Phospho-tau antibodies and methods of use

Abstract

Compositions and methods relating to improved assays for proving Alzheimer's disease are provided herein. Compositions and methods including improved antibodies for assays including immunoassays are further provided herein. Provided herein is a method for detecting phosphorylated tau in a sample from an individual, comprising performing an immunoassay on the sample using an antibody or antibody fragment comprising a variable domain heavy chain region (VH) and a variable domain light chain region (VL), wherein the VH comprises an amino acid sequence at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 30-34, and the VL comprises an amino acid sequence at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 35-40.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 242,437, filed September 9, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] background The discovery of biomarkers and screening techniques for Alzheimer's disease (AD) and other tauopathies is an area of ​​ongoing development, and these tools may be applied to screen populations to determine which non-demented individuals are at highest risk of developing AD dementia, and also to assess disease progression in patients. 42 Proteins reflecting AD pathology, including tau phosphoproteins, neurofilament light chains, and various tau isoforms, have been detected by various means. Aberrant or hyperphosphorylation of tau has been associated with the conversion of pathologically normal tau molecules into paired helical filament (PHF) tau and neurofibrillary tangles (NFTs), which are indicative of various tauopathy pathologies. Summary of the Invention [Problem to be solved by the invention]

[0003] Abstract Tau is an important microtubule-associated protein abundantly expressed in CNS neurons and plays a key role in normal cellular physiology. Tau has also been found to be dysregulated in Alzheimer's disease and other tauopathies. Six isoforms of tau protein are generated from the tau gene by alternative splicing. These isoforms differ from each other by the presence or absence of two N-terminal insertions and one repeat called R2. All six protein isoforms of tau are highly soluble under normal and healthy cellular conditions and are typically regulated by phosphorylation and dephosphorylation. Tau has been demonstrated to interact with microtubules and promote microtubule assembly. In neurons, tau promotes the formation of axonal microtubules and stabilizes them. Tau has an additional role in driving neurite outgrowth. Impaired interaction of tau with microtubules may be a key component in the pathology, development and progression of tauopathies. Hyperphosphorylation of tau is a hallmark feature of AD and other tauopathies, and the degree of hyperphosphorylation often correlates with disease progression. Hyperphosphorylation of tau protein can result in the self-assembly of insoluble tangles of paired helical and straight filaments of tau. These insoluble aggregates of tangles, called neurofibrillary tangles (NFTs), are composed of hyperphosphorylated tau and are considered pathological markers of tauopathies.

[0004] Phosphorylated tau (p-tau), total tau and Aβ detected in cerebrospinal fluid (CSF) and / or blood, respectively 42is an individual biomarker of Alzheimer's disease and several other related tauopathies. CSF ptau is increased in individuals later confirmed to have AD at both the prodromal and dementia stages compared to age- and sex-matched controls. CSF ptau levels show a strong degree of correlation with the degree of cognitive impairment in individuals with AD. In fact, CSF ptau levels can be used with some accuracy as a biomarker to predict progression from cognitively unimpaired to mild cognitive impairment (MCI) and then to AD dementia. With regard to its utility as a biomarker for predicting even relatively early stages of AD progression, CSF ptau has been shown to be significantly increased in samples from individuals with preclinical AD. Changes in the degree of ptau phosphorylation have been demonstrated in both preclinical sporadic cases of AD and early stages of autosomal dominant AD. ptau, total tau and Aβ 42 Blood levels of are generally lower than CSF levels when assayed within the same individual, and blood levels of these biomarkers, if they can be assayed with sufficient specificity and precision, may be utilized as informative biomarkers for AD and other related tauopathies.

