprising the steps of: contacting a patient sample with an antibody as described in any one of embodiments 1-8; and detecting the binding between the antibody and hTau-pT217 in the patient sample.

[0140] 35. The method of embodiment 34, further comprising the step of diagnosing the patient as one of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) in need of treatment for a neurodegenerative disease; or (iv) in need of neuroimaging if the level of hTau-pT217 detected in the patient sample exceeds a reference level.

[0141] 36. A method for diagnosing and treating a neurodegenerative disease in a patient, the method comprising the steps of: contacting a patient sample with an antibody of any one of embodiments 1-8; detecting the binding between the antibody and hTau-pT217 in the patient sample; diagnosing a patient having a neurodegenerative disease; and administering a therapeutically effective amount of an anti-human Tau antibody to the diagnosed patient.

[0142] 37. The method of embodiment 36, wherein the diagnosing step comprises diagnosing the patient as having a neurodegenerative disease when the presence of hTau-pT217 in the patient sample exceeds a reference level.

[0143] 38. The method of any one of embodiments 31 - 37, further comprising the step of quantifying hTau - pT217 in a patient sample.

[0144] 39. The method of embodiment 38, wherein the step of quantifying hTau - pT217 comprises quantifying hTau - pT217 in a patient sample relative to a reference standard.

[0145] 40. The method of any one of embodiments 30 - 39, wherein the patient sample is one of blood, plasma, serum, or CSF.

[0146] 41. The method of any one of embodiments 30 - 32 and 34 - 40, further comprising the step of contacting the patient sample with a second antibody that binds to an epitope region of hTau - pT217 that does not overlap with the antibody.

[0147] 42. The method of embodiment 41, wherein one of the antibody or the second antibody comprises a detectable label, and the detecting step comprises detecting a signal provided by the detectable label when forming a complex comprising the antibody of embodiments 1 - 8, the second antibody, and hTau - pT217.

[0148] 43. The method of any one of embodiments 41 - 42, wherein one of the antibody of embodiments 1 - 8 and the second antibody is immobilized on a substrate.

[0149] 44. The method of any one of embodiments 30 - 43, wherein the step of contacting the patient sample with the antibody of embodiments 1 - 8 and the step of contacting the patient sample with the second antibody occur simultaneously.

[0150] 45. The method of any one of embodiments 32 and 41 - 44, wherein the second antibody comprises a light - chain variable region (LCVR) and a heavy - chain variable region (HCVR), wherein the LCVR comprises complementary determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,

[0151] wherein LCDR1 has the amino acid sequence SEQ ID NO:23, LCDR2 has the amino acid sequence SEQ ID NO:24, LCDR3 has the amino acid sequence SEQ ID NO:25, HCDR1 has the amino acid sequence SEQ ID NO:20, HCDR2 has the amino acid sequence SEQ ID NO:21, and HCDR3 has the amino acid sequence SEQ ID NO:22.

[0152] 46. The method of any one of embodiments 32 and 41 - 44, wherein the second antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,

[0153] wherein LCDR1 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 23, LCDR2 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 24, LCDR3 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 25, HCDR1 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 20, HCDR2 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 21, and HCDR3 has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 22.

[0154] 47. The method of any one of embodiments 46 - 47, wherein the second antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), the LCVR has the amino acid sequence SEQ ID NO: 19, and the HCVR has the amino acid sequence SEQ ID NO: 17.

[0155] 48. The method of any one of embodiments 46 - 47, wherein the second antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR),

[0156] wherein the LCVR has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 19, and the HCVR has an amino acid sequence having at least 95% homology with the amino acid sequence SEQ ID NO: 17.

[0157] 49. A method of forming a complex between a first antibody, a second antibody, and human tau expressed in the CNS and phosphorylated at threonine at residue 217 of SEQ ID NO. 1, the method comprising: contacting a patient sample with a first antibody, wherein the first antibody is an antibody of one of embodiments 1 - 8; contacting the patient sample with a second antibody, wherein the second antibody is an antibody of one of embodiments 10 - 18.

[0158] 50. An assay for detecting human tau expressed in the CNS and phosphorylated at threonine at residue 217 of SEQ ID NO. 1, the assay comprising: an antibody of one of embodiments 1 - 8; an antibody of one of embodiments 10 - 18.

