HK40112938A - Phospho-tau antibodies and methods of use - Google Patents

Description

Phosphorylated-TAU antibody and its usage

Cross-referencing

This application claims the benefit of U.S. Provisional Patent Application No. 63/242,437, filed September 9, 2021, which is incorporated herein by reference in its entirety.

Background Technology

Biomarker discovery and screening technologies for Alzheimer's disease (AD) and other tau protein disorders are an ongoing field of development where these tools can be applied to screening populations to identify which non-demented individuals are at greatest risk of developing AD dementia and to assess disease progression in patients. Proteins reflecting AD pathology have been detected using various methods, including amyloid-β42 ( _Aβ42 ), neurofilament light chains, and various tau isoforms. Abnormal or hyperphosphorylation of tau is associated with the conversion of pathologically normal tau molecules into paired helical filaments (PHF) tau and neurofibrillary tangles (NFTs), which indicate various tau protein disorder pathologies.

Summary of the Invention

Tau is an important microtubule-associated protein, highly expressed in CNS neurons and playing a crucial role in normal cellular physiology. Tau has also been found to be dysregulated in Alzheimer's disease and other tau protein disorders. Six isoforms of tau protein are generated by the TAU gene via alternative splicing. These isoforms differ from each other by the presence or absence of two N-terminal inserts and a repeat sequence called R2. All six isoforms of tau protein are highly soluble under normal and healthy cellular conditions and are generally regulated by phosphorylation and dephosphorylation. Tau has been shown to interact with microtubules and promote microtubule assembly. In neurons, tau promotes the formation and stabilization of axonal microtubules. Tau has an additional role in driving neurite growth. Impaired interaction between tau and microtubules may be a significant component of the pathology, development, and progression of tau protein disorders. Hyperphosphorylation of tau is a hallmark feature of AD and other tau protein disorders, and the degree of hyperphosphorylation is often associated with disease progression. Hyperphosphorylation of tau protein may lead to the self-assembly of insoluble tangles of paired helical and straight filaments of tau. These insoluble tangled aggregates, known as neurofibrillary tangles (NFTs), contain hyperphosphorylated tau and are considered a pathological marker of tau protein disorders.

Phosphorylated tau (pTau), total tau, and _Aβ42 , detected in cerebrospinal fluid (CSF) and/or blood, are individual biomarkers for Alzheimer's disease and several other related tau protein disorders. In individuals subsequently diagnosed with AD, CSF pTau was increased in both the prodromal and dementia stages compared to age- and sex-matched controls. CSF pTau levels showed a strong correlation with the degree of cognitive impairment in individuals with AD. In fact, CSF pTau levels can be used with a degree of precision as a biomarker to predict progression from cognitively unimpaired to mild cognitive impairment (MCI) and then to AD dementia. Significant increases in CSF pTau have been shown in samples from preclinical AD individuals in terms of its utility as a biomarker predicting even relatively early stages of AD progression. Changes in pTau phosphorylation have been demonstrated in both preclinical sporadic cases of AD and early stages of autosomal dominant AD. When measured within the same individual, blood levels of pTau, total tau, and _Aβ42 are typically lower than CSF levels, and if blood levels of these biomarkers can be measured with sufficient specificity and accuracy, they can be used as informative biomarkers for AD and other related tau protein disorders.

Several phosphorylation sites of hyperphosphorylated tau leading to aggregation into NFTs have been identified. In the longest tau isoform, 79 potential serine or threonine phosphorylation sites are present, and at least 30 of these sites have been identified as phosphorylating in NFT aggregates. A common site used to determine the phosphorylation status of tau molecules is threonine-181. CSF fluid contains a range of tau fragments in varying abundances. Tau fragments from the N-terminal and middle regions of tau peptides are much more abundant in CSF samples than C-terminal tau fragments. Plasma samples from individuals also contain tau peptides and tau peptide fragments, but they tend to be present at lower concentrations than in matched CSF samples. The ability to detect tau phosphorylation at specific amino acid residues associated with disease pathology and progression is a key component of diagnosis, disease staging, and an indicator of the therapeutic efficacy of Alzheimer's disease (AD) and other tau protein disorders. Detecting and measuring pTau levels at specific disease-related residues in plasma samples will greatly contribute to the development of more sensitive and refined diagnostic, prognostic, and disease analyses for individuals who may be at risk of developing AD or other tau protein disorders or are in their early stages. Tau phosphorylation at threonine 217 (pTau 217) is one such residue of particular interest in the development of novel biomarkers and diagnostic assays. Changes in pTau biomarker concentrations in CSF and plasma are thought to precede measurable behavioral or cognitive changes in AD and other tau protein disorders. Developing new assays capable of producing a continuum of specific points and degrees of tau phosphorylation at certain residues will undoubtedly aid in clinically relevant medical diagnostic and treatment decisions. Comparing the results of new assays with those of existing assays can also yield further medically informative determinations. Results of plasma-based tau biomarker assays can be compared with matched CSF samples (detecting CSFpTau or CSF-soluble Aβ) and with positron emission tomography (PET) scans that detect the extent and location of Aβ aggregates, serving as indicators of their practicality, particularly for analysis in preclinical or early disease stages.

This document provides a method for detecting phosphorylated tau in samples from an individual, the method comprising: immunoassay of the sample using an antibody or antibody fragment comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises an amino acid sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:30-34, and wherein the VL comprises an amino acid sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:35-40. This document also provides a method for detecting phosphorylated tau in samples from an individual, wherein the phosphorylated tau is selected from pTau-181, pTau-212, pTau-217, pTau-231, pTau-214, and pTau-220. This document further provides a method for detecting phosphorylated tau in samples from an individual, wherein the phosphorylated tau is pTau-217. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the phosphorylated tau is pTau-231. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-217 and pTau-231. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-212 and pTau-217. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-212 and pTau-231. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-181 and pTau-217. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-181 and pTau-231. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods detect pTau-181, pTau-217, and pTau-231. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods detect pTau-212, pTau-217, and pTau-231. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods detect pTau-217 and pTau-231 in samples selected from plasma and serum samples. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods detect pTau-212 and pTau-231 in samples selected from plasma and serum samples. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods detect pTau-212 and pTau-231 in samples selected from plasma and serum samples. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-181 and pTau-217 in samples selected from plasma and serum samples. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-181 and pTau-231 in samples selected from plasma and serum samples. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method detects pTau-181, pTau-217, and pTau-231 in samples selected from plasma and serum samples. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the VH comprises an amino acid sequence according to any one of SEQ ID NO: 30-34. This document also provides a method for detecting phosphorylated tau in a sample from an individual, wherein the VL comprises an amino acid sequence according to any one of SEQ ID NO: 35-40. This document also provides a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence according to any one of SEQ ID NO: 30-34, and wherein the VL comprises an amino acid sequence according to any one of SEQ ID NO: 35-40. This document also provides a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 30, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 35. This document also provides a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 31, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 36. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the VH contains an amino acid sequence having at least about 90% identity with SEQ ID NO:31, and wherein the VL contains an amino acid sequence having at least about 90% identity with SEQ ID NO:37. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the VH contains an amino acid sequence having at least about 90% identity with SEQ ID NO:32, and wherein the VL contains an amino acid sequence having at least about 90% identity with SEQ ID NO:38. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the VH contains an amino acid sequence having at least about 90% identity with SEQ ID NO:33, and wherein the VL contains an amino acid sequence having at least about 90% identity with SEQ ID NO:39. This document also provides a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:34, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:40. This document also provides a method for detecting phosphorylated tau in a sample from an individual, wherein the antibody or antibody fragment comprises an amino acid sequence having at least about 90% identity with any one of SEQ ID NO:41-51. This document also provides a method for detecting phosphorylated tau in a sample from an individual, further comprising measuring the sample to determine the level of a biomarker selected from: Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. This article also provides methods for detecting phosphorylated tau in samples from individuals, wherein the samples are selected from blood samples, plasma samples, serum samples, and cerebrospinal fluid (CSF) samples. This article also provides methods for detecting phosphorylated tau in samples from individuals, which further includes determining the individual's Alzheimer's disease based on the detection of phosphorylated tau. This article also provides methods for detecting phosphorylated tau in samples from individuals, which further includes determining the individual's prognosis for developing Alzheimer's disease based on the detection of phosphorylated tau. This article also provides methods for detecting phosphorylated tau in samples from individuals, which also determine the individual's age, genotype, or biomarker expression. This article also provides methods for detecting phosphorylated tau in samples from individuals, wherein the biomarkers are selected from Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. This article also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods have at least about 80% specificity for detecting phosphorylated tau. This article also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods have at least about 85% specificity for detecting phosphorylated tau. This article also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods have at least about 90% specificity for detecting phosphorylated tau. This article also provides methods for detecting phosphorylated tau in samples from individuals, wherein the methods have at least about 80% sensitivity for detecting phosphorylated tau. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method has a sensitivity of at least about 85% for detecting phosphorylated tau. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method has a sensitivity of at least about 90% for detecting phosphorylated tau. This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method is capable of detecting phosphorylated tau in the sample with a detection limit of at least about 1.0 picograms/mL (pg/mL). This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method is capable of detecting phosphorylated tau in the sample with a detection limit of at least about 1.5 picograms/mL (pg/mL). This document also provides methods for detecting phosphorylated tau in samples from individuals, wherein the method is capable of detecting phosphorylated tau in the sample with a detection limit of at least about 5 picograms/mL (pg/mL).

In some embodiments, this document also provides an anti-tau antibody comprising i) a heavy chain comprising a variable heavy chain (VH) domain and ii) a light chain comprising a variable light chain (VL) domain, wherein the VH domain comprises an HCDR1 sequence having a sequence selected from SEQ ID NO: 1-5, an HCDR2 sequence having a sequence selected from SEQ ID NO: 6-9, and an HCDR3 sequence having a sequence selected from SEQ ID NO: 10-13, and the VL domain comprises an LCDR1 sequence having a sequence selected from SEQ ID NO: 14-19, an LCDR2 sequence having a sequence selected from SEQ ID NO: 20-23, and an LCDR3 sequence having a sequence selected from SEQ ID NO: 24-29. In some embodiments, the HCDR1 sequence contains SEQ ID NO:1, the HCDR2 sequence contains SEQ ID NO:6, the HCDR3 sequence contains SEQ ID NO:10, the LCDR1 sequence contains SEQ ID NO:14, the LCDR2 sequence contains SEQ ID NO:20, and the LCDR3 sequence contains SEQ ID NO:24. In some embodiments, the HCDR1 sequence contains SEQ ID NO:2, the HCDR2 sequence contains SEQ ID NO:7, the HCDR3 sequence contains SEQ ID NO:11, the LCDR1 sequence contains SEQ ID NO:15, the LCDR2 sequence contains SEQ ID NO:21, and the LCDR3 sequence contains SEQ ID NO:25. In some embodiments, the HCDR1 sequence contains SEQ ID NO:2, the HCDR2 sequence contains SEQ ID NO:7, the HCDR3 sequence contains SEQ ID NO:11, the LCDR1 sequence contains SEQ ID NO:16, the LCDR2 sequence contains SEQ ID NO:22, and the LCDR3 sequence contains SEQ ID NO:26. In some embodiments, the HCDR1 sequence contains SEQ ID NO:3, the HCDR2 sequence contains SEQ ID NO:8, the HCDR3 sequence contains SEQ ID NO:10, the LCDR1 sequence contains SEQ ID NO:17, the LCDR2 sequence contains SEQ ID NO:20, and the LCDR3 sequence contains SEQ ID NO:27. In some embodiments, the HCDR1 sequence contains SEQ ID NO:4, the HCDR2 sequence contains SEQ ID NO:7, the HCDR3 sequence contains SEQ ID NO:12, the LCDR1 sequence contains SEQ ID NO:18, the LCDR2 sequence contains SEQ ID NO:23, and the LCDR3 sequence contains SEQ ID NO:28. In some embodiments, the HCDR1 sequence contains SEQ ID NO:5, the HCDR2 sequence contains SEQ ID NO:9, the HCDR3 sequence contains SEQ ID NO:13, the LCDR1 sequence contains SEQ ID NO:19, the LCDR2 sequence contains SEQ ID NO:21, and the LCDR3 sequence contains SEQ ID NO:29. In some embodiments, this document also provides an anti-tau antibody comprising i) a heavy chain containing a variable heavy chain (VH) domain and ii) a light chain containing a variable light chain (VL) domain, wherein the VH domain has at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO:30-34. In some embodiments, this document also provides an anti-tau antibody comprising i) a heavy chain containing a variable heavy chain (VH) domain and ii) a light chain containing a variable light chain (VL) domain, wherein the VL domain has at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO:35-40. In some embodiments, the anti-tau antibody described herein is a chimeric antibody or an antigen-binding fragment thereof. In some embodiments, the anti-tau antibodies described herein include IgG-scFv, nanobodies, BiTE, double-chain antibodies, DART, TandAb, scDiabody, scDiabody-CH3, triple bodies, mini-antibodies, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, Fab’, F(ab’)2, F(ab’)3, F(ab’)2-scFv2, scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, double-chain antibody-Fc, tandem scFv-Fc, or endoantibodies. In some embodiments, the anti-tau antibodies described herein are IgG1 antibodies. In some embodiments, the anti-tau antibodies described herein are IgG2 antibodies. In some embodiments, the anti-tau antibodies described herein are IgG4 antibodies. In some embodiments, this document also provides an anti-tau antibody comprising i) a heavy chain containing a variable heavy chain (VH) domain and ii) a light chain containing a variable light chain (VL) domain, wherein the light chain is a κ chain. In some embodiments, this document also provides an anti-tau antibody comprising i) a heavy chain containing a variable heavy chain (VH) domain and ii) a light chain containing a variable light chain (VL) domain, wherein the anti-tau antibody has a binding affinity for human tau of about 100 pM to about 3 nM. In some embodiments, this document provides an anti-tau antibody comprising a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 52-56. In some embodiments, this document provides an anti-tau antibody comprising a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 57-62. In some embodiments, this document provides an anti-tau antibody comprising a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 52-56, and a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 57-62. In some embodiments, this document provides an anti-tau antibody comprising a VH domain encoded by a nucleic acid having the same sequence as SEQ ID NO: 52-56. In some embodiments, this document provides an anti-tau antibody comprising a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:57-62. In some embodiments, this document provides an anti-tau antibody comprising a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:52-56 and a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:57-62.