[0005] Several sites of phosphorylation that contribute to hyperphosphorylated tau that aggregates into NFTs have been identified. In the longest tau isoform, there are 79 potential serine or threonine phosphorylation sites, and at least 30 of these sites have been identified as phosphorylated in NFT aggregates. A common site used to assay tau molecules for phosphorylation status is at threonine-181. CSF fluids contain an array of tau fragments at various abundances. Tau fragments from the N-terminal and central regions of the tau polypeptide are significantly more abundant in CSF samples than C-terminal tau fragments. Plasma samples from individuals also contain tau polypeptides and tau polypeptide fragments, but these tend to be present at lower concentrations than in matched CSF samples. Being able to detect tau phosphorylation at specific amino acid residues associated with disease pathology and progression is an important component of diagnosis, disease staging, and as a metric for measuring treatment effectiveness for AD and other tauopathies. Detection and measurement of p-tau levels at specific disease-related residues from plasma samples will be a great help in the development of more sensitive and finely tuned diagnostic, prognostic and disease analysis for individuals who may be at risk of developing AD or other tauopathies or who are in the early stages of AD or other tauopathies. Phosphorylation of tau at threonine 217 (p-tau217) is one such residue that is of particular interest in the development of new biomarkers and diagnostic assays. It is believed that alterations in p-tau biomarker concentrations in CSF and plasma precede measurable behavioral or cognitive changes in AD and other tauopathies. The development of new assays that allow a series of specific points and degrees of tau phosphorylation at certain residues will undoubtedly be useful in clinically relevant medical diagnosis and treatment decisions. Comparison of results from new assays with results from existing assays can also yield further medically informative decisions.Results from plasma-based tau biomarker assays can be compared against matched CSF samples (detecting CSF pTau or CSF soluble Aβ) and also against positron emission tomography (PET) scans to detect the extent and location of Aβ aggregates, as a metric for their utility, particularly for analysis in preclinical or early disease stages. [Means for solving the problem]

[0006] Provided herein is a method for detecting phosphorylated tau in a sample from an individual, comprising performing an immunoassay on the sample using an antibody or antibody fragment comprising a variable domain heavy chain region (VH) and a variable domain light chain region (VL), wherein the VH comprises an amino acid sequence at least about 90% identical to a sequence set forth in any one of SEQ ID NOs: 30-34, and the VL comprises an amino acid sequence at least about 90% identical to a sequence set forth in any one of SEQ ID NOs: 35-40. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the phosphorylated tau is selected from the group consisting of pTau-181, pTau-212, pTau-217, pTau-231, pTau-214, and pTau-220. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the phosphorylated tau is pTau-217. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the phosphorylated tau is pTau-231. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the phosphorylated tau is pTau-217 and pTau-231. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the pTau-212 and pTau-217 are detected. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the pTau-212 and pTau-231 are detected. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the pTau-181 and pTau-217 are detected. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the pTau-181 and pTau-231 are detected. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-181, pTau-217 and pTau-231.Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-212, pTau-217 and pTau-231. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-217 and pTau-231 in a sample selected from the group consisting of plasma samples and serum samples. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-212 and pTau-217 in a sample selected from the group consisting of plasma samples and serum samples. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-212 and pTau-231 in a sample selected from the group consisting of plasma samples and serum samples. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-181 and pTau-217 in a sample selected from the group consisting of plasma samples and serum samples. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-181 and pTau-231 in a sample selected from the group consisting of a plasma sample and a serum sample. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-181, pTau-217 and pTau-231 in a sample selected from the group consisting of a plasma sample and a serum sample. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-212 ...Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein VH comprises an amino acid sequence according to any one of SEQ ID NOs: 30-34, and VL comprises an amino acid sequence according to any one of SEQ ID NOs: 35-40. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 30, and VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 35. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 31, and VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 36. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 31, and VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 37. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 32, and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 38. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 33, and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 39. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 34, and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 40. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the antibody or antibody fragment comprises an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 41-51.Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, further comprising carrying out an assay on 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, neurogranin, SNAP-25, synaptotagmin, alpha-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the sample is selected from the group consisting of a blood sample, a plasma sample, a serum sample, and a cerebrospinal fluid (CSF) sample. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, further comprising the step of proving Alzheimer's disease in the individual based on the detection of phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, further comprising the step of establishing the prognosis of the individual for the onset of Alzheimer's disease based on the detection of phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, further determining the age, genotype, or expression of a biomarker of the individual. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the biomarker is selected from the group consisting of Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranin, SNAP-25, synaptotagmin, alpha-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, wherein the method has a specificity of at least about 80% for detecting phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method having a specificity of at least about 85% for detecting phosphorylated tau.Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method having a specificity of at least about 90% for detecting phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method having a sensitivity of at least about 80% for detecting phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method having a sensitivity of at least about 85% for detecting phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method having a sensitivity of at least about 90% for detecting phosphorylated tau. Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method being capable of detecting phosphorylated tau in the sample with a detection limit of at least about 1.0 picogram / milliliter (pg / mL). Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method being capable of detecting phosphorylated tau in the sample with a detection limit of at least about 1.5 picograms / milliliter (pg / mL). Further provided herein is a method for detecting phosphorylated tau in a sample from an individual, the method being capable of detecting phosphorylated tau in the sample with a detection limit of at least about 5 picograms / milliliter (pg / mL).

