Compounds and methods for targeting human TAU

Abstract

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

Description

[0001] This application is a divisional application of Chinese patent application 202080039868.6. The filing date of the original application is May 22, 2020, and the name of the invention is “Compounds and methods targeting human tau”. Technical Field

[0002] The present invention belongs to the field of medicine. More specifically, the present invention relates to compounds, pharmaceutical compositions, diagnostics, and methods comprising antibodies against human tau or fragments thereof. The compounds and methods of the present invention are expected to be used 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. Background Art

[0003] Tau is an axonal microtubule-binding protein expressed in both the central nervous system ("CNS") and the periphery, promoting 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 correlate 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 also correlates with the severity of neuronal loss.

[0004] 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 (or approximately one in 10) have AD, and an additional 200,000 Americans under the age of 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 represents a significant medical expenditure; in 2019 alone, 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 personal and health impact of AD, to date, there are no approved disease-modifying treatments for AD, and such treatments remain an unmet medical need.

[0005] Furthermore, reliable and sensitive diagnostics for AD are needed to aid in the discovery and / or development of disease-modifying treatments. Approved AD diagnostic applications are Amyvid TM Flourtaucipir is currently under FDA review for AD diagnostic applications. TMBoth flotaucipir and flortaucipir are radioisotope 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 at residue 181 (residue numbering based on SEQ ID NO.1) of human tau in patient samples ("hTau-pT181") has recently been disclosed. However, hTau-pT181 diagnostic applications lack the sensitivity required for diagnostic tests, such as identifying different AD stages or patient prognosis in blood, plasma, and cerebrospinal fluid ("CSF") assays. Therefore, there is a need for a diagnostic that is suitable for blood, plasma, and / or CSF testing, which provides a lower cost and less invasive diagnostic option while also being sensitive and reliable. Preferably, such a diagnostic will be able to identify and / or differentiate AD patients (e.g., based on the stage or prognosis of AD). Such a diagnostic will also preferably be able to identify and / or differentiate effective treatment responses. In embodiments, such a diagnostic will also preferably be able to identify and / or differentiate patients who require further diagnostic evaluation, for example, patients for whom neuroimaging (such as flourtaucipir and / or amyvid) is appropriate. Summary of the Invention

[0006] Thus, in one embodiment, the present disclosure provides antibodies against human tau phosphorylated at threonine at residue 217 (residue numbering is based on SEQ ID NO. 1) ("hTau-pT217") and pharmaceutical compositions thereof, as well as methods and diagnostic applications using such antibodies and pharmaceutical compositions. In addition, according to one embodiment of the present disclosure, antibodies against human tau isoforms expressed in the CNS and pharmaceutical compositions thereof are provided (e.g., recognizing isoforms expressed in the CNS, but not recognizing human tau isoforms expressed only outside the CNS).

[0007] According to some embodiments, antibodies that specifically bind hTau-pT217 are provided. In more specific embodiments, antibodies that bind to an epitope region of human tau comprising phosphorylated threonine at residue 217 of SEQ ID NO. 1 are provided, wherein such antibodies do not bind to 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 of SEQ ID NO: 13, LCDR2 has the amino acid sequence of SEQ ID NO: 14, LCDR3 has the amino acid sequence of SEQ ID NO: 15, HCDR1 has the amino acid sequence of SEQ ID NO: 10, HCDR2 has the amino acid sequence of SEQ ID NO: 11, and HCDR3 has the amino acid sequence of SEQ ID NO: 12. In some embodiments, LCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 13, LCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 14, LCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 15, HCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 10, HCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 11, and HCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 12. According to some embodiments of the antibodies provided herein, the LCVR has the amino acid sequence of SEQ ID NO: 5 and the HCVR has the amino acid sequence of SEQ ID NO: 3. In some embodiments of the antibodies provided herein, the LCVR has the amino acid sequence of SEQ ID NO: 8 and the HCVR has the amino acid sequence of SEQ ID NO: 6. In some other embodiments, the LCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 5, and the HCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 3. In other embodiments, the LCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 8, and the HCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 6.

[0008] According to embodiments of the present disclosure, antibodies are provided that specifically bind to CNS-expressed human tau isoforms (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 CNS-expressed human tau isoforms bind to a human tau epitope region comprising residues 124 (glutamine) and 125 (alanine) of SEQ ID NO. 1. In a specific embodiment, such an antibody is provided that comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises the complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises the CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO:23, LCDR2 has the amino acid sequence of SEQ ID NO:24, LCDR3 has the amino acid sequence of SEQ ID NO:25, HCDR1 has the amino acid sequence of SEQ ID NO:20, HCDR2 has the amino acid sequence of SEQ ID NO:21, and HCDR3 has the amino acid sequence of SEQ ID NO:22. In some embodiments, LCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 23, LCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 24, LCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 25, HCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 20, HCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 21, and HCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 22. According to some embodiments of the antibodies provided herein, the LCVR has the amino acid sequence of SEQ ID NO: 17 and the HCVR has the amino acid sequence of SEQ ID NO: 19. In some other embodiments, the LCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 17, and the HCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 19.

[0009] According to some embodiments, the antibodies of the present disclosure may be humanized. In some embodiments, the antibodies of the present disclosure comprise an IgG4 heavy chain. In some embodiments, the antibodies of the present disclosure comprise a kappa light chain. According to other embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0010] According to other embodiments, the present disclosure provides a method for treating a neurodegenerative disease comprising administering to a patient in need thereof 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.

[0011] According to some embodiments, the present disclosure provides antibodies or pharmaceutical compositions thereof for use in treatment. In addition, antibodies or pharmaceutical compositions thereof are provided for use in treating a neurodegenerative disease. In some such embodiments, the neurodegenerative disease is a tauopathy. In some more specific embodiments, the tauopathy is selected from AD, PSP, and FTD.

[0012] According to some embodiments of the present disclosure, antibodies or pharmaceutical compositions thereof are provided for use in the preparation of a medicament for treating a neurodegenerative disease. In some such embodiments, the neurodegenerative disease is a tauopathy. In more specific embodiments, the tauopathy is selected from AD, PSP, and FTD.

[0013] According to other embodiments of the present disclosure, methods for detecting hTau-pT217 in a patient sample are provided. Such methods include contacting the patient sample with an antibody disclosed herein that specifically binds to hTau-pT217 and detecting a signal provided by the contacting step.

[0014] According to an embodiment, a method for detecting human tau isoforms expressed only in the CNS is provided. Such methods include contacting a patient sample with an antibody disclosed herein 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 a signal provided by the contacting step.

[0015] According to some embodiments, methods for quantifying hTau-pT217 in a patient sample are provided. Such methods include the steps of contacting the patient sample with an antibody disclosed herein that specifically binds to hTau-pT217 and detecting a signal provided by the contacting step. In some embodiments, such methods further include the steps of contacting a control standard with the antibody and detecting a signal provided by the contacting step.

[0016] In some embodiments, the present disclosure provides methods for quantifying hTau-pT217 in a patient sample. Such methods include the following steps: contacting the patient sample with an antibody disclosed herein that specifically binds to hTau-pT217, contacting the patient sample with an antibody disclosed herein that specifically binds to a CNS-expressed human tau isoform, wherein the antibody does not bind to an overlapping epitope of the antibody, and one of the antibodies comprises a detectable label; detecting a signal provided by the detectable label upon formation of a complex comprising the antibody and hTau-pT217; contacting a control standard with the antibody; and detecting a signal provided by the detectable label upon formation of a complex comprising the antibody and the control standard.