Incorporation

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually indicated to be incorporated by reference.

Attached Figure Description

This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a published copy of this patent or application document containing one or more color drawings.

Figure 1 illustrates the outline of the single-molecule array method used in this paper for the determination of the tau antibody described herein. After adding the substrate to the sample (sandwich ELISA on beads, 1.1), the sample is added to the disk (1.2). The beads are given time to settle into the microarray wells on the disk (one bead per well) (1.3). Excess beads are then removed using sealing oil for imaging (1.4). Beads with the sandwich complex (positive beads) will fluoresce through the substrate and be visible during imaging; beads without the sandwich complex (negative beads) will still be visible in the imaging but will not fluoresce (1.5). The percentage of positive beads is converted to AEB (average enzyme/bead) values.

Figures 2A-2D depict the data for antibodies 1, 2, 3, 4, 5, and 6 in the assay.

Figure 3 depicts the ELISA data.

Figures 4A-4G depict the immunohistochemical staining data for antibody 6.

Figures 5A-5G depict the immunohistochemical staining data for antibody 5.

Figures 6A-6G depict the immunohistochemical staining data for antibody 2.

Figure 7 depicts a graph of indirect ELISA assays and a graph of ELISA data for assays of antibodies binding to pTau-217 peptide.

Figure 8 depicts a graph of signal/noise (S/N) analysis of ELISA assays of antibody 2 bound to pTau-217 peptide in 120 clinical samples derived from plasma, and a graph of coefficient of variation (CV%) of ELISA assays of antibody 2 bound to pTau-217 peptide in 120 clinical samples derived from plasma.

Figure 9 depicts the calibration curves (Cal curves) of antibody 2-based pTau-217 assays for clinical samples (plates) of specified QTx derived from cerebrospinal fluid (68 CSF samples) and plasma (120 plasma samples) compared to assays using the ADx p204 antibody.

Figure 10 depicts the results of the antibody 2-based pTau assay-217 using matched samples from individual samples derived from plasma (Y-axis) or CSF (X-axis), and a statistical analysis of the relevant results for individuals with non-Alzheimer's disease clinical diagnoses, indeterminate diagnoses, or Alzheimer's disease clinical diagnoses.

Figure 11 depicts the results of the antibody 2/sample-based pTau assay-217 relative to the results of the antibody 2/sample-based pTau assay-181, along with a statistical analysis of the relevant results.

Figure 12 depicts the results of the pTau assay-217 based using antibody 2/sample versus the results of the Tau assay based using Innotest pTau 181 antibody/sample, along with a statistical analysis of the relevant results.

Figure 13 depicts the results of a pTau assay based on antibody 2 as the capture antibody, antibody ADx p204 as the detection antibody, and peptide as the calibrator, along with a statistical analysis of the results.

Figure 14 depicts the results of a pTau assay-217 based on antibody 2, grouping samples from individuals clinically diagnosed with Alzheimer's disease together with samples from control individuals derived from CSF or plasma.

Figure 15 depicts the results of the pTau assay-217 based on antibody 2 against plasma samples of different concentrations of EDTA, and lists the coefficient of variation for each sample concentration to illustrate the accuracy of the assay.

Figure 16 depicts the results of the antibody 2-based pTau assay for 217, plotted as coefficient of variation (CV%) relative to the measured concentration.

Figure 17 depicts a graph of the results of the antibody-based pTau assay-217 using antibody 2, and a statistical analysis of parallelism to determine whether real samples containing high concentrations of endogenous analyte after dilution provide a similar level of detection in the standard curve.

Figure 18 depicts the results of the antibody-based pTau assay-217 using antibody 2, and a statistical analysis of linearity to determine whether sample matrices spiked with the analyte above the detection limit can still provide reliable quantification after dilution within the standard curve range of the four sample buffer spike peaks.

Figure 19 depicts a graph of the results of the antibody-based pTau assay-217 using antibody 2, and a statistical analysis of linearity to determine whether a sample matrix spiked with an analyte above the detection limit can still provide reliable quantification after dilution within the standard curve range of three samples plus a calibration sample.

Figure 20 depicts the results of the antibody 2-based pTau assay-217 in clinical validation using a memory clinical cohort, and a plot of receiver operating characteristic (ROC) analysis mapping the sensitivity of the pTau-217 assay.

Figure 21 depicts the results of the pTau assay-217 based on antibody 2 against groups of individuals with AD dementia.

Figure 22 depicts a graph and a chart of sample stratification results using antibodies derived from the pTau assay for groups from control and AD dementia individuals.

Figure 23 depicts the results of the pTau assay-217 using antibody 2 and the pTau assay-181 using antibody 2, showing the accuracy plot with the calculated coefficient of variation.

Figure 24 depicts a graph comparing the clinical performance of various pTau-based assays in terms of sensitivity and specificity, and a chart showing the statistical analysis of the results.

Figure 25 illustrates a schematic diagram of Tau, which indicates the relative positions of various protein domains and the positions of threonine residues, and whose phosphorylation state can be determined using the methods disclosed herein.

Figure 26 illustrates the reactivity of Tau fragments with unphosphorylated T217 (Bio-pt654) and full-length Tau (Tau441) in indirect ELISA with various antibodies.

Figure 27 illustrates the reactivity of Tau fragments with phosphorylated T181 (Bio-pt126) and phosphorylated T231 (Bio-pt146) in indirect ELISA with various antibodies.

Figure 28 depicts the results of assays using pTau217 monoclonal antibody as a capture tool for various synthetic peptides, and the results of this assay using antibody 2 as a capture tool.

Figure 29 depicts Western blot analysis of brain lysate samples from AD patients or controls using various Tau antibodies.

Detailed Implementation

Alzheimer's disease (AD) is a complex disease, and effective treatment requires accurate diagnosis. This article describes improved compositions and methods for detecting AD, which contain improved antibodies for use in diagnostic and/or prognostic assays.

certain terms

Throughout this disclosure, various embodiments are presented in range form. It should be understood that the range format is for convenience and brevity only and should not be construed as a strict limitation on the range of any embodiment. Therefore, unless the context explicitly indicates otherwise, the description of a range should be considered to specifically disclose all possible subranges and individual values within that range up to one-tenth of the lower limit unit. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual values within that range, such as 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the width of the range. The upper and lower limits of these intermediate ranges may be independently included within smaller ranges and are also included within the invention, subject to any explicit exclusions within the specified range. When a range includes one or two limit values, ranges that do not include one or both of those included limit values are also included in the invention unless the context explicitly indicates otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit any embodiments. As used herein, the singular forms “a/an,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise. It will also be understood that when the terms “comprises” and/or “comprising” are used in this specification, they specify the presence of the stated features, integrals, steps, operations, elements, and/or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more associated listed items.

Unless otherwise specified or obvious from the context, as used herein, the term “about” when referring to a numerical value or range of values shall be understood to mean the numerical value and its +/- 10% values, or, for values listed in a range, to mean from 10% below the listed lower limit to 10% above the listed upper limit.

The terms “individual,” “patient,” or “object” are used interchangeably. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., physician, registered nurse, nurse practitioner, physician assistant, caregiver, or hospice worker). Furthermore, these terms refer to human or animal objects.

The term "antibody" as used herein is used in the broadest sense and includes monoclonal antibodies, including intact antibodies and their functional (antigen-binding) antibody fragments, including the following fragments: antigen-binding (Fab) fragments, F(ab’)2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (sFv or scFv)) and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). The term covers genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptide antibodies, chimeric antibodies and heteroconjugate antibodies, tandem bi-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to cover its functional antibody fragment. The term also covers intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. Antibodies may contain a rabbit IgG1 constant region. Antibodies may contain a rabbit IgG4 constant region. Antibodies include, but are not limited to, full-length antibodies and natural antibodies, as well as fragments and portions thereof that retain their binding specificity, such as any of their specific binding portions, including those having any number of immunoglobulin classes and/or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and their biologically relevant (antigen-binding) fragments or specific binding portions, including but not limited to Fab, F(ab’)2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally one type within a substantially homogeneous antibody composition; therefore, any individual antibody contained within a monoclonal antibody composition is identical, except for possibly naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies may contain a rabbit IgG1 constant region or a rabbit IgG4 constant region.

The term "complementarity-determining region" or "CDR" is a segment of the antibody variable region that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. Therefore, the CDR is sometimes referred to as a hypervariable region. A variable region contains three CDRs. CDR peptides can be obtained by constructing genes that encode the CDR of the antibody of interest. Such genes are prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using a polymerase chain reaction. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pp. 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pp. 137-185 (Wiley-Liss, Inc. 1995).

The term "Fab" refers to a protein containing a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that it has residues added at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH is the name for Fab' in this paper, where one or more cysteine residues in the constant domain are accompanied by a free thiol group. The Fab' fragment is generated by reducing the heavy chain disulfide bonds of the F(ab')2 fragment. Other chemical conjugations of antibody fragments are also known.

A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the antibody heavy chain (VH) and light chain (VL), linked by a short linker peptide of 10 to approximately 25 amino acids. The linker is typically enriched with glycine to improve flexibility, and serine or threonine to improve solubility, and may link the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody, although the constant region has been removed and the linker introduced. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96. Additionally, the antibody fragment comprises a single-chain polypeptide characterized by either a VH domain capable of assembling with the VL domain, or a VL domain capable of assembling with the VH domain, to a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

As used herein, the term "percentage of amino acid sequence identity (%)" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a given sequence, after sequence alignment and the introduction of vacancies (if necessary to achieve the maximum percentage of sequence identity), and without considering any conserved substitutions as part of sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

When ALIGN-2 is used for amino acid sequence comparison, the percentage of amino acid sequence identity of a given amino acid sequence A with respect to, and or relative to, a given amino acid sequence B (which can be alternatively phrased as a given amino acid sequence A having or containing a certain amino acid sequence identity % with respect to, and or relative to, a given amino acid sequence B) is calculated as follows: 100 × fraction X/Y, where X is the number of amino acid residues that score as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless otherwise explicitly stated, all amino acid sequence identity % values used herein were obtained using the ALIGN-2 computer program as described immediately following the previous paragraph.