[0159] The assay of embodiment 50, wherein one of the antibodies comprises a detectable label.

[0160] Sequence Listing

[0161] SEQ ID NO:1 (hTau-pT217)

[0162] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0163] SEQ ID NO:2 (HC of exemplary rabbit anti-hTau-pT217 antibody)

[0164] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAGWAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0165] SEQ ID NO:3 (Heavy chain variable region (HCVR) of an exemplary rabbit anti-hTau-pT217 antibody)

[0166] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAGWAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSL

[0167] SEQ ID NO:4 (Light chain (LC) of an exemplary rabbit anti-hTau-pT217 antibody)

[0168] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVSSHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADNTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0169] SEQ ID NO:5 (Light chain variable region (LCVR) of an exemplary rabbit anti-hTau-pT217 antibody)

[0170] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVSSHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVK

[0171] SEQ ID NO:6 (HCVR of exemplary chimeric anti-hTau-pT217 antibody)

[0172] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAGWAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSL

[0173] SEQ ID NO:7 (HC of exemplary chimeric anti-hTau-pT217 antibody)

[0174] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAGWAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSLAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0175] SEQ ID NO:8 (LCVR of exemplary chimeric anti-hTau-pT217 antibody)

[0176] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVSSHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVK

[0177] SEQ ID NO:9 (Light chain of exemplary chimeric anti-hTau-pT217 antibody)

[0178] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVSSHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0179] SEQ ID NO:10 (Exemplary HCDR1)

[0180] GLSPSWYGVH

[0181] SEQ ID NO:11 (Exemplary HCDR2)

[0182] VLRAGSHTYYAGWAKG

[0183] SEQ ID NO:12 (Exemplary HCDR3)

[0184] VGRGI

[0185] SEQ ID NO:13 (Exemplary LCDR1)

[0186] QASLAVYNNNYLA

[0187] SEQ ID NO:14 (Exemplary LCDR2)

[0188] LASSLSS

[0189] SEQ ID NO:15 (Exemplary LCDR3)

[0190] QGSYDCTIADCVA

[0191] SEQ ID NO:16 (Heavy chain of exemplary CNS-expressed human tau-binding antibody)

[0192] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSPYYWSWIRQPPDKGLEWIGEINWSGDTNYNPSLKSRVTISLDTSKNQFSLNLSSVTAADTAVYYCARSFDRWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0193] SEQ ID NO:17 (Heavy Chain Variable Region (HCVR) of an exemplary human tau-binding antibody with CNS-only expression)

[0194] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSPYYWSWIRQPPDKGLEWIGEINWSGDTNYNPSLKSRVTISLDTSKNQFSLNLSSVTAADTAVYYCARSFDRWGQGTLVTVSS

[0195] SEQ ID NO:18 (Light Chain (LC) of an exemplary human tau-binding antibody with CNS-only expression)

[0196] EIVLTQSPGTLSLSPGERATLSCRASQSVRSNYFAWYQQKPGQAPRLLIYGVSRRAFGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGASLITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0197] SEQ ID NO:19 (Light Chain Variable Region of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0198] EIVLTQSPGTLSLSPGERATLSCRASQSVRSNYFAWYQQKPGQAPRLLIYGVSRRAFGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGASLITFGQGTRLEIK

[0199] SEQ ID NO:20 (Heavy Chain CDR1 of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0200] AVYGGSFSPYYWS

[0201] SEQ ID NO:21 (Heavy Chain CDR2 of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0202] EINWSGDTN

[0203] SEQ ID NO:22 (Heavy Chain CDR3 of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0204] ARSFDR

[0205] SEQ ID NO:23 (Light Chain CDR1 of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0206] RASQSVRSNYFA

[0207] SEQ ID NO:24 (Light Chain CDR2 of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0208] YGVSRRAF

[0209] SEQ ID NO:25 (Light Chain CDR3 of an Exemplary CNS-Only Expressed Human Tau-Binding Antibody)

[0210] QQYGASLIT

[0211] SEQ ID NO:26 (Exemplary Peptide for Immunization)

[0212] RTPSLPTPPTR

[0213] Wherein T at residue 7 is phosphorylated

[0214] SEQ ID NO:27 (Exemplary Peptide for Immunization)