[0017] According to some embodiments of the present disclosure, a method is provided for diagnosing a patient as having one or more of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) being in need of neurodegenerative disease treatment; (iv) being at AD Braak stage I, II, III, IV, V, or VI; or (v) being in need of neuroimaging. According to such embodiments, such methods include the steps of contacting a patient sample with an antibody of the present disclosure that specifically binds to hTau-pT217 and detecting binding between the antibody and hTau-pT217 in the patient sample. In some such embodiments, the method further includes the steps of diagnosing the patient as having one of the following: (i) having a neurodegenerative disease; (ii) being at risk of developing a neurodegenerative disease; (iii) being in need of neurodegenerative disease treatment; (iv) being at AD Braak stage I, II, III, IV, V, or VI; or (v) being in need of neuroimaging if the level of hTau-pT217 detected in the patient sample exceeds a reference level.

[0018] In some embodiments of the present disclosure, methods for diagnosing and treating a neurodegenerative disease in a patient are provided. According to such embodiments, the method comprises the following steps: contacting a patient sample with an antibody disclosed herein that specifically binds to hTau-pT217; detecting binding between the antibody and hTau-pT217 in the patient sample; diagnosing the patient as having a neurodegenerative disease; and administering a therapeutically effective amount of the anti-human Tau antibody to the diagnosed patient. In some embodiments, the diagnostic step comprises diagnosing the patient as having a neurodegenerative disease when the presence of hTau-pT217 in the patient sample exceeds a reference level.

[0019] According to some embodiments of the methods of the present disclosure, such methods further comprise the step of quantifying hTau-pT217 in the patient sample. In such embodiments, the step of quantifying hTau-pT217 comprises quantifying hTau-pT217 in the patient sample relative to a reference standard.

[0020] According to some embodiments of the methods of the present disclosure, the patient sample is one of blood, plasma, serum, or CSF.

[0021] According to some embodiments of the methods of 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 to a CNS-expressed human tau isoform. 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 upon formation of a complex comprising the antibody that specifically binds hTau-pT217, the second antibody, and hTau-pT217. 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 methods of the present disclosure, the steps of contacting the patient sample with the antibody and contacting the patient sample with the second antibody occur simultaneously. According to some more specific embodiments, the second antibody comprises an antibody of the present disclosure that specifically binds to a CNS-expressed human tau isoform as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 ROC curves for hTau-pT217 in AD, CU-A+, and CU-A- subject groups are shown. DETAILED DESCRIPTION

[0023] As used herein, an "antibody" is an immunoglobulin molecule comprising two HCs and two LCs interconnected by disulfide bonds. The amino-terminal portion of each LC and HC comprises a variable region of approximately 100-120 amino acids, which is primarily responsible for antigen recognition through the CDRs contained therein. Interspersed within the CDRs are more conserved regions, known as framework regions ("FRs"). Each LCVR and HCVR consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxyl-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 to 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 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, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)) and the North numbering convention (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)).

[0024] According to some embodiments of the present disclosure, LC is classified as κ or λ, and each LC is characterized by a specific constant region known in the art. According to some embodiments of the present disclosure, 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. According to some embodiments, the antibody includes IgG HC, which can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4. The carboxyl-terminal portion of each HC defines the constant region primarily responsible for effector function. In specific embodiments, the antibodies of the present invention have one or more modifications in the constant region of each HC that reduce effector function.

[0025] The antibodies of the present invention are monoclonal antibodies. A monoclonal antibody is an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), rather than the method by which it is produced. For example, monoclonal antibodies can be produced by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR transplantation), or a combination of such or other technologies known in the art.

[0026] Methods for producing and purifying antibodies are well known in the art and can be found in, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY, Chapters 5-8 and 15, ISBN 0-87969-314-2. For example, a mouse or rabbit 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, phage libraries can be screened to screen thousands of Fab fragments 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, from which initial lead antibodies can be constructed. Embodiments of the antibodies disclosed herein include antibodies that have been engineered to include one or more human framework regions surrounding CDRs derived from non-human antibodies. Human framework germline sequences are available, for example, from ImMunoGeneTics (INGT) via their website http: / / imgt.cines.fr or from The Immunoglobulin Facts Book by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351.

[0027] In a specific embodiment of the invention, the antibody or nucleic acid encoding the antibody is provided in isolated form.The term "isolated" as used herein refers to a protein, peptide or nucleic acid that is free or substantially free of other macromolecular species found in the cellular environment.

[0028] The antibodies provided by the present disclosure can be used to treat patients. More specifically, embodiments of the antibodies disclosed herein can be used to treat neurodegenerative diseases or conditions, including tau diseases, 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. "Treatment" and / or "treating" and / or "treat" used interchangeably herein are intended to refer to all processes in which there may be a slowing, interruption, prevention, control, stopping 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 will benefit from at least one reduction in tau aggregation formation, NFT formation and neuronal loss, and includes: (a) inhibiting further progression of the disease, i.e., preventing its development; and (b) alleviating the disease, i.e., causing the disease or condition to subside or alleviating its symptoms or complications.

[0029] The terms "patient," "subject," and "individual," used interchangeably herein, refer to humans. In certain embodiments, the patient is further characterized by a disease, condition, or symptom (e.g., a neurodegenerative disease) that would benefit from at least one of tau aggregation formation, neurofibrillary tangle formation, and neuronal loss to reduce expansion. In another embodiment, the patient is further characterized as having a risk of developing a neurodegenerative disease, disease, or condition that would benefit from at least one of tau aggregation formation, NFT formation, and neuronal loss to reduce expansion.

[0030] As used herein, the term "specifically binds hTau-pT217" refers to the interaction of an antibody with a region of the human tau epitope comprising the phosphorylated threonine at residue 217 of SEQ ID NO. 1. This binding is dependent on the phosphorylation of the threonine at residue 217 of SEQ ID NO. 1. It will be appreciated that there are known human tau variants or isoforms, for example, arising from splice variants. It will also be appreciated that such known variants may result in changes in the residue numbering of some of the amino acid residues (including the phosphorylated threonine) of SEQ ID NO. 1, as provided herein with reference to the human tau sequence set forth in SEQ ID NO. 1.

[0031] As used herein, the term "specifically binds to a CNS-expressed human tau isoform" refers to the interaction of an antibody of the present disclosure with an epitope region common to or present on a human tau isoform expressed in the CNS, which epitope region is not present on a human tau isoform expressed only outside the CNS. Antibodies that specifically bind to a CNS-expressed human tau isoform do not bind to a human tau isoform expressed only outside the CNS (e.g., an isoform expressed only in other areas of the body, such as the peripheral nervous system). According to some embodiments, an antibody that specifically binds to a CNS-expressed human tau isoform binds to or recognizes an epitope region of a human tau isoform expressed in the CNS that comprises a glutamine at residue 124 (Q124) and an alanine at residue 125 (A125), with residue numbering referenced to SEQ ID NO.1. It will be understood that there are known human tau variants or isoforms, for example, arising from splice variants, and that such variants may result in changes 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 set forth in SEQ ID NO.1.

[0032] As used herein, the term "epitope region" refers to a discrete three-dimensional site on an antigen that is recognized in whole or in part by an antibody of the present invention. The amino acids in the epitope region provide chemically active surface groups on human tau, shape the specific three-dimensional structure of human tau, and provide specific charge characteristics. Conformational epitopes are distinguished from non-conformational / linear epitopes in that binding to the conformational epitope region is lost in the presence of denaturing solvents, whereas binding to the linear epitope region is not lost.