The terms “complementarity-determining region” and “CDR” (which are synonymous with “hypervariant region” or “HVR”) are known in the art to refer to a discontinuous amino acid sequence within the variable region of an antibody that confers antigen specificity and/or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms “frame region” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including those described in the following literature: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“…”). Chothia numbering scheme); MacCallum et al., J.Mol.Biol.262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J.Mol.Biol.262,732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T-cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J M ol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modeling antibodies on the WEB,” Protein Eng. December 2000; 13(12):819-24 (“AbM” numbering scheme). In some embodiments, the CDR of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody region sequences, with insertions (adapted by the insertion letter, e.g., "30a") and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("insertions and deletions") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions described herein pertain. Although any methods and materials similar to or equivalent to those described herein may be used to practice or test the methods and compositions described herein, representative exemplary methods and materials are described hereafter.

Tau antibody

This document provides antibodies that bind to tau. In some cases, the tau-binding antibodies are monoclonal antibodies. In some aspects, this document discloses an anti-tau antibody. In some cases, the anti-tau antibody specifically binds to mammalian tau. In some cases, the anti-tau antibody specifically binds to human tau. In some cases, the anti-tau antibody specifically binds to the N-terminal portion of tau. In some cases, the anti-tau antibody specifically binds to the N-terminal portion of human tau. In some cases, the anti-tau antibody specifically binds to the portion of tau containing the protein domain P2. In some cases, the anti-tau antibody specifically binds to the portion of human tau containing the protein domain P2. In some cases, the anti-tau antibody specifically binds to the portion of tau containing the protein domain P1. In some cases, the anti-tau antibody specifically binds to the portion of human tau containing the protein domain P1. In some cases, the anti-tau antibody specifically binds to the portions of tau containing both protein domains P1 and P2. In some cases, the anti-tau antibody binds specifically to portions of human tau containing protein domains P1 and P2.

In some embodiments, the anti-tau antibody comprises i) a heavy chain comprising a variable heavy chain (VH) domain and ii) a light chain comprising a variable light chain (VL) domain. In some embodiments, the VH domain comprises a heavy chain CDR1 (HCDR1) sequence having sequences selected from SEQ ID NO:1-5, a heavy chain CDR2 (HCDR2) sequence having sequences selected from SEQ ID NO:6-9, and a heavy chain CDR3 (HCDR3) sequence having sequences selected from SEQ ID NO:10-13. In some embodiments, the VL domain comprises a light chain CDR1 (LCDR1) sequence having sequences selected from SEQ ID NO:14-19, a light chain CDR2 (LCDR2) sequence having sequences selected from SEQ ID NO:20-23, and a light chain CDR3 (LCDR3) sequence having sequences selected from SEQ ID NO:24-29.

In some implementations, the VH region of the anti-tau antibody contains HCDR1, HCDR2 and HCDR3 sequences selected from Table 1.

Table 1. Amino acid sequence of HCDR

SEQ ID NO: HCDR1 sequence 1 SQKVG 2 SYAMI 3 NYKVG 4 NYAMS 5 THAMT SEQ ID NO: HCDR2 sequence 6 IINNYGSTYYASWAKG 7 FISRSGITYYASWAKG 8 IINYYSQTYYASWAKG 9 VINPSGSAYYATWVNG SEQ ID NO: HCDR3 sequence 10 DPDGSIVFDI 11 EFGAVGSDYYRDAFNL 12 EFGAVGSDYYRDALRL 13 DYITAGDYYMDAFDP

In some embodiments, the VH region comprises the HCDR1 sequence having SEQ ID NO:1; the HCDR2 sequence having SEQ ID NO:6; and the HCDR3 sequence having SEQ ID NO:10. In some embodiments, the VH region comprises the HCDR1 sequence having SEQ ID NO:2; the HCDR2 sequence having SEQ ID NO:7; and the HCDR3 sequence having SEQ ID NO:11. In some embodiments, the VH region comprises the HCDR1 sequence having SEQ ID NO:3; the HCDR2 sequence having SEQ ID NO:8; and the HCDR3 sequence having SEQ ID NO:10. In some embodiments, the VH region comprises the HCDR1 sequence having SEQ ID NO:4; the HCDR2 sequence having SEQ ID NO:7; and the HCDR3 sequence having SEQ ID NO:12. In some implementations, the VH region includes the HCDR1 sequence having SEQ ID NO:5; the HCDR2 sequence having SEQ ID NO:9; and the HCDR3 sequence having SEQ ID NO:13.

In some implementations, the VL region of the anti-tau antibody contains sequences selected from LCDR1, LCDR2, and LCDR3 in Table 2.

Table 2. LCDR amino acid sequence

SEQ ID NO: LCDR1 sequence 14 QSSQSVVYNNRLS 15 QASESINSWLS 16 QASQNIYSNLA 17 QSSQSVYSNKRLA 18 QASQSIGSNLA 19 QASQSISNQLS SEQ ID NO: LCDR2 sequence 20 GASTLAS twenty one RASTLAS twenty two GASNLAS twenty three GASTLES SEQ ID NO: LCDR3 sequence twenty four LGSYDCSSGDCHA 25 QSYYEEDGIGYA 26 QGYDYSTAGAYP 27 AGGYDCSTGDCWT 28 QSYYEGSDIGYA 29 QQGYNRDNVDNL

In some embodiments, the VL region comprises the LCDR1 sequence having SEQ ID NO:14; the LCDR2 sequence having SEQ ID NO:20; and the LCDR3 sequence having SEQ ID NO:24. In some embodiments, the VL region comprises the LCDR1 sequence having SEQ ID NO:15; the LCDR2 sequence having SEQ ID NO:21; and the LCDR3 sequence having SEQ ID NO:25. In some embodiments, the VL region comprises the LCDR1 sequence having SEQ ID NO:16; the LCDR2 sequence having SEQ ID NO:22; and the LCDR3 sequence having SEQ ID NO:26. In some embodiments, the VL region comprises the LCDR1 sequence having SEQ ID NO:17; the LCDR2 sequence having SEQ ID NO:20; and the LCDR3 sequence having SEQ ID NO:27. In some embodiments, the VL region comprises the LCDR1 sequence having SEQ ID NO:18; the LCDR2 sequence having SEQ ID NO:23; and the LCDR3 sequence having SEQ ID NO:28. In some embodiments, the VL region comprises the LCDR1 sequence having SEQ ID NO:19; the LCDR2 sequence having SEQ ID NO:21; and the LCDR3 sequence having SEQ ID NO:29.

In some embodiments, the anti-tau antibody is its antigen-binding fragment. In some embodiments, the anti-tau antibody is a chimeric antibody or its antigen-binding fragment. In some embodiments, the anti-tau antibody includes IgG-scFv, nanobodies, microbodies, microantibodies, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, Fab’, F(ab’)2, F(ab’)3, F(ab’)2-scFv2, scFv, scFv-KIH, Fab-scFv-Fc, or endoantibodies. In some embodiments, the anti-tau antibody includes bispecific antibodies. In some embodiments, the anti-tau antibody includes multispecific antibodies. In some embodiments, the anti-tau antibody is an IgG1 antibody. In some embodiments, the anti-tau antibody is an IgG2 antibody. In some embodiments, the anti-tau antibody is an IgG4 antibody. In some embodiments, the anti-tau antibody comprises a light chain, wherein the light chain is a κ chain.

In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to about 3 nM for human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to 300 pM for human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to 500 pM for human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to 800 pM for human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 300 pM to 600 pM for human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 300 pM to 900 pM for human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 400 pM to 1 nM for human tau. In some embodiments, the anti-tau antibody has a binding affinity for human tau of about 500 pM to 1.5 nM. In some embodiments, the anti-tau antibody has a binding affinity for human tau of about 500 pM to 2 nM. In some embodiments, the anti-tau antibody has a binding affinity for human tau of about 600 pM to 3 nM. In some embodiments, the anti-tau antibody has a binding affinity for human tau of about 100 pM to about 3 nM.

In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to 300 pM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to 500 pM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 100 pM to 800 pM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 300 pM to 600 pM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 300 pM to 900 pM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 400 pM to 1 nM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 500 pM to 1.5 nM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 500 pM to 2 nM for phosphorylated human tau. In some embodiments, the anti-tau antibody has a binding affinity of about 600 pM to 3 nM for phosphorylated human tau.

This article describes antibodies containing any of the sequences shown in Table 3 or Table 4.

Table 3. Variable structural domains of heavy chains

Table 4. Variable domains in light chains

In some embodiments, the variable domain (VH) of the heavy chain region comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of any one of SEQ ID NO: 30-34.

In some embodiments, the VH comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 consecutive amino acid residues of any one of SEQ ID NO: 30-34.

In some embodiments, the VH comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 80% sequence identity with at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 consecutive amino acid residues of any one of SEQ ID NO:30-34, and has at least 80% sequence identity with at least 120 consecutive amino acid residues of any one of SEQ ID NO:30-34.

In some embodiments, the VH comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 90% sequence identity with at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 consecutive amino acid residues of any one of SEQ ID NO:30-34, and has at least 90% sequence identity with at least 120 consecutive amino acid residues of any one of SEQ ID NO:30-34.

In some embodiments, the VH comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 50 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 60 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 70 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 80 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 90 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 95% sequence identity with at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 consecutive amino acid residues of any one of SEQ ID NO:30-34, and has at least 95% sequence identity with at least 120 consecutive amino acid residues of any one of SEQ ID NO:30-34.

In some embodiments, the VH comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 99% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 99% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 99% sequence identity with at least 110 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 99% sequence identity with at least 115 consecutive amino acid residues of any one of SEQ ID NO: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 consecutive amino acid residues of any one of SEQ ID NO: 30-34, and has at least 99% sequence identity with at least 120 consecutive amino acid residues of any one of SEQ ID NO: 30-34.

In some embodiments, the light chain variable domain (VL) comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of any one of SEQ ID NO: 35-40.

In some embodiments, the VL comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 35-40.

In some embodiments, the VL comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 80% sequence identity with at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 80% sequence identity with at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 80% sequence identity with at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 80% sequence identity with at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 80% sequence identity with at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 80% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO:35-40, and has at least 80% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO:35-40.

In some embodiments, the VL comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 90% sequence identity with at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 90% sequence identity with at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 90% sequence identity with at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 90% sequence identity with at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 90% sequence identity with at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 90% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO:35-40, and has at least 90% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO:35-40.

In some embodiments, the VL comprises an amino acid sequence of at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 50 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 60 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 70 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 80 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 90 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 95% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 99% sequence identity with at least 100 consecutive amino acid residues of any one of SEQ ID NO: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 consecutive amino acid residues of any one of SEQ ID NO: 35-40, and has at least 99% sequence identity with at least 105 consecutive amino acid residues of any one of SEQ ID NO: 35-40.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NO: 30-34; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of any one of SEQ ID NO: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO:30-34; and the VL comprises an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NO:35-40.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:30; and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:35.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:36.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:37. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:37.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:38. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:32; and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:38.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:39. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:33; and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:39.

In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to SEQ ID NO:40. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:34; and the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to SEQ ID NO:40.

In some embodiments, this document describes antibodies or antibody fragments comprising a heavy chain sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO: 41, 43, 46, 48, and 50. In some cases, the antibody or antibody fragment comprises a heavy chain sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 41, 43, 46, 48, and 50.

In some embodiments, this document describes antibodies or antibody fragments comprising a light chain sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:42, 44, 45, 47, 49, and 51. In some cases, the antibody or antibody fragment comprises a light chain sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO:42, 44, 45, 47, 49, and 51.

In some embodiments, this document describes an antibody or antibody fragment comprising a heavy chain sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:41, 43, 46, 48 and 50, and a light chain sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:42, 44, 45, 47, 49 and 51. In some cases, the antibody or antibody fragment comprises a heavy chain sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 41, 43, 46, 48, and 50, and a light chain sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 42, 44, 45, 47, 49, and 51.

Table 5. Heavy chain and light chain sequences

In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO:52-56. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO:57-62. The nucleic acid sequences of the VH domain of the anti-tau antibody described herein are listed in Table 6, and the nucleic acid sequences of the VL domain of the anti-tau antibody described herein are listed in Table 7. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:52. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:53. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:54. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:55. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:56. In some embodiments, the anti-tau-tau antibody comprises a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:57. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:62. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:52; and a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:57. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:53; and a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:53; and a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:54; and a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:55; and a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:563; and a VL domain encoded by a nucleic acid having at least 90% sequence identity with SEQ ID NO:62. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:52. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:53. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:54. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:55. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:56. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:57. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:62. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:52; and a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:57. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:53; and a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:53; and a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:54; and a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:55; and a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:56; and a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO:62.