[0007] Also provided herein in certain embodiments is an anti-tau antibody comprising: i) a heavy chain comprising a variable heavy (VH) domain; and ii) a light chain comprising a variable light (VL) domain, wherein the VH domain comprises an HCDR1 sequence comprising a sequence selected from SEQ ID NOs: 1-5, an HCDR2 sequence comprising a sequence selected from SEQ ID NOs: 6-9, and an HCDR3 sequence comprising a sequence selected from SEQ ID NOs: 10-13, and the VL domain comprises an LCDR1 sequence comprising a sequence selected from SEQ ID NOs: 14-19, an LCDR2 sequence comprising a sequence selected from SEQ ID NOs: 20-23, and an LCDR3 sequence comprising a sequence selected from SEQ ID NOs: 24-29. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.Further provided herein in some embodiments is an anti-tau antibody comprising i) a heavy chain comprising a variable heavy (VH) domain and ii) a light chain comprising a variable light (VL) domain, wherein the VH domain comprises at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 30-34. Further provided herein in some embodiments is an anti-tau antibody comprising i) a heavy chain comprising a variable heavy (VH) domain and ii) a light chain comprising a variable light (VL) domain, wherein the VL domain comprises at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 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 antibody described herein comprises an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, a sc diabody, a sc diabody-CH3, a triple body, a mini-antibody, a minibody, a TriBi minibody, a scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a Fab', a F(ab')2, a F(ab')3, a F(ab')2-scFv2, a scFv, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a sc diabody-Fc, a diabody-Fc, a tandem scFv-Fc, or an intrabody. In some embodiments, the anti-tau antibody described herein is an IgG1 antibody. In some embodiments, the anti-tau antibody described herein is an IgG2 antibody. In some embodiments, the anti-tau antibody described herein is an IgG4 antibody. Further provided herein, in some embodiments, is an anti-tau antibody comprising i) a heavy chain comprising a variable heavy (VH) domain and ii) a light chain comprising a variable light (VL) domain, wherein the light chain is a kappa chain. Further provided herein, in some embodiments, is an anti-tau antibody comprising i) a heavy chain comprising a variable heavy (VH) domain and ii) a light chain comprising a variable light (VL) domain, wherein the light chain has a binding affinity for human tau of about 100 pM to about 3 nM.Further provided herein in some embodiments is an anti-tau antibody comprising a VH domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 52-56. Further provided herein in some embodiments is an anti-tau antibody comprising a VL domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 57-62. Further provided herein in some embodiments is an anti-tau antibody comprising a VH domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 52-56 and a VL domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 57-62. Further provided herein in some embodiments is an anti-tau antibody comprising a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NOs: 52-56. Further provided herein, in some embodiments, is an anti-tau antibody comprising a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 57-62. Further provided herein, in some embodiments, is an anti-tau antibody comprising a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 52-56 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 57-62.

[0008] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows a schema of the single molecule array (Simoa®) method used herein to assay the tau antibodies described herein. After the substrate is added to the sample (sandwich ELISA on beads, 1.1), the sample is added to the Simoa® 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 to allow imaging (1.4). Beads with sandwich complexes (positive beads) will fluoresce with the substrate and show up clearly during imaging; beads without sandwich complexes (negative) will still show up clearly in imaging but will not fluoresce (1.5). The percentage of positive beads is converted to an AEB (average enzyme / bead) value.

[0011] [Figure 2A-2B] 2A-2D show data for Antibody 1, Antibody 2, Antibody 3, Antibody 4, Antibody 5 and Antibody 6 in the Simoa® assay. [Fig. 2C-2D] 2A-2D show data for Antibody 1, Antibody 2, Antibody 3, Antibody 4, Antibody 5 and Antibody 6 in the Simoa® assay.