[0033] The antibodies of the present invention can be incorporated into pharmaceutical compositions that can be prepared by methods well known in the art and comprise an antibody 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, Loyd V. ed., Pharmaceutical Press, 2012, which provides an overview of formulation techniques generally understood by practitioners). Carriers suitable for use in pharmaceutical compositions include any material that retains the activity of the molecule when combined with the antibody of the present invention and is non-reactive with the patient's immune system. Pharmaceutical compositions comprising the antibodies of the present invention can be administered parenterally (e.g., subcutaneously, intravenously, intraperitoneally, intramuscularly, or transdermally) to patients at risk of or showing a disease or condition described herein. The pharmaceutical compositions of the present invention comprise an "effective" or "therapeutically effective" amount (used interchangeably herein) of an antibody of the present invention. An effective amount refers to the amount (dose, time, and mode of administration) necessary to achieve the desired therapeutic effect. The effective amount of an antibody may vary depending on factors such as the disease state, age, sex, and weight of the subject, and the ability of the antibody to elicit the desired response in the subject. An effective amount is also one in which any toxic or detrimental effects of the antibody of the invention are outweighed by the therapeutic benefits.

[0034] The percentage homology mentioned in the context of two or more amino acid sequences of the present disclosure refers to that, when using a sequence comparison algorithm (for example, BLASTP and BLASTN or other algorithms available to the technician) or by visual inspection to compare and align to obtain maximum correspondence, two or more sequences have the identical amino acid residues of a specified percentage.Depending on the application, the percentage homology can be present in a certain area of ​​the sequence being compared, such as a functional domain, or, be present in the total length of the two sequences being compared.As an example, the percentage homology of a sequence can be compared with a reference sequence.For example, when using a sequence comparison algorithm, test sequence and 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 percentage sequence identity or the homology of the test sequence relative to the reference sequence according to the specified program parameters. Exemplary sequence alignment and / or homology algorithms are available 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 Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally 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 analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0035] 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 may also refer to lymph fluid, biopsy aspirate, ascites, body fluid extracts, solid tissue, external parts of the skin, respiratory tract, nasal cavity, intestinal and genitourinary tracts, tears, saliva, milk, tumors, organs, cell cultures, and / or cell culture components.

[0036] The present disclosure also relates to methods for clinical diagnosis, prognosis or treatment of a subject by a medical professional 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 of the present disclosure. For example, a medical professional can recommend treatment, change treatment, or perform additional diagnostic assessments (e.g., neuroimaging).

[0037] The anti-tau antibodies disclosed herein 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. Such assays can be used to detect and / or assess the abundance and / or pattern of hTau-pT217 expression for diagnostic, prognostic, or therapeutic purposes, such as to monitor polypeptide levels in serum, plasma, blood, or CSF, as part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen.

[0038] The anti-tau antibodies of the present invention 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. This assay 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 various 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 present invention may also be used for pharmacogenomic analysis. Such embodiments may be used to identify subjects who may benefit from a specific or improved treatment modality and / or to monitor the efficacy of current treatment regimens.

[0039] The level or measurement of hTau-pT217 provided by the assays of the present 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, if the method for detecting hTau-pT217 indicates that the level or concentration of hTau-pT217 in the patient sample is higher than a reference value, then hTau-pT217 is said to be "increased" in the patient sample. Conversely, if the level or concentration of hTau-pT217 in the patient sample is lower than a reference value or, for example, a previously measured hTau-pT217 value in the patient sample, then hTau-pT217 is said to be "decreased" in the patient sample.

[0040] As used herein, a "reference value" refers to a known or approximate concentration of hTau-pT217 associated with a particular condition. The concentration level in a reference value can be an absolute or relative amount, a range of amounts, or a minimum, mean, and / or median amount of hTau-pT217. A reference value can also serve as a baseline for hTau-pT217 to which patient samples are compared.

[0041] As used herein, a "control standard" refers to a sample that can be used to compare the results obtained from a patient sample in the methods of the present 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, mean, and / or median amount. The control standard can also serve as a baseline for hTau-pT217 to which patient samples are compared. The control standard can include concentration values ​​from the same patient or a known normal reference for hTau-pT217. In addition, in some embodiments, the control standard can represent the hTau-pT217 concentration in the form of a standard curve.

[0042] As used herein, the term "capture antibody" refers to an antibody that binds to hTau-pT217. In such embodiments, the capture antibody is capable of binding to and capturing hTau-pT217 in a patient sample under appropriate conditions, e.g., specifically binding to hTau-pT217 (e.g., not binding to 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 rest of the sample. In some embodiments, the capture antibody can be an antibody that specifically binds to a CNS-expressed human tau isoform (e.g., which can include hTau phosphorylated at threonine at residue 217), and the antibody that specifically binds to hTau-pT217 is used as a "secondary (or detection) antibody." In some embodiments, the capture antibody is immobilized. In some embodiments, the detection antibody is labeled with a detectable label. In some embodiments, the capture antibody is immobilized in a "sandwich" immunoassay, and the capture antibody or first antibody specifically binds to an epitope region of human tau comprising 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 label. 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 first antibody. In such embodiments, the detection antibody binds to hTau-pT217 at a second epitope region that does not overlap with the first or capture antibody and can be labeled with a detectable label. In some such embodiments, the second antibody is an antibody of the present invention that specifically binds to a CNS-expressed human tau isoform.

[0043] As used herein, a "detectable label" is a moiety, composition, or technology that can be used to detect the formation of a complex between an antibody that specifically binds hTau-pT217 and hTau-pT217 of the present invention. According to some embodiments, the detectable label can be conjugated directly or indirectly to the antibody (capture or detection, as the case may be). Exemplary embodiments of detectable labels include biotin; radioisotopes; fluorophores or other fluorescent moieties; and enzyme moieties.

[0044] The terms "diagnosis" or "diagnosing" as used interchangeably herein refer to methods by which one skilled 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, as well as identifying a patient for, for example, the presence or development of a neurological disease or condition or for which treatment is needed, or the effectiveness of a treatment for a neurological disease in a patient. According to the present invention, diagnosis can be based on a combination of other clinical indicators understood by a medical professional to arrive at a diagnosis. According to some embodiments of the present invention, the diagnostic applications of the present invention can be used to diagnose a patient in AD Braak stage I, II, III, IV, V, or VI. AD Braak staging is known in the art, as described in Braak et al., (2006) Acta Neuropathol 112(4):389-404.