Table 6. Nucleic acid sequences encoding the VH domain

Table 7. Nucleic acid sequences encoding the VL domain

In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing a sequence identical to that selected from SEQ ID NO:63-67. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing a sequence identical to that selected from SEQ ID NO:68-73. The nucleic acid sequences of the heavy chain of the anti-tau antibody described herein are listed in Table 8, and the nucleic acid sequences of the light chain of the anti-tau antibody described herein are listed in Table 9. The nucleic acid sequences listed in Tables 8 and 9 can be used in the in vitro production process of the antibody described herein. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing a sequence identical to that of SEQ ID NO:63. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing a sequence identical to that of SEQ ID NO:64. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing a sequence identical to that of SEQ ID NO:65. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:66. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:67. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:68. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:69. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:70. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:71. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:72. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:73. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:63; and a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:68. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:64; and a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:69. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:64; and a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:70. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:65; and a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:71. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:66; and a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:72. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:67; and a light chain encoded by a nucleic acid containing the same sequence as SEQ ID NO:73.

Table 8. Nucleic acid sequences encoding the heavy chain

Table 9. Nucleic acid sequences encoding the light chain

The method disclosed herein

This document discloses a method for detecting phosphorylated tau in samples from an individual using the antibodies described herein. In some embodiments, the phosphorylated tau is selected from pTau-212, pTau-217, pTau-231, pTau-214, and pTau-220. In some embodiments, the method for detecting phosphorylated tau in samples from an individual using the antibodies described herein includes improved specificity and sensitivity.

This document describes a method for detecting phosphorylated tau in a sample from an individual, the method comprising: measuring the sample using an antibody or antibody fragment that binds to phosphorylated tau. In some embodiments, the phosphorylated tau is selected from pTau-212, pTau-217, pTau-231, pTau-214, pTau-220, and pTau-181. In some embodiments, the phosphorylated tau is selected from pTau-212, pTau-217, pTau-231, pTau-214, and pTau-220. In some embodiments, the phosphorylated tau is pTau-217. In some embodiments, the phosphorylated tau is pTau-231. In some embodiments, the phosphorylated tau is pTau-181. In some embodiments, the phosphorylated tau is pTau-212. In some embodiments, the phosphorylated tau is pTau-217. In some embodiments, the phosphorylated tau is pTau-214. In some embodiments, the phosphorylated tau is pTau-220. In some embodiments, the phosphorylated tau is pTau-181 and pTau-217. In some embodiments, the phosphorylated tau is pTau-181 and pTau-231. In some embodiments, the phosphorylated tau is pTau-217 and pTau-231. In some embodiments, the phosphorylated tau is pTau-181, pTau-217, and pTau-231.

This document also describes a method for detecting phosphorylated tau in samples from individuals, the method comprising: measuring the sample using an antibody or antibody fragment that binds to a variety of phosphorylated tau proteins. In some embodiments, the method detects pTau-217 and pTau-231. In some embodiments, the method detects pTau-212 and pTau-217. In some embodiments, the method detects pTau-212 and pTau-231. In some embodiments, the method detects pTau-212, pTau-217, and pTau-231.

This document describes a method for detecting phosphorylated tau in samples from an individual, wherein the method detects pTau-217 and pTau-231 in samples selected from plasma and serum samples. In some embodiments, the method detects pTau-212 and pTau-217 in samples selected from plasma and serum samples. In some embodiments, the method detects pTau-212, pTau-217, and pTau-231 in samples selected from plasma and serum samples.

The methods described herein may include measuring a sample, wherein the sample is selected from plasma samples and serum samples. In some cases, the sample is a blood sample. In some cases, the sample is a cerebrospinal fluid sample. The sample may be a blood sample obtained by venous blood collection. The sample may be a blood sample obtained by finger puncture. The sample may be obtained from a healthcare provider or the subject. The method may include obtaining the sample from the subject. In some cases, the sample is obtained from the subject during a visit to a clinic or hospital.

In some embodiments, methods for determining the levels of biomarkers selected from Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof are also described herein. In some cases, the biomarker is Aβ42. In some cases, the biomarker is Aβ40. In some cases, the biomarker is both Aβ42 and Aβ40. In some cases, the biomarker is APOE. In some cases, the biomarker is selected from APOE2, APOE3, and APOE4. In some cases, the biomarker is APOE4.

In some embodiments, methods for detecting phosphorylated tau in a sample include immunoassays or ligand assays using antibodies or antibody fragments described herein. In some cases, the assay is selected from enzyme-linked immunosorbent assay (ELISA), colorimetric immunoassay, homogeneous immunoassay, non-optical immunoassay, fluorescence immunoassay, chemiluminescence immunoassay, electrochemiluminescence immunoassay, fluorescence resonance energy transfer (FRET) immunoassay, time-resolved fluorescence immunoassay, lateral flow immunoassay, microspot immunoassay, surface plasmon resonance assay, ligand assay, agglutination assay, chromatographic assay, and immunocapture-binding mass spectrometry. In some cases, the assay includes an immunoassay. In some cases, the assay is selected from Western blotting, enzyme-linked immunosorbent assay (ELISA), and chromatography. In some cases, the immunoassay is singlet. In some cases, the immunoassay is multiplex.

The methods described herein may include multiple immunoassays using the antibodies or antibody fragments described herein. In some cases, the multiple immunoassays are the same immunoassay (e.g., four or more ELISA assays). When the multiple immunoassays are the same immunoassay, each of the multiple immunoassays may detect different phosphorylated tau. When the multiple immunoassays are the same immunoassay, each of the multiple immunoassays may be performed in the same reaction chamber or in different reaction chambers. The reaction chamber may be any suitable space for performing the immunoassay. Examples of reaction chambers include, but are not limited to, wells in a microplate, Eppendorf tubes, or droplets.

In some cases, the multiple immunoassays are different immunoassays. When the multiple immunoassays are different immunoassays, each of the multiple immunoassays can detect different phosphorylated tau. When the multiple immunoassays are different immunoassays, each of the multiple immunoassays can be performed in the same reaction chamber or in different reaction chambers.

In some cases, the assay includes non-immunological assays. In some cases, the assay is selected from high-performance liquid chromatography (HPLC), high-performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), immunohistochemistry (IHC), polymerase chain reaction (PCR), quantitative PCR (qPCR), and combinations thereof.

The method described herein, using the antibodies described herein, can be used to determine an individual's Alzheimer's disease based on the detection of phosphorylated tau. In some embodiments, Alzheimer's disease is determined if pTau-212, pTau-217, pTau-231, pTau-214, pTau-220, or combinations thereof, are detected in a sample from an individual.

The method described herein, using the antibodies described herein, can be used to predict the prognosis of an individual's development of Alzheimer's disease based on the detection of phosphorylated tau. In some embodiments, the prognosis of an individual's development of Alzheimer's disease is determined if pTau-212, pTau-217, pTau-231, pTau-214, pTau-220, or combinations thereof, are detected in a sample from an individual.

Compared to diseases or conditions or neurologically and cognitively unimpaired symptom, the method described herein using the antibodies described herein can be used to accurately and specifically determine an individual's Alzheimer's disease (AD) by comparison with: non-Alzheimer's disease (AD) neurodegenerative disease, Aβ-negative non-AD neurodegenerative disease, Aβ-positive non-AD neurodegenerative disease, behavioral variant frontotemporal dementia (BvFTD), primary progressive aphasia (PPA), vascular dementia (VaD), Parkinson's disease (PD), PD with dementia (PDD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), Aβ-negative cognitively impaired or unimpaired controls, and combinations thereof. In some embodiments, the method described herein using the antibodies described herein includes at least a 70%, 80%, 90%, 95%, 99%, or greater improvement in accuracy or specificity in determining AD compared to diseases or conditions or neurologically and cognitively unimpaired symptom.

Compared to neuropathological examination or clinical diagnosis, the methods described herein, using the antibodies described herein, can be used to accurately and specifically identify an individual's Alzheimer's disease (AD). In some embodiments, the methods described herein, using the antibodies described herein, include at least a 70%, 80%, 90%, 95%, 99%, or greater improvement in accuracy or specificity in identifying AD compared to neuropathological examination or clinical diagnosis. In some embodiments, neuropathological examination or clinical diagnosis includes neurological tests, psychological examinations, or brain imaging (e.g., MRI, CT, or PET scans).

The method described herein, using the antibodies described herein, can detect phosphorylated tau in a sample at a detection limit. In some embodiments, the method described herein, using the antibodies described herein, can detect phosphorylated tau in the sample at a detection limit of at least about 1.5 picograms per milliliter (pg/mL). In some embodiments, the method described herein, using the antibodies described herein, can detect phosphorylated tau in the sample at a detection limit of at least about 5 picograms per milliliter (pg/mL). In some embodiments, the method described herein, using the antibodies described herein, can detect phosphorylated tau in the sample at detection limits in the following ranges: about 0.5 pg/mL to about 10 pg/mL, about 1 pg/mL to about 8 pg/mL, about 1.5 pg/mL to about 7 pg/mL, about 2 pg/mL to about 6 pg/mL, or about 3 pg/mL to about 5 pg/mL.

Production of Tau antibodies

In some embodiments, the antibodies or antibody fragments described herein are generated using any method known in the art for synthesizing antibodies or antibody fragments, specifically by chemical synthesis or by recombinant expression, and preferably by recombinant expression technology.

In some cases, the antibody or its binding fragment is recombinantly expressed, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242). This involves synthesizing overlapping oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, the nucleic acid molecule encoding the antibody may be generated by PCR amplification using synthetic primers that are hybridizable to the 3′ and 5′ ends of the sequence, or by cloning from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing an immunoglobulin) using oligonucleotide probes that are specific to the specific gene sequence.

In some cases, antibodies or their conjugates are optionally generated by immunizing animals (such as mice) to produce polyclonal antibodies or more preferably by generating monoclonal antibodies, for example, as described by Kohler and Milstein (1975, Nature 256:495-497) or by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, clones encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) to obtain clones of Fab fragments that bind to a specific antigen, or by screening antibody libraries (see, for example, Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

In some implementations, techniques have been developed to generate “chimeric antibodies” by splicing genes from mouse antibody molecules with appropriate antigen specificity and genes from human antibody molecules with appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454). A chimeric antibody is a molecule in which different parts are derived from different animal species, such as those molecules having a variable region derived from mouse monoclonal antibodies and a constant region of human immunoglobulins.

In some embodiments, techniques described for the generation of single-chain antibodies (US Patent No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are suitable for generating single-chain antibodies. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv region via amino acid bridges, thereby generating single-chain polypeptides. Alternatively, techniques for assembling functional Fv fragments in *E. coli* (Skerra et al., 1988, Science 242:1038-1041) are also optionally used.

In some embodiments, an expression vector containing the antibody's nucleotide sequence or the antibody's nucleotide sequence is transferred into host cells using conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured using conventional techniques to produce the antibody. In specific embodiments, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

In some implementations, a variety of host expression vector systems are used to express the antibodies or their binding fragments described herein. Such host expression systems represent both a medium for generating and subsequently purifying the coding sequence of the antibody and cells that express the antibody or its binding fragment in situ when transformed or transfected with an appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., *Escherichia coli* and *Bacillus subtilis*) transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing sequences encoding antibodies or their binding fragments; yeast (e.g., *Pichia pastoris*) transformed with recombinant yeast expression vectors containing sequences encoding antibodies or their binding fragments; insect cell systems infected with recombinant viral expression vectors (e.g., baculoviruses); plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors containing sequences encoding antibodies or their binding fragments (e.g., Ti plasmids); or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) carrying recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters).

For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines stably expressing antibodies are optionally engineered. Host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers, instead of using expression vectors containing viral origins of replication. After the introduction of foreign DNA, the engineered cells are then grown in enrichment media for 1–2 days and then transferred to selective media. Selectable markers in the recombinant plasmid confer selective resistance and allow cells to stably integrate the plasmid into their chromosomes, and grow to form loci, which then clone and expand into cell lines. This method can be advantageously used for the engineering of cell lines expressing antibodies or their binding fragments.