[0012] [Diagram 3] FIG. 3 shows the ELISA data.

[0013] [Figure 4A] 4A-4G show data for immunohistochemical staining of Antibody 6. [Figure 4B] 4A-4G show data for immunohistochemical staining of Antibody 6. [Figure 4C] 4A-4G show data for immunohistochemical staining of Antibody 6. [Figure 4D]4A-4G show data for immunohistochemical staining of Antibody 6. [Figure 4E] 4A-4G show data for immunohistochemical staining of Antibody 6. [Figure 4F] 4A-4G show data for immunohistochemical staining of Antibody 6. [Figure 4G] 4A-4G show data for immunohistochemical staining of Antibody 6.

[0014] [Figure 5A] 5A-5G show data for immunohistochemical staining of Antibody 5. [Figure 5B] 5A-5G show data for immunohistochemical staining of Antibody 5. [Figure 5C] 5A-5G show data for immunohistochemical staining of Antibody 5. [Figure 5D] 5A-5G show data for immunohistochemical staining of Antibody 5. [Figure 5E] 5A-5G show data for immunohistochemical staining of Antibody 5. [Figure 5F] 5A-5G show data for immunohistochemical staining of Antibody 5. [Figure 5G] 5A-5G show data for immunohistochemical staining of Antibody 5.

[0015] [Figure 6A] 6A-6G show data for immunohistochemical staining of Antibody 2. [Figure 6B] 6A-6G show data for immunohistochemical staining of Antibody 2. [Figure 6C] 6A-6G show data for immunohistochemical staining of Antibody 2. [Figure 6D] 6A-6G show data for immunohistochemical staining of Antibody 2. [Figure 6E] 6A-6G show data for immunohistochemical staining of Antibody 2. [Figure 6F] 6A-6G show data for immunohistochemical staining of Antibody 2. [Figure 6G] 6A-6G show data for immunohistochemical staining of Antibody 2.

[0016] [Figure 7] FIG. 7 shows a diagram of the indirect ELISA assay and a graph of the ELISA data assaying antibody binding to the pTau-217 peptide.

[0017] [Figure 8] FIG. 8 shows a graph of the signal / noise (S / N) analysis of the ELISA assay for Antibody 2 binding to pTau-217 peptide for 120 clinic samples derived from plasma and a graph of the coefficient of variation (CV%) for the ELISA assay for Antibody 2 binding to pTau-217 peptide for 120 clinic samples derived from plasma.

[0018] [Figure 9] FIG. 9 shows a graph of the calibration curve (Cal Curve) for the Simoa®-based pTau-217 assay using Antibody 2 for the QTx indicated groups (plates) of clinical samples derived from cerebrospinal fluid (68 CSF samples) and plasma (120 plasma samples) compared to an assay using ADx p204 antibody.

[0019] [Figure 10] FIG. 10 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 in matched samples from individuals with a clinical diagnosis of either non-Alzheimer's disease, indeterminate diagnosis or Alzheimer's disease, and statistical analysis of the correlation results.

[0020] [Figure 11]FIG. 11 shows a graph of Simoa®-based pTau Assay-217 results using Antibody 2 vs. Simoa®-based pTau Assay-181 results using Antibody 2 by sample and a statistical analysis of the correlation results.

[0021] [Figure 12] FIG. 12 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 vs. Simoa®-based Tau assay results using Innotest pTau181 antibody by sample, and a statistical analysis of the correlation results.

[0022] [Figure 13] FIG. 13 shows a graph of the Simoa®-based p-tau assay results using Antibody 2 as the capture antibody, antibody ADx p204 as the detector antibody and peptide as a calibrator, as well as a statistical analysis of the correlation results.

[0023] [Figure 14] FIG. 14 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2, grouping together samples from individuals with a clinical diagnosis of Alzheimer's disease and samples from control individuals, derived from either CSF or plasma.

[0024] [Figure 15] FIG. 15 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 for EDTA plasma samples of various concentrations, and a chart listing the coefficient of variation for each sample concentration to demonstrate the precision of the assay.

[0025] [Figure 16] FIG. 16 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 graphed as coefficient of variation (CV%) versus measured concentration.

[0026] [Figure 17] FIG. 17 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 and a statistical analysis of parallelism to determine whether actual samples containing high endogenous analyte concentrations provide a similar degree of detection in the standard curve after dilution.