[0045] Example

[0046] anti-hTau-pT217 antibody

[0047] Anti-hTau-pT217 antibodies disclosed herein, or antibodies that specifically bind to hTau-pT217, are produced using 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 comprising a phosphorylated threonine and four or more amino acids N- and C-terminal to this threonine, as set forth in SEQ ID NO. 1 (SEQ ID NO. 26 provides an example of a peptide that can be used for immunization). Lymphocytes that produce antibodies that bind to 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, pp. 59-103 (Academic Press, 1986)). Hybridomas are seeded and cultured in a suitable culture medium (preferably containing one or more substances that inhibit the survival of unfused myeloma cells). The binding specificity of the monoclonal antibodies produced by the hybridomas is then determined by in vitro binding assays (e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)) using hTau-pT217 and recombinant tau that is not phosphorylated at threonine residue 217 (numbering is based on reference to SEQ ID NO. 1). Antibodies that specifically bind to hTau-pT217 (e.g., do not bind to human tau that is not phosphorylated at residue 217, numbering is based on SEQ ID NO. 1) are identified. Preferably, the hybridomas can be subcloned by limiting dilution procedures and cultured by standard methods, including in animals as ascites tumors (Goding, Monoclonal Antibodies: Principles and Practice, pp. 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-Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0048] cDNA encoding the antibodies of the present invention is sequenced using conventional protocols. An exemplary rabbit anti-hTau-pT217 antibody ("mAb A"), generated essentially according to the protocols described herein, comprises a heavy chain of SEQ ID NO. 2 and a light chain of SEQ ID NO. 4. The complementarity determining regions (CDRs) or variable regions of the sequenced antibodies can be used to convert into chimeric or humanized antibodies, and / or into other mammalian IgG formats. For example, clones can be converted into murine IgG chimeric antibodies, such as the exemplary rabbit variable region, murine IgG constant region chimeric anti-hTau-pT217 antibody ("mAb B") having a heavy chain variable region of SEQ ID NO. 6 and a heavy chain of SEQ ID NO. 7, and a light chain variable region of SEQ ID NO. 8 and a light chain of SEQ ID NO. 9. Binding specificity can then be reassessed. The cDNA sequences encoding the heavy and light chains can be cloned and engineered into a GS (glutamine synthetase) expression vector. The engineered immunoglobulin expression vector can then be stably transfected into CHO cells. Those skilled in the art will appreciate that mammalian expression of antibodies will result in glycosylation, typically at highly conserved N-glycosylation sites in the Fc region. Stable clones can be verified for expression of antibodies that specifically bind to hTau-pT217. Positive clones can be expanded into serum-free medium and used to produce antibodies in a bioreactor. The medium into which the antibodies have 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 equilibrated with a compatible buffer (e.g., phosphate-buffered saline). The column is washed to remove non-specifically bound components. For example, bound antibodies are eluted by a pH gradient, and the antibody fractions are detected, such as by SDS-PAGE, and then pooled. The antibodies can be concentrated and / or sterile filtered using conventional techniques. Soluble aggregates and multimers can be effectively removed by conventional techniques, including size exclusion, hydrophobic interactions, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, for example, at -70°C, or can be freeze-dried.

[0049] Antibodies specific for hTau isoforms expressed only in the CNS

[0050] Antibodies that specifically bind to CNS-expressed human tau isoforms disclosed herein can be produced using a hybridoma method (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 rabbit) can be immunized with a human tau protein (e.g., hTau as set forth in SEQ ID NO. 1) comprising glutamine at residue 124 and alanine at residue 125 (numbering as shown in SEQ ID NO. 1) or a peptide thereof. 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 fusing agent to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Hybridomas can be seeded and cultured in a suitable culture medium (preferably containing one or more substances that inhibit the survival of unfused myeloma cells). The binding specificity of the monoclonal antibodies produced by the hybridomas is then determined by in vitro binding assays (e.g., immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA)) for CNS-expressed human tau isoforms (e.g., peptides having the sequence of SEQ ID NO. 1) and / or peripherally expressed human tau isoforms (e.g., peptides having the sequence of SEQ ID NO. 27, which do not contain the glutamine at residue 124 adjacent to the alanine at residue 125 of SEQ ID NO. 1). Antibodies that specifically bind to CNS-expressed human tau isoforms (e.g., do not bind to peripherally expressed human tau) can be identified. Preferably, the hybridomas can be subcloned by limiting dilution procedures and cultured by standard methods, including in animals as ascites tumors (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Monoclonal antibodies secreted by hybridomas (and / or subclones) are purified according to conventional procedures, such as affinity chromatography (eg, protein A or protein G-agarose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0051] The cDNA encoding the antibodies of the present invention was sequenced using conventional procedures. An exemplary murine antibody of the present disclosure that specifically binds to a CNS-expressed human tau isoform ("mAb C"), produced essentially according to the procedures 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 antibodies can be used to convert into chimeric or humanized antibodies, and / or into other mammalian IgG formats, and express their components in host cells such as CHO cells.

[0052] Binding kinetics and affinity

[0053] Octet available from ForteBio The binding of the hTau-pT217-specifically binding antibodies of the present disclosure to the recombinant hTau-pT217 protein having the amino acid sequence of SEQ ID NO: 1 was measured by a biolayer interferometry (BLI) assay measured using an ELISA instrument (performed 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)).

[0054] Unless otherwise noted, all reagents and materials were obtained from ForteBio (Freemont, CA). Protein A biosensors were used to immobilize the antibody of interest for analysis. An exemplary antibody sample (mAb A) of the present invention was prepared at 5 μg / mL by dilution into running buffer. 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 consisted of: (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) monitoring the association phase by incubating the antibody-loaded biosensor with serial dilutions of recombinant hTau-pT217 protein for 300 seconds; and (4) monitoring the dissociation phase by returning the biosensor to running buffer.

[0055] Binding data were processed using standard double referencing and fitted to a 1:1 binding model using Data Analysis v9.0 evaluation software to determine the association rates (k on, M -1 s -1 units) and dissociation rate (koff, s -1 Unit). From the relation K D = koff / kon Calculate the equilibrium dissociation constant (K D The results are given in Table 1.

[0056] Table 1: BLI binding data to recombinant hTau-pT217.

[0057]

[0058] *K D The results are considered relative as they were not normalized for the effects of avidity.

[0059] Binding specificity to hTau-pT217

[0060] Use Octet available from ForteBio The specificity of exemplary antibodies (mAb A and mAb B) of the present invention that specifically bind to hTau-pT217 was determined using synthetic peptides using a BLI assay measured by an instrument (using HBS-EP + running buffer (GE Healthcare, 10 mM Hepes pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% surfactant P20) at 25°C). 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 peptides were incubated with 5 μg / mL of IgG from mAb A and mAb B diluted in running buffer for 300 seconds, followed by 300 seconds of dissociation. Binding data were determined using Data Analysis v9.0 evaluation software. The binding signal (nm) for each peptide at the end of dissociation is provided in Table 2.

[0061] Table 2: BLI binding data of mAb A and mAb B to recombinant hTau with and without pT217

[0062] Exemplary antibodies Phospho-threonine (nm unit) Non-phosphorylated threonine (nm unit) mAb A 4.4178 -0.0024 mAb B 4.7715 0.3389

[0063] hTau-pT217 immunoassay

[0064] The 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 small streptavidin spot plate using the Meso Scale Discovery (MSD) platform. mAb A or mAb B is used as the capture antibody and biotinylated. A SULFO-TAG secondary antibody, such as mAb C (an antibody disclosed herein that specifically binds to a CNS-expressed human tau isoform), is used as the detection antibody. The antibody is conjugated with Sulfo-NHS-Biotin (Thermo Scientific, catalog number: 21329) or MSDGOLD 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 that has been phosphorylated in vitro with glycogen synthase kinase-3 and characterized by mass spectrometry. Samples were thawed on wet ice, vortexed briefly, and plasma diluted 1:4 in sample buffer: for mAb A (phosphate buffered saline (PBS), 0.5% bovine serum albumin (BSA), 0.5% Tween 20, 5mM EDTA, 5mM EGTA); for mAb 2 (50mM HEPES, 300mM NaCl, 5mM EDTA, 5mM EGTA, 1% Triton X-100, 1% MSD Blocker A, 2% PEG). Heterophilic Blocker 1 (Scantibodies Inc, catalog number: 3KC533) was added to a concentration of 200ug / mL. Calibration diluents were made by mixing sample buffer 50 / 50 with Knock-Out Serum Replacement (Gibco, 10828-010).