In some cases, multiple selection systems are used, including but not limited to the use of herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes in tk-, hgprt-, or aprt- cells, respectively. In addition, antimetabolite resistance was used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and neo, which confers resistance to aminoglycoside G-418 (Clinical Pharmacy 12:488-505). ; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Commonly known methods in the field of recombinant DNA technology that can be used are described in the following: Ausubel et al. (ed., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Chapters 12 and 13, Dracopoli et al. (ed.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

In some cases, antibody expression levels are increased by vector amplification (for a review, see Bebbington and Hentschel, The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol. 3. (Academic Press, New York, 1987)). When the marker in the antibody-expressing vector system is amplifiable, an increase in the level of inhibitors present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the antibody's nucleotide sequence, the production of said antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

In some cases, any method known in the art for antibody purification is used, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly by affinity for a specific antigen following protein A) and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins.

expression carrier

In some implementations, the vector includes any suitable vector derived from eukaryotic or prokaryotic sources. In some cases, the vector is obtained from bacteria (e.g., *Escherichia coli*), insects, yeast (e.g., *Pichia pastoris*), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, the pBAD vector series, the pBADM vector series, the pET vector series, the pETM vector series, the pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, the pQE vector series, pRSETA, pRSET B, pRSET C, the pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

In some cases, yeast vectors include pDEST ^™ 14, pDEST ^™ 15, pDEST ^™ 17, pDEST ^™ 24, pYES-DEST52, pBAD-DEST49, pAO815, pFLD1, pGAPZ A, B and C, pPIC3.5K, pPIC6 A, B and C, pPIC9K, pTEF1/Zeo, pYES2, pYES2/CT, pYES2/NTA, B and C, or pYES3/CT.

Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

Examples of mammalian vectors include transient expression vectors or stable expression vectors. Transient mammalian expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Stable mammalian expression vectors may include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

In some cases, cell-free systems are mixtures of cytoplasmic and/or nuclear components derived from cells and are used for in vitro nucleic acid synthesis. In other cases, cell-free systems utilize prokaryotic or eukaryotic cell components. Sometimes, nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, E. coli S30 extraction systems, E. coli T7 S30 systems, or...

host cells

In some implementations, the host cell includes any suitable cell, such as cells of natural origin or genetically modified cells. In some cases, the host cell is a production host cell. In some cases, the host cell is a eukaryotic cell. In other cases, the host cell is a prokaryotic cell. In some cases, eukaryotic cells include fungi (e.g., yeast cells), animal cells, or plant cells. In some cases, prokaryotic cells are bacterial cells. Examples of bacterial cells include Gram-positive bacteria or Gram-negative bacteria. Sometimes, Gram-negative bacteria are anaerobic, rod-shaped, or both.

In some cases, Gram-positive bacteria include phyla Actinobacteria, Firmicutes, or Soft-walled Bacteria. In other cases, Gram-positive bacteria include phyla Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi/Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia/Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. Other bacteria can be from the phyla Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. Bacterial cells can be *Escherichia coli*, *Clostridium botulinum*, or *Coli bacilli*.

Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1 ^™ , DH10B ^™ , TOP10, DH5α, DH10Bac ^™ , OmniMax ^™ , MegaX ^™ , DH12S ^™ , INV110, TOP10F', INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2 ^™ , Stbl3 ^™ , or Stbl4 ^™ .

In some cases, animal cells include cells derived from vertebrates or invertebrates. In others, animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In still others, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

Fungi include ascomycetes such as yeasts, molds, filamentous fungi, basidiomycetes, or zygomycetes. In some cases, yeasts include either the phylum Ascomycetes or Basidiomycetes. In some cases, the phylum Ascomycetes includes the subphylum Saccharomycotina (true yeasts, such as Saccharomyces cerevisiae (baker's yeast)) or the subphylum Taphrinomycotina (e.g., Schizomycota). In some cases, the phylum Basidiomycetes includes the subphylum Agaricomycotina (e.g., Tremellomycetes) or the subphylum Pucciniomycotina (e.g., Microbotryomycetes).

Exemplary yeasts or filamentous fungi include, for example, the following genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, for example, the following species: *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Candida utilis*, *Candida boidini*, *Candida albicans*, *Candida tropicalis*, *Candida stellatoidea*, *Candida glabrata*, *Candida krusei*, and *Candida parapsilosis*. Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichiametanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis *Yarrowia lipolytica*, *Zygosaccharomyces rouxii*, *Yarrowia lipolytica*, *Trichosporon pullulans*, *Rhodosporidium toru-Aspergillus niger*, *Aspergillus nidulans*, *Aspergillus awamori*, *Aspergillus oryzae*, *Trichoderma mareesei*, *Yarrowia lipolytica* Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

Exemplary yeast host cells include, but are not limited to, Pichia pastoris strains such as GS115, KM71H, SMD1168, SMD1168H and X-33; and Saccharomyces cerevisiae strains such as INVSC1.

In some cases, the additional animal cells include cells obtained from mollusks, arthropods, lizards, or sponges. In others, the additional animal cells are mammalian cells, such as those from primates, apes, horses, cattle, pigs, canines, felines, or rodents. In still others, rodents include mice, rats, hamsters, gerbils, squirrels, fancy rats, or guinea pigs.

Exemplary mammalian host cells include, but are not limited to, the 293A cell line, 293FT cell line, 293F cell line, 293H cell line, CHO DG44 cell line, CHO-S cell line, CHO-K1 cell line, FUT8 KO CHOK1 cell line, Expi293F ^™ cell line, Flp-In ^™ T-REx ^™ 293 cell line, Flp-In ^™ -293 cell line, Flp-In ^™ -3T3 cell line, Flp-In ^™ -BHK cell line, Flp-In ^™ -CHO cell line, Flp-In ^™ -CV-1 cell line, Flp-In ^™ -Jurkat cell line, FreeStyle ^™ 293-F cell line, FreeStyle ^™ CHO-S cell line, GripTite ^™ 293MSR cell line, GS-CHO cell line, HepaRG ^™ cell line, and T-REx ^™ cell line. Jurkat cell line, Per.C6 cell line, T-REx ^™ -293 cell line, T-REx ^™ -CHO cell line and T-REx ^™ -HeLa cell line.

In some cases, mammalian host cells are stable cell lines, or cell lines that have integrated the genetic material of interest into their own genome and are able to express the products of that genetic material after multiple generations of cell division. In other cases, mammalian host cells are transient cell lines, or cell lines that have not integrated the genetic material of interest into their own genome and are unable to express the products of that genetic material after multiple generations of cell division.

Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five ^™ cells, and other cells.

In some cases, plant cells include cells derived from algae. Exemplary insect cell lines include, but are not limited to, strains from *Chlamydomonas reinhardtii* 137c or *Synechococcus elongatus* PPC 7942.

Numbering Implementation Plan

Embodiment 1 includes a method for detecting phosphorylated tau in a sample from an individual, the method comprising: immunoassay of the sample using an antibody or antibody fragment comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises an amino acid sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO: 30-34, and wherein the VL comprises an amino acid sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO: 35-40. Embodiment 2 includes the method of Embodiment 1, wherein the phosphorylated tau is selected from pTau-181, pTau-212, pTau-217, pTau-231, pTau-214, and pTau-220. Embodiment 3 includes the method of Embodiments 1-2, wherein the phosphorylated tau is pTau-217. Embodiment 4 includes the method of Embodiments 1-2, wherein the phosphorylated tau is pTau-231. Embodiment 5 includes the method as described in Embodiment 2, wherein the method detects pTau-217 and pTau-231. Embodiment 6 includes the method as described in Embodiment 2, wherein the method detects pTau-212 and pTau-217. Embodiment 7 includes the method as described in Embodiment 2, wherein the method detects pTau-212 and pTau-231. Embodiment 8 includes the method as described in Embodiment 2, wherein the method detects pTau-181 and pTau-217. Embodiment 9 includes the method as described in Embodiment 2, wherein the method detects pTau-181 and pTau-231. Embodiment 10 includes the method as described in Embodiment 2, wherein the method detects pTau-181, pTau-217, and pTau-231. Embodiment 11 includes the method as described in Embodiment 2, wherein the method detects pTau-212, pTau-217, and pTau-231. Embodiment 12 includes the method as described in Embodiment 5, wherein the method detects pTau-217 and pTau-231 in a sample selected from plasma and serum samples. Embodiment 13 includes the method as described in Embodiment 6, wherein the method detects pTau-212 and pTau-217 in a sample selected from plasma and serum samples. Embodiment 14 includes the method as described in Embodiment 7, wherein the method detects pTau-212 and pTau-231 in a sample selected from plasma and serum samples. Embodiment 15 includes the method as described in Embodiment 11, wherein the method detects pTau-181 and pTau-217 in a sample selected from plasma and serum samples. Embodiment 16 includes the method as described in Embodiment 11, wherein the method detects pTau-181 and pTau-231 in a sample selected from plasma and serum samples. Embodiment 17 includes the method as described in Embodiment 11, wherein the method detects pTau-181, pTau-217, and pTau-231 in a sample selected from plasma and serum samples. Embodiment 18 includes the method as described in Embodiment 11, wherein the method detects pTau-212, pTau-217, and pTau-231 in a sample selected from plasma and serum samples. Embodiment 19 includes the method as described in Embodiments 1-18, wherein the VH comprises an amino acid sequence according to any one of SEQ ID NO: 30-34. Embodiment 20 includes the method as described in Embodiments 1-19, wherein the VL comprises an amino acid sequence according to any one of SEQ ID NO: 35-40. Embodiment 21 includes the method as described in Embodiments 1-20, wherein the VH comprises an amino acid sequence according to any one of SEQ ID NO: 30-34, and wherein the VL comprises an amino acid sequence according to any one of SEQ ID NO: 35-40. Embodiment 22 includes the method as described in Embodiments 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:30, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:35. Embodiment 23 includes the method as described in Embodiments 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:31, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:36. Embodiment 24 includes the method as described in Embodiments 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:31, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:37. Embodiment 25 comprises the method as described in Embodiments 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:32, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:38. Embodiment 26 comprises the method as described in Embodiments 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:33, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:39. Embodiment 27 comprises the method as described in Embodiments 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:34, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:40. Embodiment 28 comprises the method as described in Embodiments 1-27, wherein the antibody or antibody fragment comprises an amino acid sequence having at least about 90% identity with any one of SEQ ID NO:41-51. Implementation scheme 29 includes the method as described in implementation schemes 1-28, further comprising measuring the sample to determine the level of a biomarker selected from: Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. Implementation scheme 30 includes the method as described in implementation schemes 1-29, wherein the sample is selected from blood samples, plasma samples, serum samples, and cerebrospinal fluid (CSF) samples. Implementation scheme 31 includes the method as described in implementation schemes 1-30, further comprising determining the individual's Alzheimer's disease based on the detection of phosphorylated tau. Implementation scheme 32 includes the method as described in implementation schemes 1-31, further comprising determining the individual's prognosis for developing Alzheimer's disease based on the detection of phosphorylated tau. Implementation scheme 33 includes the method as described in implementation scheme 32, further determining the individual's age, genotype, or biomarker expression. Implementation scheme 34 includes the method as described in implementation scheme 33, wherein the biomarker is selected from Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. Implementation scheme 35 includes the method as described in implementation schemes 1-34, wherein the method has at least about 80% specificity for detecting phosphorylated tau. Implementation scheme 36 includes the method as described in implementation schemes 1-34, wherein the method has at least about 85% specificity for detecting phosphorylated tau. Implementation scheme 37 includes the method as described in implementation schemes 1-34, wherein the method has at least about 90% specificity for detecting phosphorylated tau. Embodiment 38 includes the method as described in Embodiments 1-37, wherein the method has a sensitivity of at least about 80% for detecting phosphorylated tau. Embodiment 39 includes the method as described in Embodiments 1-37, wherein the method has a sensitivity of at least about 85% for detecting phosphorylated tau. Embodiment 40 includes the method as described in Embodiments 1-37, wherein the method has a sensitivity of at least about 90% for detecting phosphorylated tau. Embodiment 41 includes the method as described in Embodiments 1-40, wherein the method is capable of detecting phosphorylated tau in the sample at a detection limit of at least about 1.0 picograms per milliliter (pg/mL). Embodiment 42 includes the method as described in Embodiments 1-40, wherein the method is capable of detecting phosphorylated tau in the sample at a detection limit of at least about 1.5 picograms per milliliter (pg/mL). Embodiment 43 includes the method as described in Embodiments 1-40, wherein the method is capable of detecting phosphorylated tau in the sample at a detection limit of at least about 5 picograms per milliliter (pg/mL).