[0027] [Figure 18] FIG. 18 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 to determine whether a sample matrix spiked with a detection analyte above the upper limit of detection can still provide reliable quantification after dilution within the standard curve range for four samples plus buffer spikes, and a statistical analysis of linearity.

[0028] [Figure 19] FIG. 19 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 to determine whether a sample matrix spiked with a detection analyte above the upper limit of detection can still provide reliable quantification after dilution within the standard curve range for three samples plus a calibration sample, and a statistical analysis of linearity.

[0029] [Figure 20] FIG. 20 shows a graph of Simoa®-based pTau-217 assay results using Antibody 2 in a clinical validation of a memory loss clinic cohort, and a graph of the Receiver Operating Characteristic (ROC) analysis graphed against pTau-217 assay sensitivity.

[0030] [Figure 21] FIG. 21 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 for groups from control and AD dementia individuals.

[0031] [Figure 22]FIG. 22 shows a graph of Simoa®-based p-tau assay-181 results using antibodies from Quanterix® for groups from control and AD dementia individuals, and a chart of sample stratification.

[0032] [Diagram 23] FIG. 23 shows a graph of Simoa®-based pTau assay-217 results using Antibody 2 and Simoa®-based pTau assay-181 results using antibodies from Quanterix® showing precision plots with calculated coefficients of variation.

[0033] [Figure 24] FIG. 24 shows graphs of the clinical performance of various p-Tau Simoa®-based assays comparing sensitivity and specificity, and charts with statistical analyses of the results.

[0034] [Diagram 25] FIG. 25 shows a schematic diagram of tau, indicating the relative positions of the various protein domains and the location of threonine residues that can be assayed for phosphorylation status using the methods disclosed herein.

[0035] [Figure 26] FIG. 26 shows a graph of reactivity of various antibodies against tau fragments, using non-phosphorylated T217 (Bio-pt654) and full length tau (tau441) in an indirect ELISA.

[0036] [Figure 27] FIG. 27 shows a graph of the reactivity of various antibodies to tau fragments with phosphorylated T181 (Bio-pt126) and phosphorylated T231 (Bio-pt146) in an indirect ELISA.

[0037] [Figure 28]FIG. 28 shows a diagram of an assay utilizing pTau217 monoclonal antibody as a capture tool against various synthetic peptides and a graph of the results for this assay using Antibody 2 as the capture tool.

[0038] [Figure 29] FIG. 29 shows Western blot analysis using various tau antibodies on brain lysate samples from AD patients or control subjects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description Alzheimer's disease (AD) is a complex disease and effective treatment requires accurate diagnosis. Described herein are improved compositions and methods for detecting AD, including improved antibodies for use in diagnostic and / or prognostic assays.

[0040] Specific Terminology

[0041] Throughout this disclosure, various embodiments are described in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as a rigid limitation on the scope of any embodiment. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range, to the tenth of the unit of the lower limit, unless the context clearly indicates otherwise. For example, the description of a range, e.g., 1-6, should be considered to have specifically disclosed subranges, e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual values ​​within that range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may be independently included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention unless the context clearly indicates otherwise.

[0042] The terminology used herein is for the purpose of describing particular embodiments only and is not to be construed as limiting any embodiment. As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless the context clearly indicates otherwise. It is further understood that the terms "comprises" and / or "comprising" as used herein specify the presence of stated features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0043] Unless specifically stated or clear from the context, as used herein, the term "about" in reference to a number or range of numbers is understood to mean the stated number and that number + / - 10%, or 10% below the recited lower limit and 10% above the recited upper limit for the values ​​recited for the range.

[0044] The terms "individual," "patient," or "subject" are used interchangeably. None of these terms require or are limited to a situation characterized by the supervision (e.g., regular or intermittent) of a health care worker (e.g., a physician, registered nurse, nurse practitioner, physician's assistant, hospital clerk, or hospice worker). Additionally, these terms refer to either a human or an animal subject.