[0065] Block MSD small-spot streptavidin (MSD, L45SA)-coated plates with 200 μL of PBS containing 3% bovine serum albumin at 650 rpm on a shaker at room temperature for 1 hour. Wash the plates three times with 200 μL of wash buffer (PBS + 0.05% Tween 20). For hTau-pT217 plates, 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 at room temperature for 1 hour on a shaker at 650 rpm. Wash the plates again three times with 200 μL of wash buffer. Add 50 μL of diluted calibrators or samples to the plates and incubate at room temperature for 2 hours on a shaker at 650 rpm. The plates were then washed three times with 200 uL of wash buffer. For the hTau-pT217 plate, 25 uL of 0.25 ug / mL (diluted in MSD diluent 35, for mAb 2, 2% PEG was added) of SULFO-tagged detection antibody (mAb C) was added and incubated at room temperature for 1 hour with shaking at 650 rpm on a plate shaker. The plates were washed three times with 200 uL of wash buffer for the final time. 150 uL of 2X MSD plate reading buffer T (MSD, R92TC) containing surfactant was added to each plate and the plate was read on an MSD SQ120 within 10 minutes of adding the plate reading buffer. The results were analyzed by MSD software using the 4PL, 1 / y 2 The weights are calculated for interpolation using the following equation: Y = b1 + ((b2 – b1) / (1 + (x / b3) b4 )).

[0066] hTau-pT217 immunoassay as a prognostic assay for neuroimaging

[0067] Levels of hTau-pT217 and hTau-pT181 were assessed in the blood of subjects enrolled in AD clinical trials. The hTau-pT217 immunoassay described herein was used to assess 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) scanning, which measures tau 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 measured in the neocortical region of interest based on a reference signal in the white matter. Correlations between flortaucipir SUVR and plasma pTau (pT181 and pT217, respectively) were assessed using Spearman rank correlation. Receiver operating characteristic (ROC) curve analysis was performed using a logistic regression model with age and sex as covariates and a flortaucipir-positive cutoff of SUVR>1.1. A mixed-effects model was used to assess the predictive value of baseline pTau for future cognitive decline, with pTau quartiles evaluated.

[0068] As shown in Table 3, in both studies, the hTau-pT217 immunoassay showed statistically significantly higher correlation than Flortaucipir PET (p-value < 0.05).

[0069] Table 3. Correlation of hTau-pT217 and hTau-pT181 Immunoassays with Flortaucipir PET

[0070] hTau-pT217 immunoassay hTau-pT181 immunoassay Study 1 0.783 0.332 Study 2 0.463 0.308

[0071] In both studies, the area under the ROC curve for flortaucipir positivity showed higher values ​​for hTau-pT217 than for hTau-pT181 (Study 1: 0.88 vs. 0.79; Study 2: 0.83 vs. 0.81). Furthermore, an 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 for cognitive decline caused by neurodegenerative diseases, and exhibits significant improvement over the hTau-pT181 immunoassay.

[0072] hTau-pT217 immunoassay as a diagnostic marker for AD and disease progression

[0073] The levels of hTau-pT217 in CSF were assessed using the described immunoassays and compared with the results of the hTau-pT181 immunoassay. Briefly, CSF samples from the Swedish BioFINDER study of intact 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) were assessed using the hTau-pT217 and hTau-p181 immunoassays. 184 participants received 18 F-Flortaucipir positron emission tomography (PET) Quantification in a priori defined regions associated with tau pathology in AD (including tau Braak stages I-II, III-IV, and V-VI and worse) 18 Uptake of F-Flortaucipir.

[0074] In CU patients, both hTau-pT217 immunoassay and hTau-pT181 immunoassay were associated with Braak stage I-II 18 F-Flortaucipir was associated with AD; in AD patients, hTau-pT217 immunoassay and hTau-pT181 immunoassay were both associated with Braak stages III-IV and V-VI; in MCI patients, hTau-pT217 was associated with Braak stages I-II, III-IV, and V-VI. 18 F-Flortaucipir was associated with the region, while hTau-pT181 was associated only in the Braak stage I-II region. Importantly, in all three diagnostic groups (CU, MCI, and AD) and all regions (Braak stages I-II, III-IV, and V-VI), the region 18 The correlation between F-Flortaucipir and hTau-pT217 immunoassays was statistically significantly better than that between hTau-pT181 immunoassays and 18 Correlation between F-Flortaucipir (p<0.001-0.016).

[0075] Compared with the hTau-pT181 immunoassay, the hTau-pT217 immunoassay showed similar 18 The correlation coefficients of F-Flortaucipir were statistically significantly higher (all p<0.001): hTau-pT217 (0.698-0.752) vs. hTau-pT181 (0.572-0.706). In addition, hTau-pT217 immunoassay proved to be pathological in all regions.18 F-Flortaucipir status was statistically significantly (p < 0.001) more accurate predictor (hTau-pT217: AUC 0.890-0.929; hTau-pT181 immunoassay: AUC 0.859-0.904). In addition, the hTau-pT217 immunoassay showed statistically significant improvement (p = 0.026) in differentiating AD from non-AD neurodegenerative diseases (hTau-pT217: AUC 0.943; hTau-pT181: AUC 0.914) compared to the hTau-pT181 immunoassay. These results indicate that the hTau-pT217 immunoassay is more accurate than the hTau-pT181 immunoassay in differentiating AD from non-AD neurodegenerative diseases. 18 F-Flortaucipir neuroimaging correlates with AD and is able to differentiate AD from other neurological diseases and stages and shows significant improvement compared with hTau-pT181 immunoassays.

[0076] hTau-pT217 immunoassay correlates with Tau PET SUVr

[0077] Tau PET SUVr has been shown to correlate with tau pathology in the literature. In a study measuring hTau-pT217 in plasma, serum, and CSF of patients with mild AD, the hTau-pT217 immunoassay disclosed herein correlated with Tau PET SUVr. Briefly, 190 subjects underwent an hTau-pT217 immunoassay in plasma at baseline. Of these 190 subjects, 185 underwent a Tau PET SUVr assay. Data were analyzed using a Spearman test, and a significant correlation was observed with a Spearman p = 0.49 and an uncorrected p-value of < 0.001. Additionally, 187 subjects underwent serum hTau-pT217 measurement at baseline. Of these 187 subjects, 182 underwent a Tau PET SUVr assay. Data were analyzed using a Spearman test, and a significant correlation was observed with a Spearman p = 0.41 and an uncorrected p-value of < 0.001. In addition, 86 subjects had hTau-pT217 measured in CSF at baseline. Of these 86 subjects, 29 underwent Tau PET SUVr assay. The data were analyzed using the Spearman test, and a significant correlation was observed with Spearman ρ = 0.70 and an uncorrected p-value < 0.001. The results support the use of pTau217 levels measured in CSF, plasma, or serum to identify Tau pathology.

[0078] hTau-pT217 immunoassay correlates with cognitive status in mild AD

[0079] The mean values ​​of hTau-pT217 in plasma, serum, and CSF of subjects with mild AD were calculated according to the immunoassay described above. The results in each matrix are provided in Table 4.