Embodiment 44 includes an anti-tau antibody comprising i) a heavy chain comprising a variable heavy chain (VH) domain and ii) a light chain comprising a variable light chain (VL) domain, wherein the VH domain comprises an HCDR1 sequence having a sequence selected from SEQ ID NO: 1-5, an HCDR2 sequence having a sequence selected from SEQ ID NO: 6-9, and an HCDR3 sequence having a sequence selected from SEQ ID NO: 10-13, and the VL domain comprises an LCDR1 sequence having a sequence selected from SEQ ID NO: 14-19, an LCDR2 sequence having a sequence selected from SEQ ID NO: 20-23, and an LCDR3 sequence having a sequence selected from SEQ ID NO: 24-29. Embodiment 45 includes an anti-tau antibody as described in Embodiment 44, wherein the HCDR1 sequence comprises SEQ ID NO:1, the HCDR2 sequence comprises SEQ ID NO:6, the HCDR3 sequence comprises SEQ ID NO:10, the LCDR1 sequence comprises SEQ ID NO:14, the LCDR2 sequence comprises SEQ ID NO:20, and the LCDR3 sequence comprises SEQ ID NO:24. Embodiment 46 includes an anti-tau antibody as described in Embodiment 44, wherein the HCDR1 sequence comprises SEQ ID NO:2, the HCDR2 sequence comprises SEQ ID NO:7, the HCDR3 sequence comprises SEQ ID NO:11, the LCDR1 sequence comprises SEQ ID NO:15, the LCDR2 sequence comprises SEQ ID NO:21, and the LCDR3 sequence comprises SEQ ID NO:25. Embodiment 47 includes an anti-tau antibody as described in Embodiment 44, wherein the HCDR1 sequence comprises SEQ ID NO:2, the HCDR2 sequence comprises SEQ ID NO:7, the HCDR3 sequence comprises SEQ ID NO:11, the LCDR1 sequence comprises SEQ ID NO:16, the LCDR2 sequence comprises SEQ ID NO:22, and the LCDR3 sequence comprises SEQ ID NO:26. Embodiment 48 includes an anti-tau antibody as described in Embodiment 44, wherein the HCDR1 sequence comprises SEQ ID NO:3, the HCDR2 sequence comprises SEQ ID NO:8, the HCDR3 sequence comprises SEQ ID NO:10, the LCDR1 sequence comprises SEQ ID NO:17, the LCDR2 sequence comprises SEQ ID NO:20, and the LCDR3 sequence comprises SEQ ID NO:27. Embodiment 49 includes an anti-tau antibody as described in Embodiment 44, wherein the HCDR1 sequence comprises SEQ ID NO:4, the HCDR2 sequence comprises SEQ ID NO:7, the HCDR3 sequence comprises SEQ ID NO:12, the LCDR1 sequence comprises SEQ ID NO:18, the LCDR2 sequence comprises SEQ ID NO:23, and the LCDR3 sequence comprises SEQ ID NO:28. Embodiment 50 includes an anti-tau antibody as described in Embodiment 44, wherein the HCDR1 sequence comprises SEQ ID NO:5, the HCDR2 sequence comprises SEQ ID NO:9, the HCDR3 sequence comprises SEQ ID NO:13, the LCDR1 sequence comprises SEQ ID NO:19, the LCDR2 sequence comprises SEQ ID NO:21, and the LCDR3 sequence comprises SEQ ID NO:29. Embodiment 51 includes an anti-tau antibody as described in Embodiment 44, wherein the VH domain has at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO: 30-34. Embodiment 52 includes an anti-tau antibody as described in Embodiment 44, wherein the VL domain has at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO: 35-40.

Embodiment 53 includes anti-tau antibodies as described in Embodiments 44-52, wherein the anti-tau antibody is a chimeric antibody or its antigen-binding fragment. Embodiment 54 includes anti-tau antibodies as described in Embodiments 44-53, wherein the anti-tau antibody includes IgG-scFv, nanobodies, BiTE, double-chain antibodies, DART, TandAb, scDiabody, scDiabody-CH3, trisomy, microantibodies, microantibodies, TriBi microantibodies, scFv-CH3KIH, Fab-scFv-FcKIH, Fab-scFv, scFv-CH-CL-scFv, Fab’, F(ab’)2, F(ab’)3, F(ab’)2-scFv2, scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, double-chain antibody-Fc, tandem scFv-Fc, or endoantibodies. Embodiment 55 includes an anti-tau antibody as described in Embodiments 44-54, wherein the anti-tau antibody is an IgG1 antibody. Embodiment 56 includes an anti-tau antibody as described in Embodiments 44-55, wherein the anti-tau antibody is an IgG2 antibody. Embodiment 57 includes an anti-tau antibody as described in Embodiments 44-56, wherein the anti-tau antibody is an IgG4 antibody. Embodiment 58 includes an anti-tau antibody as described in Embodiments 44-57, wherein the light chain is a κ chain. Embodiment 59 includes an anti-tau antibody as described in Embodiments 44-58, wherein the anti-tau antibody has a binding affinity for human tau of about 100 pM to about 3 nM. Embodiment 60 includes an anti-tau antibody as described in Embodiments 44-59, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO: 52-56. Embodiment 61 includes an anti-tau antibody as described in Embodiments 44-60, wherein the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO: 57-62. Embodiment 62 includes an anti-tau antibody as described in Embodiments 44-61, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO: 52-56, and a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO: 57-62. Embodiment 63 includes an anti-tau antibody as described in Embodiments 44-62, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO: 52-56. Embodiment 64 includes an anti-tau antibody as described in Embodiments 44-63, wherein the anti-tau antibody comprises a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO: 57-62. Embodiment 65 includes an anti-tau antibody as described in Embodiments 44-64, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid containing the same sequence as SEQ ID NO: 52-56 and a VL domain encoded by a nucleic acid containing the same sequence as SEQ ID NO: 57-62.

Example

The following embodiments are provided for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. The embodiments of the invention, together with the methods described herein, currently represent preferred embodiments, are exemplary, and are not intended to be limiting of the scope of the invention. Variations and other uses thereof will be apparent to those skilled in the art as encompassed within the spirit of the invention as defined in the claims.

Example 1: Tau antibody screening

According to the manufacturer's instructions, the Tau antibody for detecting phosphorylated Tau is determined using a 2-step or 3-step assay in the bead assay. See Figure 1.

The antibodies tested were antibody 1, antibody 2, antibody 3, antibody 4, antibody 5, and antibody 6. The capture results are shown in Figures 2A-2D. Figure 2A shows data from a 2-step protocol assay where all capture antibodies were tested against a Tau-12 detector (detecting N-terminal Tau). The data indicate similar results for all captures, with improved sensitivity observed with antibody 6. Figure 2B shows data from a 2-step protocol assay where all capture antibodies were tested against an HT7-BT2 detector (detecting Tau in the middle domain region). Two biotinylated antibodies were used. The data show an approximately 10-fold increase in background compared to the Tau-12 detector. No capture signal was detected at 1000 pg/mL. Figure 2C shows data from a 3-step protocol assay where all capture antibodies were tested against a Tau-12 detector. The data indicate decreased sensitivity compared to the 2-step protocol, with improved sensitivity observed with antibody 6. Figure 2D shows data from a 3-step protocol assay where all capture antibodies were tested against an HT7-BT2 detector. Two biotinylated antibodies were used. Data showed that the background increased by approximately 10-fold compared to the Tau-12 detector. Based on these results, the sensitivity of the two-step protocol was further optimized.

Example 2. Pharmacokinetics of Tau antibody

The pharmacokinetic profile of the antibody was tested.

Antigen information for the antibodies is shown in Table 10.

Table 10.

Antibodies were generated and purified. The antibodies were assayed using a standard indirect ELISA protocol. Briefly, the peptide antigens corresponding to SEQ ID NO:74-81 were diluted to 1 μg/ml in PBS and plated onto Greiner Bio One Microlon 96-well plates. The peptide antigens were generated by the manufacturer Abcam. WZN-1A and WZN-1B served as targets. WZN-1C, WZN-1D, WZN-1E, WZN-1F, WZN-1G, and WZN-1H served as negative controls. In the peptide antigen sequence, phosphorylated residues indicated phosphorylation of threonine by (pT) or phosphorylation of serine by (pS). After blocking with 1% BSA in PBS at pH 7.4, the antibodies were serially diluted 1 to 4 times to an initial concentration of 1 μg/ml. After incubation, unbound antibodies were washed away with 1X TBST, and HRP-labeled goat anti-rabbit secondary antibody was applied according to the manufacturer's instructions. Unbound secondary antibody was then washed away with 1X TBST, and 3,3',5,5'-tetramethylbenzidine (TMB) was applied for 5 minutes at room temperature, with the plate read at 650 nm. The data are shown in Figure 3. Figure 3 illustrates the screening data for different monoclonal antibodies at different peptide concentrations.

Example 3. Tau antibody for immunohistochemistry

The Tau antibody described herein was tested in an immunohistochemical assay.

In summary, all antibodies were optimized using a range of concentrations (0.01–3.00 μg/ml) and stained using the Leica BondRX automated IHC platform: ER1 antigen retrieval (sodium citrate, pH 6) for 20 min at 100 °C; primary antibody at RT for 15 min; IVD-grade Leica Polymer Refine HRP detection for 8 min at room temperature; DAB chromophore detection for 10 min at room temperature; and finally, hematoxylin counterstaining for 5 min at room temperature. Antibodies stained with alkaline IHC were further stained with alkaline phosphatase (AP) after treatment (200 U/ml, 37 °C, for 60 min). A mediator-only control (AP-free buffer) was also used. Positive antigen control tissues were cerebral cortex from healthy FFPE patients and cerebral cortex from Alzheimer's disease patients. Negative antigen control tissues were liver, skeletal muscle, and cardiac muscle from healthy FFPE patients. All tissues were organized into tissue microarrays to simplify the IHC staining process. A negative reagent (detection system only) control was used and displayed as negative. The baseline antibodies used for staining along with the test antibodies were rabbit monoclonal antibodies against Tau [EPR22524-95] (ab254256, Abcam plc) and rabbit monoclonal antibodies against Tau (S214 phosphate) [EPR1884(2)] (ab170892, Abcam plc).

The baseline antibody demonstrated negative staining in negative control tissues and positive staining in positive control tissues (data not shown). Data for the Tau antibody are shown in Figures 4A-4G (antibody 6), 5A-5G (antibody 5), and 6A-6G (antibody 2). Antibodies 5 and 6 showed similar data to the baseline antibody, exhibiting negative staining in negative control tissues, including normal heart, normal liver, normal skeletal muscle, and normal cerebral cortex, and positive staining in positive control tissues, including Alzheimer's disease cerebral cortex. Antibodies 1-4 did not show similar data to the baseline antibody.

Example 4. Detection of Tau peptide using Tau antibody

The Tau antibody for detecting phosphorylated Tau, as described herein, was tested in an ELISA assay. First, the pTau 217 reactivity of the Tau antibody was tested by an indirect ELISA. Figure 7 shows a diagram depicting the format of the indirect ELISA assay used. Briefly, streptavidin beads were bound to a plate, and a biotinylated peptide was added to the plate under conditions allowing biotin-streptavidin binding. The biotinylated peptide is a synthetic peptide containing a portion of Tau and having a phosphorylated threonine residue at position 217 (pT217). This is the target peptide. After binding the synthetic peptide to the plate via the formation of a biotin-streptavidin complex, the Tau antibody was added to the plate under conditions allowing antibody-target peptide binding. After binding, the plate was washed to remove any unbound antibody, and then a second antibody or tracer antibody against the species of the derived Tau antibody (a goat anti-mouse antibody conjugated to peroxidase or a donkey anti-rabbit antibody conjugated to peroxidase) was added to the plate. After binding, the plate was washed to remove any unbound tracer antibody. Next, TMB ELISA peroxidase chromogenic substrate (3,3',5,5'-tetramethylbenzidine) was added to the plate to visualize antibody reactivity in an indirect ELISA assay. Antibody sample binding was quantified using an ELISA microplate reader.