[0045] The term "antibody" herein is used in the broadest sense and includes monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment 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), or single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, and heteroconjugate antibodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may include a rabbit IgG1 constant region. The antibody may include a rabbit IgG4 constant region. Antibodies include, but are not limited to, full-length and native antibodies, as well as fragments and portions thereof that retain their binding specificity, such as any specific binding portion thereof, including those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entities). Monoclonal antibodies are generally antibodies within a composition of substantially homogeneous antibodies; thus, any individual antibodies contained within a monoclonal antibody composition are identical, except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibody may comprise a rabbit IgG1 constant region or a rabbit IgG4 constant region.

[0046] The term "complementarity determining region" or "CDR" refers to a segment of the variable region of an antibody that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. Thus, CDRs are sometimes called hypervariable regions. A variable region contains three CDRs. CDR peptides can be obtained by constructing a gene that encodes the CDRs of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from the RNA of antibody-producing cells. See, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

[0047] The term "Fab" refers to a protein comprising the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. Fab' fragments are produced by reducing the heavy chain disulfide bridges of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0048] A "single chain variable fragment (scFv)" is a fusion protein of the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an antibody, connected by a short linker peptide of 10 to about 25 amino acids. The linker is usually glycine-rich for flexibility, serine- or threonine-rich for solubility, and can connect 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 despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Furthermore, antibody fragments include single chain polypeptides that have the characteristics of a VH domain, i.e., the ability to assemble with a VL domain, or the characteristics of a VL domain, i.e., the ability to assemble with a VH domain, to become a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0049] As used herein, the term "percent (%) amino acid sequence identity" in relation to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, without considering any conservative substitutions as part of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the skill 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 the appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

[0050] In the situation where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B in that program, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0051] The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, 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 "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, 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 determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. 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, 2003 Jan;27(1):55-77 (the "IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70 (the "Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. 2000 Dec;13(12):819-24 (the "AbM" numbering scheme). In certain embodiments, the CDRs of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0052] The boundaries of a given CDR or FR may vary depending on the scheme used for identification.For example, Kabat scheme is based on structural alignment, while Chothia scheme is based on structural information.The numbering for both Kabat scheme and Chothia scheme is based on the most common antibody region sequence length, and insertions are provided with insertion letters, such as "30a", and deletions occur in some antibodies.These two schemes place certain insertions and deletions ("indels") at different positions to produce differential numbering.The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the methods and compositions described herein belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions described herein, representative exemplary methods and materials are now described. Tau Antibody

[0054] Antibodies that bind to tau are provided herein. In some examples, the antibodies that bind to tau are monoclonal antibodies. In certain embodiments, anti-tau antibodies are disclosed herein. In some examples, the anti-tau antibodies specifically bind to mammalian tau. In some examples, the anti-tau antibodies specifically bind to human tau. In some examples, the anti-tau antibodies specifically bind to the N-terminal portion of tau. In some examples, the anti-tau antibodies specifically bind to the N-terminal portion of human tau. In some examples, the anti-tau antibodies specifically bind to a portion of tau that includes protein domain P2. In some examples, the anti-tau antibodies specifically bind to a portion of human tau that includes protein domain P2. In some examples, the anti-tau antibodies specifically bind to a portion of tau that includes protein domain P1. In some examples, the anti-tau antibodies specifically bind to a portion of human tau that includes protein domain P1. In some examples, the anti-tau antibodies specifically bind to a portion of tau that includes protein domains P1 and P2. In some examples, the anti-tau antibodies specifically bind to a portion of human tau that includes protein domains P1 and P2.

[0055] In some embodiments, the anti-tau antibody comprises i) a heavy chain comprising a variable heavy (VH) domain and ii) a light chain comprising a variable light (VL) domain. In some embodiments, the VH domain comprises a heavy chain CDR1 (HCDR1) sequence comprising a sequence selected from SEQ ID NOs: 1-5, a heavy chain CDR2 (HCDR2) sequence comprising a sequence selected from SEQ ID NOs: 6-9, and a heavy chain CDR3 (HCDR3) sequence comprising a sequence selected from SEQ ID NOs: 10-13. In some embodiments, the VL domain comprises a light chain CDR1 (LCDR1) sequence comprising a sequence selected from SEQ ID NOs: 14-19, a light chain CDR2 (LCDR2) sequence comprising a sequence selected from SEQ ID NOs: 20-23, and a light chain CDR3 (LCDR3) sequence comprising a sequence selected from SEQ ID NOs: 24-29.