[0080] Table 4. Mean values ​​of hTau-pT217 in plasma, serum, and CSF of subjects with mild AD

[0081] body fluid average value Standard deviation N plasma 14.2 6.2 190 serum 13.4 6.2 187 CSF 684.9 531.1 86

[0082] The hTau-pT217 immunoassay disclosed herein correlates with cognitive status (based on the Mini-Mental State Examination (MMSE)) and the change from baseline in MMSE relative to placebo in patients with mild AD. hTau-pT217 was assessed by immunoassay in plasma, serum, and CSF of patients with mild AD. Briefly, for each matrix (plasma, serum, and CSF), the significance of subjects with hTau-pT217 measurements (as described herein) and MMSE assessments and assessments of changes from baseline was evaluated using a Spearman test. The hTau-pT217 immunoassay showed statistically significant correlations with cognitive status and changes from baseline in plasma and serum (the number of CSF samples was too small to achieve statistical significance, but the data showed correlations. As the number of samples increases, such as assessments of serum and plasma, it is expected that CSF will have statistically significant correlations with MMSE and changes from baseline in MMSE). The results are provided in Table 5.

[0083] Table 5. Correlation of hTau-pT217 Immunoassay Results with MMSE and MMSE Baseline Change in Plasma, Serum, and CSF in Subjects Diagnosed with Mild AD

[0084]

[0085]

[0086] *Changes from baseline in the MMSE were not considered statistically significant due to the small sample size of patients.

[0087] These results demonstrate a cross-sectional association of hTau-pT217 with the cognitive measure MMSE and support the utility of hTau-pT217 in determining the risk of future cognitive decline.

[0088] hTau-pT217 immunoassay correlates with amyloid status

[0089] The hTau-pT217 immunoassay disclosed herein is associated with amyloid status. In short, 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 people (CUY-A-). Samples from each group were analyzed using the hTau-pT217 immunoassay described herein. The results are provided in Table 6.

[0090] Table 6. hTau-pT217 is associated with AD and amyloid status

[0091]

[0092]

[0093] The evaluation of the hTau-pT217 immunoassay for identifying amyloid-positive subjects was determined by evaluating the results for the different groups presented in Table 6 using a student's t-test. The results are presented in Table 7.

[0094] Table 7. Students t-test for differences in mean plasma hTau-pT217 values ​​associated with AD and amyloid status

[0095]

[0096] 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 that 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 identifying amyloid-positive subjects. The area under the ROC curve was 0.94, as shown in Table 6 and Table 7. Figure 1 shown.

[0097] The data presented herein demonstrate that the hTau-pT217 assay of the present disclosure is capable of identifying amyloid-positive subjects; serving as a diagnosis of AD and determining a subject's cognitive status associated with AD; identifying subjects at risk for AD and / or in the earliest stages of AD; and serving as a diagnosis of AD progression. The data also demonstrate that the hTau-pT217 assay of the present disclosure correlates with neuroimaging, is functional in serum, plasma, and CSF matrices, and outperforms the known hTau-pT181 assay.

[0098] Exemplary embodiments of the present disclosure

[0099] 1. An antibody that specifically binds to human tau phosphorylated at threonine at residue 217 of SEQ ID NO. 1 ("hTau-pT217").

[0100] 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,

[0101] Among them, LCDR1 has the amino acid sequence of SEQ ID NO: 13, LCDR2 has the amino acid sequence of SEQ ID NO: 14, LCDR3 has the amino acid sequence of SEQ ID NO: 15, HCDR1 has the amino acid sequence of SEQ ID NO: 10, HCDR2 has the amino acid sequence of SEQ ID NO: 11, and HCDR3 has the amino acid sequence of SEQ ID NO: 12.

[0102] 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,

[0103] Wherein LCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 13, LCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 14, LCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 15, HCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 10, HCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 11, and HCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 12.

[0104] 4. The antibody of embodiment 2 or 3, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) selected from the group consisting of:

[0105] a. LCVR having the amino acid sequence of SEQ ID NO: 5 and HCVR having the amino acid sequence of SEQ ID NO: 3; and

[0106] b. LCVR having the amino acid sequence of SEQ ID NO: 8 and HCVR having the amino acid sequence of SEQ ID NO: 6.

[0107] 5. The antibody of embodiment 2 or 3, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR) selected from the group consisting of:

[0108] a. a LCVR having an amino acid sequence having at least 95% homology to the amino acid sequence of SEQ ID NO: 5, a HCVR having an amino acid sequence having at least 95% homology to the amino acid sequence of SEQ ID NO: 3; and

[0109] b. A LCVR having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 8, and a HCVR having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 6.

[0110] 6. The antibody of any one of embodiments 1-5, wherein the antibody is humanized.

[0111] 7. The antibody of any one of embodiments 1-6, wherein the antibody comprises an IgG4 heavy chain.

[0112] 8. The antibody of any one of embodiments 1-7, wherein the antibody comprises a kappa light chain.

[0113] 9. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 8 and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0114] 10. An antibody that specifically binds to a CNS-expressed human tau isoform.

[0115] 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.

[0116] 12. 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,

[0117] Among them, LCDR1 has the amino acid sequence of SEQ ID NO:23, LCDR2 has the amino acid sequence of SEQ ID NO:24, LCDR3 has the amino acid sequence of SEQ ID NO:25, HCDR1 has the amino acid sequence of SEQ ID NO:20, HCDR2 has the amino acid sequence of SEQ ID NO:21, and HCDR3 has the amino acid sequence of SEQ ID NO:22.

[0118] 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,

[0119] wherein LCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 23, LCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 24, LCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 25, HCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 20, HCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 21, and HCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 22.

[0120] 14. The antibody of embodiment 12 or 13, comprising a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 19.

[0121] 15. The antibody of embodiment 12 or 13, comprising a light chain variable region (LCVR) having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 17, and a heavy chain variable region (HCVR) having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 19.

[0122] 16. The antibody of any one of embodiments 10-15, wherein the antibody is humanized.

[0123] 17. The antibody of any one of embodiments 10-16, wherein the antibody comprises an IgG4 heavy chain.

[0124] 18. The antibody of any one of embodiments 10-17, wherein the antibody comprises a kappa light chain.

[0125] 19. A pharmaceutical composition comprising the antibody of any one of embodiments 10-18 and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0126] 20. A method for treating a neurodegenerative disease, comprising administering an effective amount of the antibody or pharmaceutical composition thereof according to any one of embodiments 1 to 19 to a patient in need thereof.

[0127] 21. The method of embodiment 20, wherein the neurodegenerative disease is a tauopathy.

[0128] 22. The method of embodiment 21, wherein the tauopathy is one of AD, PSP and FTD.

[0129] 23. The antibody or pharmaceutical composition thereof according to any one of embodiments 1 to 19, for use in therapy.

[0130] 24. The antibody or pharmaceutical composition thereof according to any one of embodiments 1 to 19, for use in treating a neurodegenerative disease.

[0131] 25. The antibody or pharmaceutical composition thereof according to embodiment 24, wherein the neurodegenerative disease is a tauopathy.

[0132] 26. The antibody or pharmaceutical composition thereof according to embodiment 25, wherein the tauopathies are selected from AD, PSP and FTD.

[0133] 27. The antibody or pharmaceutical composition thereof according to any one of embodiments 1 to 19, for use in the preparation of a medicament for treating a neurodegenerative disease.

[0134] 28. The antibody or pharmaceutical composition thereof according to embodiment 27, wherein the neurodegenerative disease is a tauopathy.

[0135] 29. The antibody or pharmaceutical composition thereof according to embodiment 28, wherein the tauopathy is selected from AD, PSP and FTD.