[0215] AEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPAAAPRGKPVSRVPQLKARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNRPCLSPKHPTPGSSDPLIQPSSPAVCPEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

Claims

1. An antibody that specifically binds to hTau-pT217, wherein hTau-pT217 is human tau phosphorylated at threonine at residue 217 of SEQ ID NO.1; the antibody that specifically binds to hTau-pT217 comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of LCDR1 is SEQ ID NO:13, the amino acid sequence of LCDR2 is SEQ ID NO:14, the amino acid sequence of LCDR3 is SEQ ID NO:15, the amino acid sequence of HCDR1 is SEQ ID NO:10, the amino acid sequence of HCDR2 is SEQ ID NO:11, and the amino acid sequence of HCDR3 is SEQ ID NO:

12.

2. The antibody according to claim 1, which comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of the LCVR is SEQ ID NO:5 and the amino acid sequence of the HCVR is SEQ ID NO:

3.

3. The antibody according to claim 1 or 2, wherein the antibody is humanized.

4. The antibody according to claim 1 or 2, wherein the antibody comprises an IgG4 heavy chain.

5. The antibody according to claim 1 or 2, wherein the antibody comprises a κ light chain.

6. A pharmaceutical composition comprising the antibody according to any one of claims 1-5 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

7. Use of the antibody according to any one of claims 1-5 for the preparation of a reagent for detecting hTau-pT217 in a patient sample, wherein the detection comprises the following steps: contacting the patient sample with the antibody according to any one of claims 1-5; contacting the patient sample with a second antibody that binds to an epitope region of hTau-pT217 that does not overlap with the antibody according to any one of claims 1-5; and detecting the binding of the antibody according to any one of claims 1-5 to hTau-pT217. Wherein the second antibody specifically binds to human tau isoforms expressed in the CNS, and the second antibody binds to a human tau epitope region comprising glutamine at residue 124 and alanine at residue 125 of SEQ ID NO.

1. The second antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementary determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3. The amino acid sequence of the LCDR1 is SEQ ID NO:23, the amino acid sequence of the LCDR2 is SEQ ID NO:24, the amino acid sequence of the LCDR3 is SEQ ID NO:25, the amino acid sequence of the HCDR1 is SEQ ID NO:20, the amino acid sequence of the HCDR2 is SEQ ID NO:21, and the amino acid sequence of the HCDR3 is SEQ ID NO:

22.

8. The use according to claim 7, wherein the detection further comprises the step of quantifying hTau-pT217 in a patient sample.

9. The use according to claim 7 or 8, wherein the detection comprises diagnosing the patient as one or more of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) in need of treatment for a neurodegenerative disease; or (iv) in need of neuroimaging if the level of hTau-pT217 detected in the patient sample exceeds a reference level.

10. The use according to claim 9, wherein the detection further comprises the step of diagnosing the patient as having a neurodegenerative disease, wherein the neurodegenerative disease is a tauopathy.

11. The use according to claim 10, wherein the tauopathy is selected from Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD).

12. The use according to claim 7, wherein the patient sample is one of blood, plasma, serum, or CSF.

13. The use according to claim 7, wherein one of the antibodies or the second antibody according to any one of claims 1-5 comprises a detectable label, and the detection comprises detecting a signal provided by the detectable label when forming a complex comprising the antibody, the second antibody, and hTau-pT217 according to any one of claims 1-5.

14. The use according to claim 10, wherein the tauopathy is Alzheimer's disease (AD).

15. The use according to claim 12, wherein the patient sample is CSF.

16. The use according to claim 12, wherein the patient sample is plasma.

17. The use according to claim 7 or 8, wherein one of the antibody and the second antibody according to any one of claims 1-5 is immobilized on a substrate.

18. The use according to claim 17, wherein the substrate is selected from a microplate or a bead.

19. The use according to claim 7 or 8, wherein the steps of contacting the patient sample with the antibody according to any one of claims 1-5 and contacting the patient sample with a second antibody occur simultaneously.

20. The use according to claim 13, wherein the detectable label is a chemiluminescent label or an enzyme label.

21. The use according to claim 9, wherein the detection further comprises the step of diagnosing the patient as in need of neuroimaging.

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