[0056] In some embodiments, the VH region of the anti-tau antibody comprises HCDR1, HCDR2 and HCDR3 sequences selected from Table 1. [Table 1]

[0057] In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:1; an HCDR2 sequence comprising SEQ ID NO:6; and an HCDR3 sequence comprising SEQ ID NO:10. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:2; an HCDR2 sequence comprising SEQ ID NO:7; and an HCDR3 sequence comprising SEQ ID NO:11. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:3; an HCDR2 sequence comprising SEQ ID NO:8; and an HCDR3 sequence comprising SEQ ID NO:10. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:4; an HCDR2 sequence comprising SEQ ID NO:7; and an HCDR3 sequence comprising SEQ ID NO:12. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO:5; an HCDR2 sequence comprising SEQ ID NO:9; and an HCDR3 sequence comprising SEQ ID NO:13.

[0058] In some embodiments, the VL region of the anti-tau antibody comprises LCDR1, LCDR2 and LCDR3 sequences selected from Table 2. [Table 2]

[0059] In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 14; an LCDR2 sequence comprising SEQ ID NO: 20; and an LCDR3 sequence comprising SEQ ID NO: 24. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 15; an LCDR2 sequence comprising SEQ ID NO: 21; and an LCDR3 sequence comprising SEQ ID NO: 25. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 16; an LCDR2 sequence comprising SEQ ID NO: 22; and an LCDR3 sequence comprising SEQ ID NO: 26. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 17; an LCDR2 sequence comprising SEQ ID NO: 20; and an LCDR3 sequence comprising SEQ ID NO: 27. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 18; an LCDR2 sequence comprising SEQ ID NO: 23; and an LCDR3 sequence comprising SEQ ID NO: 28. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 19; an LCDR2 sequence comprising SEQ ID NO: 21; and an LCDR3 sequence comprising SEQ ID NO: 29.

[0060] In some embodiments, the anti-tau antibody is an antigen-binding fragment thereof. In some embodiments, the anti-tau antibody is a chimeric antibody or an antigen-binding fragment thereof. In some embodiments, the anti-tau antibody comprises an IgG-scFv, a nanobody, a mini-antibody, a minibody, a scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a Fab', a F(ab')2, a F(ab')3, a F(ab')2-scFv2, a scFv, a scFv-KIH, a Fab-scFv-Fc, or an intrabody. In some embodiments, the anti-tau antibody comprises a bispecific antibody. In some embodiments, the anti-tau antibody comprises a multispecific antibody. 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, and the light chain is a kappa chain.

[0061] In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 100 pM to about 3 nM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 100 pM to 300 pM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 100 pM to 500 pM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 100 pM to 800 pM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 300 pM to 600 pM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 300 pM to 900 pM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 400 pM to 1 nM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 500 pM to 1.5 nM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 500 pM to about 2 nM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 600 pM to about 3 nM. In some embodiments, the anti-tau antibody has a binding affinity to human tau of about 100 pM to about 3 nM.

[0062] In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 100 pM to 300 pM. In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 100 pM to 500 pM. In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 100 pM to 800 pM. In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 300 pM to 600 pM. In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 300 pM to 900 pM. In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 400 pM to 1 nM. In some embodiments, the anti-tau antibody has a binding affinity to phosphorylated human tau of about 500 pM to 1.5 nM. In some embodiments, the anti-tau antibody has a binding affinity for phosphorylated human tau of about 500 pM to 2 nM, hi some embodiments, the anti-tau antibody has a binding affinity for phosphorylated human tau of about 600 pM to 3 nM.

[0063] Antibodies comprising any of the sequences shown in Table 3 or Table 4 are described herein. [Table 3] [Table 4-1] [Table 4-2]

[0064] In some embodiments, the variable domain heavy chain region (VH) comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence according to any one of SEQ ID NOs: 30-34.

[0065] In some embodiments, the VH comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.

[0066] In some embodiments, the VH comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 80% sequence identity with at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.In some embodiments, the VH comprises an amino acid sequence of at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34, and has at least 80% sequence identity with at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34, and has at least 80% sequence identity with at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.

[0067] In some embodiments, the VH comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 90% sequence identity with at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.In some embodiments, the VH comprises an amino acid sequence of at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34, and has at least 90% sequence identity with at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34, and has at least 90% sequence identity with at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.