[0136] 30. A method for detecting hTau-pT217 in a patient sample, comprising the steps of:

[0137] contacting the patient sample with the antibody of any one of embodiments 1-8; and detecting a signal provided by the contacting step.

[0138] 31. A method for quantifying hTau-pT217 in a patient sample, comprising the steps of:

[0139] contacting the patient sample with the antibody of any one of embodiments 1-8; and detecting a signal provided by the contacting step.

[0140] 32. The method of embodiment 31, further comprising the steps of: contacting a control standard with the antibody; and detecting a signal provided by the step of contacting the control standard.

[0141] 33. A method for quantifying hTau-pT217 in a patient sample, comprising the steps of: contacting the 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 a signal provided by the detectable label upon formation of 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, wherein one of the antibody of embodiments 1-8 and the second antibody comprises a detectable label; detecting a signal provided by the detectable label upon formation of a complex comprising the antibody of embodiments 1-8, the second antibody, and the control standard.

[0142] 34. A method for diagnosing a patient as one or more of: (i) having a neurodegenerative disease; (ii) being at risk for developing a neurodegenerative disease; (iii) being in need of neurodegenerative disease treatment; or (iv) being in need of neuroimaging, comprising the steps of: contacting a patient sample with the antibody of any one of embodiments 1-8; and detecting binding between the antibody and hTau-pT217 in the patient sample.

[0143] 35. The method of embodiment 34, further comprising the step of diagnosing the patient as one of: (i) having a neurodegenerative disease; (ii) being at risk for developing a neurodegenerative disease; (iii) requiring neurodegenerative disease treatment; or (iv) requiring neuroimaging if the level of hTau-pT217 detected in the patient sample exceeds a reference level.

[0144] 36. A method for diagnosing and treating a neurodegenerative disease in a patient, the method comprising the steps of: contacting a patient sample with the antibody of any one of embodiments 1-8; detecting binding between the antibody and hTau-pT217 in the patient sample; diagnosing the patient with a neurodegenerative disease; and administering a therapeutically effective amount of the anti-human Tau antibody to the diagnosed patient.

[0145] 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.

[0146] 38. The method of any one of embodiments 31-37, further comprising the step of quantifying hTau-pT217 in the patient sample.

[0147] 39. The method of embodiment 38, wherein the step of quantifying hTau-pT217 comprises quantifying hTau-pT217 in the patient sample relative to a reference standard.

[0148] 40. The method of any one of embodiments 30-39, wherein the patient sample is one of blood, plasma, serum or CSF.

[0149] 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.

[0150] 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 upon formation of a complex comprising the antibody of embodiments 1-8, the second antibody, and hTau-pT217.

[0151] 43. The method according to any one of embodiments 41-42, wherein the antibody according to embodiments 1-8 and one of the second antibodies are immobilized on a substrate.

[0152] 44. The method of any one of embodiments 30-43, wherein the steps of contacting the patient sample with the antibody and contacting the patient sample with the second antibody occur simultaneously.

[0153] 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 complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3,

[0154] Among them, LCDR1 has the amino acid sequence of SEQ ID NO:23, LCDR2 has the amino acid sequence of SEQ ID NO:24, LCDR3 has the amino acid sequence of SEQ ID NO:25, HCDR1 has the amino acid sequence of SEQ ID NO:20, HCDR2 has the amino acid sequence of SEQ ID NO:21, and HCDR3 has the amino acid sequence of SEQ ID NO:22.

[0155] 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,

[0156] wherein LCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 23, LCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 24, LCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 25, HCDR1 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 20, HCDR2 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 21, and HCDR3 has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 22.

[0157] 47. The method of any one of embodiments 46-47, wherein the second antibody comprises a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 17.

[0158] 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),

[0159] Among them, LCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 19, and HCVR has an amino acid sequence that is at least 95% homologous to the amino acid sequence of SEQ ID NO: 17.

[0160] 49. A method for 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 the first antibody, wherein the first antibody is an antibody of one of embodiments 1-8; contacting the patient sample with the second antibody, wherein the second antibody is an antibody of one of embodiments 10-18.

[0161] 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: the antibody of any one of embodiments 1-8; the antibody of any one of embodiments 10-18.

[0162] 51. The assay of embodiment 50, wherein one of the antibodies comprises a detectable label.

[0163] Sequence Listing

[0164] SEQ ID NO: 1 (hTau-pT217)

[0165] MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEP

[0166] GSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGH

[0167] VTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKT

[0168] PPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQT

[0169] APVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSV

[0170] QIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGG

[0171] NKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLA

[0172] DEVSASLAKQGL

[0173] SEQ ID NO: 2 (HC of an exemplary rabbit anti-hTau-pT217 antibody)

[0174] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAG

[0175] WAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSLGQPKAPSVFPLAP

[0176] CCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSS

[0177] QPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCV

[0178] VVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKV

[0179] HNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGK

[0180] AEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGKSEQ IDNO:3 (Heavy chain variable region of exemplary rabbit anti-hTau-pT217 antibody)

[0181] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAG

[0182] WAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSL

[0183] SEQ ID NO:4 (Light chain of exemplary rabbit anti-hTau-pT217 antibody)

[0184] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVS

[0185] SHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVKGDPVAPTVLI

[0186] FPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADNTYNLSSTLT

[0187] LTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0188] SEQ ID NO:5 (LCVR of exemplary rabbit anti-hTau-pT217 antibody)

[0189] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVS

[0190] SHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVK

[0191] SEQ ID NO:6 (HCVR of exemplary chimeric anti-hTau-pT217 antibody)

[0192] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAG

[0193] WAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSL

[0194] SEQ ID NO:7 (HC of exemplary chimeric anti-hTau-pT217 antibody)

[0195] QSVEESGGRLVTPGTPLTLTCTVSGLSPSWYGVHWVRQAPGKGLEWIGVLRAGSHTYYAG

[0196] WAKGRFAISKTSTTVALKITSPTTEDTAIYFCGSVGRGIWGPGTLVTVSLAKTTPPSVYPLAP

[0197] GSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSST

[0198] WPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTC

[0199] VVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCR

[0200] VNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWN

[0201] GQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS

[0202] PGK

[0203] SEQ ID NO:8 (LCVR of exemplary chimeric anti-hTau-pT217 antibody)

[0204] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVS

[0205] SHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVK

[0206] SEQ ID NO:9 (LC of exemplary chimeric anti-hTau-pT217 antibody)

[0207] AQVLTQTASPVSATVGGTVTINCQASLAVYNNNYLAWYQQKPGQPPKRLIYLASSLSSGVS

[0208] SHFKGSGSGTQFTLTISDVQADDAATYFCQGSYDCTIADCVAFGGGTEVVVKRADAAPTVS

[0209] IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS

[0210] TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0211] SEQ ID NO:10 (Exemplary HCDR-1)

[0212] GLSPSWYGVH

[0213] SEQ ID NO: 11 (Exemplary HCDR2)

[0214] VLRAGSHTYYAGWAKG

[0215] SEQ ID NO: 12 (Exemplary HCDR3)

[0216] VGRGI

[0217] SEQ ID NO: 13 (Exemplary LCDR1)

[0218] QASLAVYNNNYLA

[0219] SEQ ID NO: 14 (Exemplary LCDR2)

[0220] LASSLSS

[0221] SEQ ID NO: 15 (Exemplary LCDR3)

[0222] QGSYDCTIADCVA

[0223] SEQ ID NO: 16 (HC of an exemplary CNS-expressed-only human tau-binding antibody)