As shown in Figure 7, the ability of five antibodies to detect phosphorylated Tau was tested using this indirect ELISA technique. IBA493 mAb corresponds to a rabbit anti-Tau antibody (Eli Lilly and Company) capable of binding to Tau phosphorylated at threonine residue 217 (pTau 217). PT3 corresponds to a mouse anti-phosphorylated (T212/T217) Tau selective antibody (Janssen Biotech Inc.). 30H10L2 corresponds to antibody 2 described herein. 71H1L2 corresponds to antibody 6 described herein. 62H10L7 corresponds to antibody 5 described herein. The reactivity of all five antibodies with pTau 217 was determined in two separate ELISA instruments at the following concentrations: ^10⁻² , ^10⁻¹ , ^10⁻⁰ , ^10¹ , ^10² , ^10³ , and ^10⁴ ng/mL/plate. As can be seen in the Bio-pt655 (phosphorylated T217) and Bio-pt660 (phosphorylated T217) plots, both IBA493 mAb and PT3 showed robust concentration-dependent reactivity to pTau 217. Antibody 2 showed a more moderate concentration-dependent reactivity to pTau 217, as revealed at ^10⁴ ng/mL/plate. Antibodies 5 and 6 did not show reactivity to pTau 217 in these assays. Figure 7, showing the results of this ELISA assay against phosphatase-treated pTau using five test antibodies, demonstrates the specificity of antibodies IBA493 mAb, PT3, and antibody 2 in detecting phosphorylated Tau.

The Tau antibody for detecting phosphorylated Tau, as described herein, was tested in a Tau-based assay. Figures 8-24 illustrate the results of the assay, which was designed to test the sensitivity of the Tau reactivity of the antibody described herein. In some respects, the Tau-based assay can detect a given analyte with approximately 1000X greater sensitivity compared to detecting the same analyte in an indirect ELISA assay. This increased sensitivity of the Tau-based assay allows for the development and use of biomarkers that could not previously generate detectable signals using conventional assays, such as indirect ELISA. The increased sensitivity of the Tau-based assay compared to conventional immunoassays, such as indirect ELISA, is due to the fact that the method is able to detect a single target molecule, whereas conventional immunoassays typically require large reaction volumes and millions of fluorophores, or millions of antibody conjugates reacting with a chromogenic substrate, before a light signal can be detected. For the Tau-based assay, the average enzyme/bead (AEB) represents the raw signal output.

In Figure 8, antibody 2, capable of detecting pTau 217 as described herein, was used in the assay to detect analyte levels in plasma samples derived from individuals. The signal-to-noise (S/N) ratio for each sample was determined and plotted in the figure. 120 plasma samples were taken from individuals and assayed. The plotted S/N ratio indicates that all but one tested sample produced a signal within the expected concentration range. When the plasma samples were diluted 1:3 and then assayed again, only 3 of the 120 samples produced results below the blank control measurement, and only 5 samples showed an S/N of 1.5, which was determined as the limit of detection (LOD). In Figure 8, for each of the 120 plasma samples assayed, the coefficient of variation (CV%) was calculated to determine each sample, and the results were plotted against the measured concentration. Ten of the 120 samples produced CV% greater than 20, and based on this analysis, the estimated limit of quantitation (LLOQ) was determined to be 0.08 pg/mL. This LLOQ value represents the minimum amount of analyte (phosphorylated Tau at T217) that can be quantified with an acceptable level of accuracy. These results in Figure 8 indicate the sensitivity of the method using antibody 2 to detect phosphorylated Tau at T217.

In Figure 9, calibration curves for the pTau-217 assay were generated and plotted [AEB vs. log(CAL) pg/mL] using 68 CSF samples and 120 plasma samples (allotted into 4 plates using antibody 2 and 4 plates using ADx neuroscience antibody ADx204). In another plot of this assay [AEB vs. log(CAL) pg/mL] performed on a separate instrument, the AEB plotted on a logarithmic scale indicates that data fitting allows for accurate analyte quantification when measuring samples.

In Figure 10, 38 paired CSF and EDTA plasma samples were measured using the pTau-217 assay with antibody 2. This assay is also known as the ALZpath Dx. The results were plotted, and samples were indicated by clinical diagnosis (non-AD, indeterminate, or AD). These results and their statistical analysis indicate a strong correlation between CSF and plasma pTau levels, as measured by the pTau-217 assay with antibody 2 (R-value for a two-tailed t-test between non-AD and AD is ~0.7 and P-value is <0.0001).

In Figure 11, 42 CSF samples were measured using the pTau-217 assay (using antibody 2) and the pTau-181 assay (using pTau-181 antibody from Corp., item number 103714) and plotted against each other. This indicates that the pTau-217 assay using antibody 2 shows the expected relationship with the AD-related analyte (pTau-181) detected in CSF. Statistical analysis indicated that the R-value for the two-tailed t-test was ~0.8 and the P-value was <0.0001.

In Figure 12, 42 CSF samples were measured using the pTau-217 assay (using antibody 2) and the pTau assay (using Innotest pTau-181 antibody) and plotted against each other. This indicates that the pTau-217 assay using antibody 2 shows the expected relationship with the AD-related analyte (pTau) detected in CSF. Statistical analysis indicated that the R-value for the two-tailed t-test was ~0.77 and the P-value was <0.0001.

In Figure 13, 42 CSF samples were measured using the HD-X assay, with antibody 2 as the capture antibody, ADx204 antibody as the detector, and a peptide as the calibrator. This indicates that the assay using known AD biomarkers shows the expected relationship. Statistical analysis indicated that the R-value of the two-tailed t-test was ~0.9 and the P-value was <0.0001.

In Figure 14, CSF and plasma samples were measured using antibody 2 with the pTau-217 assay and plotted separately. Clinically diagnosed AD or controls without an AD diagnosis were used as classifiers for each sample. Analysis of the plotted results indicated a significant difference between individuals with a clinical AD diagnosis and controls for both CSF and plasma samples. The calculated area under the curve (AUC) was 0.94 for CSF samples and 0.86 for plasma samples. These results indicate that the pTau-217 assay using antibody 2 can distinguish AD cases in CSF and plasma.

In Figure 15, four EDTA plasma samples with high pTau levels served as quality controls (labeled QC_L1, QC_L2, QC_M, and QC_H). Using antibody 2, the pTau-217 assay was performed in duplicate to measure and calculate pTau levels. The results from the replicate tests demonstrate the accuracy and reproducibility of the pTau-217 assay using antibody 2.

In Figure 16, using antibody 2, the pTau-217 assay was performed on the control sample from Figure 15 and additional measurement samples, and plotted in two separate experiments to generate the precision profile. The precision profile was based on the measured sample concentration and the internal run CV% of the four QC samples. Based on this experiment, the functional LLOQ of pTau-217 in this assay was determined to be 0.26 pg/mL.

In Figure 17, parallelism was assessed in the pTau-217 assay using antibody 2. The determination of parallelism is also important because it indicates whether the signal is specific. Parallelism is determined whether actual samples containing high concentrations of endogenous analyte in the diluted standard curves provide the same level of detection in the assay. This can represent the difference in binding affinity of the antibody to the endogenous analyte and the standard or calibration analyte. This ensures that the recombinant standard is parallel to the natural identification of the endogenous analyte. In this experiment, starting at a dilution of 3X, four plasma samples were diluted with factor 2 in five steps, each from a different donor, with relatively high concentrations of pTau-217 for detection and spiked dilution buffer (sample 5). Starting at a dilution factor of 12X, the concentrations of all four plasma samples were reduced to below LLOD. In the graph of log(measured pg/mL) versus log[dilution factor (DF)], the relationship between plasma measurements and peak measurements indicates that the detection is linear at various dilutions. Three of the four plasma samples were identified as falling within the acceptable range of parallelism, while sample 4 fell just outside the acceptable range. The acceptable range of parallelism was <15%. These results demonstrate the practicality of using antibody 2 to determine pTau-217 levels in plasma samples at different concentrations, producing consistent and accurate pTau-217 level calculations.

In Figures 18-19, dilution linearity was demonstrated using the pTau-217 assay with antibody 2 to show that samples with peak concentrations at the upper bound of the agreed-upon amount (ULOQ) could be diluted to concentrations within the working range while still producing reliable assay results. In Figure 18, measurements were performed on three spiked samples (s1, s2, and s3) and a calibration sample, and plotted as log(measured pg/mL) relative to log(DF) to determine dilution linearity. In Figure 19, measurements were performed on three spiked samples (s1, s2, and s3) and a calibration sample, and plotted as log(measured pg/mL) relative to log(DF) to determine dilution linearity. The highest peak in s1, s2, and s3 was omitted because it was outside the calibration range of 50 pg/mL.

In Figure 20, the pTau-217 assay with antibody 2 was used to evaluate samples collected from the memory clinical cohort. Plasma samples were measured and plotted against the calculated pTau217 concentration. The clinical diagnosis of AD was used as the classifier. An AUC of 0.916 was calculated, indicating that the pTau-217 assay with antibody 2 successfully distinguished AD+ from AD- in this cohort. Also in Figure 20, receiver operating characteristic (ROC) curves were plotted to illustrate the diagnostic capability (AD+ or AD-) of this binary classifier system, as the discrimination thresholds differ between classifiers.

In Figures 21-22, the clinical performance of the pTau-217 assay using antibody 2 is compared with that of the pTau-181 assay using antibody P-tau181. In Figure 21, antibody 2 was able to distinguish plasma samples taken from individuals with Alzheimer's disease from controls (P-value for antibody 2: 1.3e ^-12 ). In Figure 22, the commercially available P-tau181 assay (Corp., item number 103714) was also able to distinguish plasma samples taken from individuals with Alzheimer's disease from controls (P-value: 9.6e ^-08 ). The data from the individuals from whom the samples in Figures 21-22 originated are listed in Figure 22.

Figure 23 shows the accuracy plots for the assays generated using P-tau217 antibody 2 and P-tau181- antibody (Corp., item number 103714). The calculated LLOD values were 0.55 pg/mL and 0.24 pg/mL, respectively. No inverse calculations were performed on the concentrations, and therefore no inverse calculations were performed on the LLOD values.

Figure 24 shows the ROC curves for P-tau181-antibody (Corp., item number 103714) and P-tau217 antibody 2. Data analysis indicates that P-tau217 antibody 2 exhibits superior sensitivity and specificity compared to the assay using P-tau181-antibody in distinguishing Alzheimer's disease from controls. When tested on plasma samples, antibody 2 showed a diagnostic accuracy of 92.5% for Alzheimer's disease using this method. When tested on plasma samples, antibody 2 showed a diagnostic specificity of 85% for Alzheimer's disease using this method.

Figure 25 depicts a schematic diagram of a Tau polypeptide indicating the relative positions of various protein domains and the positions of threonine residues, which can be determined using the methods disclosed herein to determine its phosphorylation state. The position of pT217 within the P2 domain of Tau is indicated. pT181 is located within the P1 domain, and pT231 is located near the boundary between the P2 and R1 domains.

Figure 26 shows the assays of various Tau antibodies using indirect ELISA, plotting their reactivity to the Tau fragment with unphosphorylated T217 (Bio-pt654) and full-length Tau (Tau441). IBA493 mAb and PT3 both showed concentration-dependent reactivity to both Bio-pt654 and Tau441. Antibodies 2, 5, and 6 described in this paper showed no reactivity to either Bio-pt654 or Tau441 in this assay, demonstrating the accuracy and specificity of pTau-217 detection by antibodies 2, 5, and 6.

Figure 27 shows the assays performed using indirect ELISA on various Tau antibodies and plots the reactivity to Tau fragments containing phosphorylated T181 (Bio-pt126) and phosphorylated T231 (Bio-pt146). IBA493 mAb and antibody 2, described herein, exhibited concentration-dependent reactivity to Bio-pt126. IBA493 mAb was the only antibody tested to show concentration-dependent reactivity to Bio-pt146. This indicates that IBA493 mAb, PT3, and antibody 2 are distinguishable based on which analytes each antibody interacts with via indirect ELISA. IBA493 mAb interacts with pTau-217, non-phosphorylated T217, full-length Tau, pTau-181, and pTau-231. PT3 interacts with pTau-217, non-phosphorylated T217, and full-length Tau. Antibody 2 interacts with pTau-217 and pTau-181. It was also shown that the interaction between IBA493 mAb and unphosphorylated T217 was significantly less than the interaction between PT3 and unphosphorylated T217.

Figure 28 depicts an assay for detecting the capture of specific Tau peptides using antibody 2. In this assay, antibody 2 binds to the plate, and the sample wells from the plate are subjected to various biotinylated peptides under conditions favorable for the formation of specific antibody-ligand interactions. The sample is then washed to remove excess unbound biotinylated peptides. Streptavidin beads conjugated with peroxidase are then added to the sample, allowing the biotin-streptavidin complex to form on the peptide-bound antibody. TMB is then added _{to reduce} the substrate, and colorimetric development of the sample is measured using an ELISA plate reader. Plotting the results shows that Adx-pt655 exhibits specific dose-dependent reactivity for the various pTau-217, pTau-231, and pTau-181 peptides tested. These results demonstrate the specificity of antibody 2 for specific characteristics of pTau (i.e., Tau phosphorylated at threonine 217). Under the same conditions, antibodies 5 and 6, tested by indirect ELISA with the same Tau peptide, did not produce a specific dose-dependent response to the tested Tau peptide.