[0224] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSPYYWSWIRQPPDKGLEWIGEINWSGDTNYN

[0225] PSLKSRVTISLDTSKNQFSLNLSSVTAADTAVYYCARSFDRWGQGTLVTVSSASTKGPSVFPL

[0226] APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS

[0227] SLGTKTYTCNVDHKPSNTKVDKRVEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMI

[0228] SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW

[0229] MSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP

[0230] EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLH

[0231] NHHTTKSFSRTPGK

[0232] SEQ ID NO: 17 (HCVR of an exemplary CNS-expressed-only human tau-binding antibody)

[0233] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSPYYWSWIRQPPDKGLEWIGEINWSGDTNYN

[0234] PSLKSRVTISLDTSKNQFSLNLSSVTAADTAVYYCARSFDRWGQGTLVTVSS

[0235] SEQ ID NO: 18 (LC of an exemplary CNS-expressed-only human tau binding antibody)

[0236] EIVLTQSPGTLSLSPGERATLSCRASQSVRSNYFAWYQQKPGQAPRLLIYGVSRRAFGIPDRF

[0237] SGSGSGTDFTLTISRLEPEDFAVYYCQQYGASLITFGQGTRLEIKRTVAAPSVFIFPPSDEQLK

[0238] SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY

[0239] EKHKVYACEVTHQGLSSPVTKSFNRGEC

[0240] SEQ ID NO: 19 (LCVR of an exemplary CNS-expressed-only human tau-binding antibody)

[0241] EIVLTQSPGTLSLSPGERATLSCRASQSVRSNYFAWYQQKPGQAPRLLIYGVSRRAFGIPDRF

[0242] SGSGSGTDFTLTISRLEPEDFAVYYCQQYGASLITFGQGTRLEIK

[0243] SEQ ID NO: 20 (HCDR1 of an exemplary CNS-expressed-only human tau-binding antibody)

[0244] AVYGGSFSPYYWS

[0245] SEQ ID NO: 21 (HCDR2 of an exemplary CNS-expressed-only human tau-binding antibody)

[0246] EINWSGDTN

[0247] SEQ ID NO: 22 (HCDR3 of an exemplary CNS-expressed-only human tau-binding antibody)

[0248] ARSFDR

[0249] SEQ ID NO: 23 (LCDR1 of an exemplary CNS-expressed-only human tau binding antibody)

[0250] RASQSVRSNYFA

[0251] SEQ ID NO: 24 (LCDR2 of an exemplary CNS-expressed-only human tau binding antibody)

[0252] YGVSRRAF

[0253] SEQ ID NO: 25 (LCDR3 of an exemplary CNS-expressed-only human tau binding antibody)

[0254] QQYGASLIT

[0255] SEQ ID NO: 26 (Exemplary peptide for immunization)

[0256] RTPSLPTPPTR

[0257] The phosphorylation of T at residue 7

[0258] SEQ ID NO: 27 (Exemplary peptide for immunization)

[0259] AEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPG

[0260] SETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHV

[0261] TQEPESGKVVQEGFLREPGPPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEGGRH

[0262] APELLKHQLLGDLHQEGPPLKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSKASPAQDGR

[0263] PPQTAAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGRAKGQDAPLEFTFHVEITP

[0264] NVQKEQAHSEEHLGRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQPAAPRGKPVSR

[0265] VPQLKARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNRPCLSPKHPTPGSSDPLIQPSSAPVC

[0266] PEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPP

[0267] APKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKKVAVVRTPKPPSSAKS

[0268] RLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPG

[0269] GGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHV

[0270] PGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQ

[0271] LATLADEVSASLAKQGL

Claims

1. A method for detecting hTau-pT217 in a sample taken from a patient, comprising the following steps: contacting the sample with a first antibody that specifically binds to human tau phosphorylated at threonine at residue 217 of SEQ ID NO. 1 ("hTau-pT217"); contacting the sample with a second antibody that binds to an epitope region of hTau-pT217 that does not overlap with the first antibody; and detecting the binding of the first antibody to hTau-pT217, wherein the second antibody specifically binds to a CNS-expressed human tau isoform, and wherein 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.

2. The method according to claim 1, wherein the method is characterized in that it further comprises a step of quantifying hTau-pT217 in the sample.

3. The method according to claim 1 or 2, wherein the sample is taken from one of blood, plasma, serum or CSF.

4. The method of any one of claims 1 to 3, wherein one of the first antibody or the second antibody comprises a detectable label, and the detecting step comprises detecting a signal provided by the detectable label upon formation of a complex comprising the first antibody, the second antibody, and hTau-pT217.

5. The method of any one of claims 1-4, wherein the first 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 sequences of the CDRs are selected from the group consisting of: (a) LCDR1 has the amino acid sequence of SEQ ID NO: 13, LCDR2 has the amino acid sequence of SEQ ID NO: 14, LCDR3 has the amino acid sequence of SEQ ID NO: 15, HCDR1 has the amino acid sequence of SEQ ID NO: 10, HCDR2 has the amino acid sequence of SEQ ID NO: 11, and HCDR3 has the amino acid sequence of SEQ ID NO: 12; or (b) LCDR1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:13, LCDR2 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14, LCDR3 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:15, HCDR1 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:10, HCDR2 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:11, and HCDR3 has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

12.

6. The method of claim 5, wherein the LCVR and HCVR of the first antibody are selected from: a. LCVR having the amino acid sequence of SEQ ID NO: 5 and HCVR having the amino acid sequence of SEQ ID NO: 3; b. LCVR having the amino acid sequence of SEQ ID NO: 8 and HCVR having the amino acid sequence of SEQ ID NO: 6; c. a LCVR having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, and a HCVR having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; and d. A LCVR having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 8, a HCVR having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

6. The method of claim 3 , wherein the sample is taken from CSF.

8. The method of claim 3, wherein the sample is taken from plasma.

9. The method according to any one of claims 1 to 8, wherein one of the first antibody and the second antibody is immobilized on a substrate.

10. The method of claim 9, wherein the substrate is selected from one of a microplate or beads.

11. The method of any one of claims 1-7, wherein the steps of contacting the sample with the first antibody and contacting the sample with the second antibody occur simultaneously.

12. An antibody that specifically binds to a CNS-expressed human tau isoform, 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, wherein the 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, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 23, LCDR2 has the amino acid sequence of SEQ ID NO: 24, LCDR3 has the amino acid sequence of SEQ ID NO: 25, HCDR1 has the amino acid sequence of SEQ ID NO: 20, HCDR2 has the amino acid sequence of SEQ ID NO: 21, and HCDR3 has the amino acid sequence of SEQ ID NO:

22.

13. The antibody of claim 12, 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, wherein LCDR1 has an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 23, LCDR2 has an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 24, LCDR3 has an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 25, HCDR1 has an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 20, HCDR2 has an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 21, and HCDR3 has an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO:

22.

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

19.

15. The antibody of any one of claims 12-14, comprising a light chain variable region (LCVR) having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (HCVR) having an amino acid sequence at least 95% homologous to the amino acid sequence of SEQ ID NO:

19.

16. The antibody of any one of claims 12-15, wherein the antibody is humanized.

17. The antibody of any one of claims 12-16, wherein the antibody comprises an IgG4 heavy chain.

18. The antibody of any one of claims 12-17, wherein the antibody comprises a kappa light chain.

19. A pharmaceutical composition comprising the antibody of any one of claims 12 to 18 and one or more pharmaceutically acceptable carriers, diluents or excipients.

Images