Various pTau-217 antibodies corresponding to Antibody 1, Antibody 2, Antibody 3, Antibody 4, Antibody 5, and Antibody 5 described herein were also evaluated on a mesoscale discovery technology platform as capture antibodies directly coated onto or coated onto streptavidin-coated plates. This system uses non-radioactive electrochemiluminescent labeling, thus providing significant advantages compared to conventional ELISA assays. These advantages include lower background signal, improved sensitivity, and dynamic detection range.

In Figure 29, Western blotting was used to evaluate the binding of various antibodies to brain lysate samples from AD patients and controls. Samples were loaded according to the same sample key shown in Figure 29 for the five Western blotting results. Protein ladders were run in lanes 1 and 10. Phosphatase-treated pTau was loaded at a dose of 0.05 μg in lane 2. Full-length Tau (Tau411) was loaded at a dose of 0.05 μg in lane 3. Lanes 4–6 contained samples with a dilution factor of 5 from different controls. Lanes 7–9 contained samples with a dilution factor of 5 from different AD subjects. Results indicated that both IBA394 mAb and PT3 bound to and immunoprecipitated different lengths of Tau isoforms in both control and AD samples, and immunoprecipitated significantly more Tau in the AD samples, while showing no interaction with either synthetic full-length Tau or phosphatase-treated pTau. Antibody 2 (30H2L10) bound to and immunoprecipitated Tau isoforms of varying lengths in AD samples, but did not immunoprecipitate significant amounts of Tau in samples from control individuals. Antibodies 5 and 6 did not produce detectable Western blot signals in this assay.

While preferred embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments of this disclosure described herein may be employed in practice. The scope of this disclosure is intended to be defined by the appended claims, and thereby to cover the methods and structures within the scope of those claims and their equivalents.

Claims (65)

1. A method for detecting phosphorylated tau in a sample from an individual, the method comprising: immunoassay of the sample using an antibody or antibody fragment comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises an amino acid sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:30-34, and wherein the VL comprises an amino acid sequence having at least about 90% identity with a sequence shown in any one of SEQ ID NO:35-40. 2. The method of claim 1, wherein the phosphorylated tau is selected from pTau-181, pTau-212, pTau-217, pTau-231, pTau-214 and pTau-220. 3. The method according to any one of claims 1-2, wherein the phosphorylated tau is pTau-217. 4. The method according to any one of claims 1-2, wherein the phosphorylated tau is pTau-231. 5. The method of claim 2, wherein the method detects pTau-217 and pTau-231. 6. The method of claim 2, wherein the method detects pTau-212 and pTau-217. 7. The method of claim 2, wherein the method detects pTau-212 and pTau-231. 8. The method of claim 2, wherein the method detects pTau-181 and pTau-217. 9. The method of claim 2, wherein the method detects pTau-181 and pTau-231. 10. The method of claim 2, wherein the method detects pTau-181, pTau-217, and pTau-231. 11. The method of claim 2, wherein the method detects pTau-212, pTau-217 and pTau-231. 12. The method of claim 5, wherein the method detects pTau-217 and pTau-231 in a sample selected from plasma samples and serum samples. 13. The method of claim 6, wherein the method detects pTau-212 and pTau-217 in a sample selected from plasma samples and serum samples. 14. The method of claim 7, wherein the method detects pTau-212 and pTau-231 in a sample selected from plasma samples and serum samples. 15. The method of claim 8, wherein the method detects pTau-181 and pTau-217 in a sample selected from plasma samples and serum samples. 16. The method of claim 9, wherein the method detects pTau-181 and pTau-231 in a sample selected from plasma samples and serum samples. 17. The method of claim 10, wherein the method detects pTau-181, pTau-217 and pTau-231 in a sample selected from plasma samples and serum samples. 18. The method of claim 11, wherein the method detects pTau-212, pTau-217 and pTau-231 in a sample selected from plasma samples and serum samples. 19. The method of any one of claims 1-18, wherein the VH comprises an amino acid sequence according to any one of SEQ ID NO:30-34. 20. The method of any one of claims 1-19, wherein the VL comprises an amino acid sequence according to any one of SEQ ID NO: 35-40. 21. The method of any one of claims 1-20, wherein the VH comprises an amino acid sequence according to any one of SEQ ID NO:30-34, and wherein the VL comprises an amino acid sequence according to any one of SEQ ID NO:35-40. 22. The method of any one of claims 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:30, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:35. 23. The method of any one of claims 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:31, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:36. 24. The method of any one of claims 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:31, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:37. 25. The method of any one of claims 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:32, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:38. 26. The method of any one of claims 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:33, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:39. 27. The method of any one of claims 1-21, wherein the VH comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:34, and wherein the VL comprises an amino acid sequence having at least about 90% identity with SEQ ID NO:40. 28. The method of any one of claims 1-27, wherein the antibody or antibody fragment comprises an amino acid sequence having at least about 90% identity with any one of SEQ ID NO:41-51. 29. The method of any one of claims 1-28, further comprising measuring the sample to determine the level of a biomarker selected from the group consisting of: Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. 30. The method of any one of claims 1-29, wherein the sample is selected from blood samples, plasma samples, serum samples, and cerebrospinal fluid (CSF) samples. 31. The method of any one of claims 1-30, further comprising determining Alzheimer's disease in the individual based on the detection of phosphorylated tau. 32. The method of any one of claims 1-31, further comprising determining the prognosis of the individual's development of Alzheimer's disease based on the detection of phosphorylated tau. 33. The method of claim 32, further comprising determining the individual’s age, genotype, or biomarker expression. 34. The method of claim 33, wherein the biomarker is selected from Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranulin, SNAP-25, synaptokinin, α-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. 35. The method of any one of claims 1-34, wherein the method has at least about 80% specificity for detecting phosphorylated tau. 36. The method of any one of claims 1-34, wherein the method has at least about 85% specificity for detecting phosphorylated tau. 37. The method of any one of claims 1-34, wherein the method has at least about 90% specificity for detecting phosphorylated tau. 38. The method of any one of claims 1-37, wherein the method has a sensitivity of at least about 80% for detecting phosphorylated tau. 39. The method of any one of claims 1-37, wherein the method has a sensitivity of at least about 85% for detecting phosphorylated tau. 40. The method of any one of claims 1-37, wherein the method has a sensitivity of at least about 90% for detecting phosphorylated tau. 41. The method of any one of claims 1-40, wherein the method is capable of detecting phosphorylated tau in the sample at a detection limit of at least about 1.0 picograms per milliliter (pg/mL). 42. The method of any one of claims 1-40, wherein the method is capable of detecting phosphorylated tau in the sample at a detection limit of at least about 1.5 picograms per milliliter (pg/mL). 43. The method of any one of claims 1-40, wherein the method is capable of detecting phosphorylated tau in the sample at a detection limit of at least about 5 picograms per milliliter (pg/mL). 44. An anti-tau antibody comprising i) a heavy chain comprising a variable heavy chain (VH) domain and ii) a light chain comprising a variable light chain (VL) domain, wherein the VH domain comprises an HCDR1 sequence having a sequence selected from SEQ ID NO: 1-5, an HCDR2 sequence having a sequence selected from SEQ ID NO: 6-9, and an HCDR3 sequence having a sequence selected from SEQ ID NO: 10-13, and the VL domain comprises an LCDR1 sequence having a sequence selected from SEQ ID NO: 14-19, an LCDR2 sequence having a sequence selected from SEQ ID NO: 20-23, and an LCDR3 sequence having a sequence selected from SEQ ID NO: 24-29. 45. The anti-tau antibody of claim 44, wherein the HCDR1 sequence comprises SEQ ID NO:1, the HCDR2 sequence comprises SEQ ID NO:6, the HCDR3 sequence comprises SEQ ID NO:10, the LCDR1 sequence comprises SEQ ID NO:14, the LCDR2 sequence comprises SEQ ID NO:20, and the LCDR3 sequence comprises SEQ ID NO:24. 46. The anti-tau antibody of claim 44, wherein the HCDR1 sequence comprises SEQ ID NO:2, the HCDR2 sequence comprises SEQ ID NO:7, the HCDR3 sequence comprises SEQ ID NO:11, the LCDR1 sequence comprises SEQ ID NO:15, the LCDR2 sequence comprises SEQ ID NO:21, and the LCDR3 sequence comprises SEQ ID NO:25. 47. The anti-tau antibody of claim 44, wherein the HCDR1 sequence comprises SEQ ID NO:2, the HCDR2 sequence comprises SEQ ID NO:7, the HCDR3 sequence comprises SEQ ID NO:11, the LCDR1 sequence comprises SEQ ID NO:16, the LCDR2 sequence comprises SEQ ID NO:22, and the LCDR3 sequence comprises SEQ ID NO:26. 48. The anti-tau antibody of claim 44, wherein the HCDR1 sequence comprises SEQ ID NO:3, the HCDR2 sequence comprises SEQ ID NO:8, the HCDR3 sequence comprises SEQ ID NO:10, the LCDR1 sequence comprises SEQ ID NO:17, the LCDR2 sequence comprises SEQ ID NO:20, and the LCDR3 sequence comprises SEQ ID NO:27. 49. The anti-tau antibody of claim 44, wherein the HCDR1 sequence comprises SEQ ID NO:4, the HCDR2 sequence comprises SEQ ID NO:7, the HCDR3 sequence comprises SEQ ID NO:12, the LCDR1 sequence comprises SEQ ID NO:18, the LCDR2 sequence comprises SEQ ID NO:23, and the LCDR3 sequence comprises SEQ ID NO:28. 50. The anti-tau antibody of claim 44, wherein the HCDR1 sequence comprises SEQ ID NO:5, the HCDR2 sequence comprises SEQ ID NO:9, the HCDR3 sequence comprises SEQ ID NO:13, the LCDR1 sequence comprises SEQ ID NO:19, the LCDR2 sequence comprises SEQ ID NO:21, and the LCDR3 sequence comprises SEQ ID NO:29. 51. The anti-tau antibody of claim 44, wherein the VH domain has at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO:30-34. 52. The anti-tau antibody of claim 44, wherein the VL domain has at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with a sequence selected from SEQ ID NO:35-40. 53. The anti-tau antibody according to any one of claims 44-52, wherein the anti-tau antibody is a chimeric antibody or an antigen-binding fragment thereof. 54. The anti-tau antibody according to any one of claims 44-53, wherein the anti-tau antibody comprises IgG-scFv, nanobody, BiTE, double-chain antibody, DART, TandAb, scDiabody, scDiabody-CH3, trisomy, microantibody, microantibody, TriBi microantibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, Fab’, F(ab’)2, F(ab’)3, F(ab’)2-scFv2, scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, double-chain antibody-Fc, tandem scFv-Fc, or internal antibody. 55. The anti-tau antibody according to any one of claims 44-54, wherein the anti-tau antibody is an IgG1 antibody. 56. The anti-tau antibody according to any one of claims 44-55, wherein the anti-tau antibody is an IgG2 antibody. 57. The anti-tau antibody according to any one of claims 44-56, wherein the anti-tau antibody is an IgG4 antibody. 58. The anti-tau antibody according to any one of claims 44-57, wherein the light chain is a κ chain. 59. The anti-tau antibody according to any one of claims 44-58, wherein the anti-tau antibody has a binding affinity for human tau of about 100 pM to about 3 nM. 60. The anti-tau antibody according to any one of claims 44-59, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO:52-56. 61. The anti-tau antibody according to any one of claims 44-60, wherein the anti-tau antibody comprises a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO:57-62. 62. The anti-tau antibody according to any one of claims 44-61, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 52-56 and a VL domain encoded by a nucleic acid having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a sequence selected from SEQ ID NO: 57-62. 63. The anti-tau antibody according to any one of claims 44-62, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:52-56. 64. The anti-tau antibody according to any one of claims 44-63, wherein the anti-tau antibody comprises a VL domain encoded by a nucleic acid, the nucleic acid comprising the same sequence as SEQ ID NO:57-62. 65. The anti-tau antibody according to any one of claims 44-64, wherein the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising the same sequence as SEQ ID NO:52-56 and a VL domain encoded by a nucleic acid comprising the same sequence as SEQ ID NO:57-62.