[0068] In some embodiments, the VH comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 95% sequence identity with at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.In some embodiments, the VH comprises an amino acid sequence of at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34, and has at least 95% sequence identity with at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34, and has at least 95% sequence identity with at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.

[0069] In some embodiments, the VH comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 99% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 99% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 99% sequence identity with at least 110 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 99% sequence identity with at least 115 contiguous amino acid residues of any one of SEQ ID NOs: 30-34. In some embodiments, the VH comprises an amino acid sequence of at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34 and has at least 99% sequence identity to at least 120 contiguous amino acid residues of any one of SEQ ID NOs: 30-34.

[0070] In some embodiments, the variable domain light chain region (VL) comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 91% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 92% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 93% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence according to any one of SEQ ID NOs: 35-40.

[0071] In some embodiments, the VL comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40.

[0072] In some embodiments, the VL comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 80% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40.

[0073] In some embodiments, the VL comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 90% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40.

[0074] In some embodiments, the VL comprises an amino acid sequence of at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 50 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 60 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 70 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 80 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 90 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 95% sequence identity with at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40. In some embodiments, the VL comprises an amino acid sequence of at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 99% sequence identity with at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 35-40.In some embodiments, the VL comprises an amino acid sequence of at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40 and has at least 99% sequence identity to at least 105 contiguous amino acid residues of any one of SEQ ID NOs: 35-40.

[0075] In some embodiments, the VH comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 91% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 91% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 92% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 92% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 93% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 30-34; and the VL comprises an amino acid sequence having at least 93% sequence identity to an amino acid sequence according to any one of SEQ ID NOs: 35-40.In some embodiments, the VH comprises an amino acid sequence having at least 94% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 94% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 35-40. In some embodiments, the VH comprises an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 30-34; the VL comprises an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 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 NOs: 30-34; 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 NOs: 35-40.

[0076] 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 set forth in SEQ ID NO: 30; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in 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.

[0077] 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 set forth in SEQ ID NO:31; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in 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.

[0078] 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 set forth in SEQ ID NO: 31; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in 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.

[0079] 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 of SEQ ID NO: 32; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of 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.

[0080] 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; 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; 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; 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; 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 set forth in SEQ ID NO: 33; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in 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.

[0081] 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 of SEQ ID NO: 34; and the VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of 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.

[0082] In some embodiments, described herein are antibodies or antibody fragments that comprise a heavy chain sequence at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 41, 43, 46, 48, and 50. In some examples, the antibody or antibody fragment comprises a heavy chain sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 41, 43, 46, 48, and 50.

[0083] In some embodiments, described herein are antibodies or antibody fragments that comprise a light chain sequence at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 42, 44, 45, 47, 49, and 51. In some examples, the antibody or antibody fragment comprises a light chain sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 42, 44, 45, 47, 49, and 51.

[0084] In some embodiments, an antibody or antibody fragment is described herein that comprises a heavy chain sequence that is at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 41, 43, 46, 48, and 50, and a light chain sequence that is at least about 90% identical to the sequence set forth in any one of SEQ ID NOs: 42, 44, 45, 47, 49, and 51. In some examples, the antibody or antibody fragment comprises a heavy chain sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 41, 43, 46, 48, and 50, and a light chain sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 42, 44, 45, 47, 49, and 51. [Table 5-1] [Table 5-2]

[0085] In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 52-56. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 57-62. The nucleic acid sequences of the VH domains of the anti-tau-tau antibodies described herein are listed in Table 6, and the nucleic acid sequences of the VL domains of the anti-tau-tau antibodies described herein are listed in Table 7. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:54. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:55. In some embodiments, the anti-tau-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:56. In some embodiments, the anti-tau-tau antibody comprises a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:57. In some embodiments, the anti-tau-tau antibody comprises a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid that comprises at least 90% sequence identity to SEQ ID NO: 61. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid that comprises at least 90% sequence identity to SEQ ID NO: 62.In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:52 and a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:57. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:53 and a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:53 and a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:54 and a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:55 and a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:563 and a VL domain encoded by a nucleic acid comprising at least 90% sequence identity to SEQ ID NO:62. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:52. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:53. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:54. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:55. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:56. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:57.In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:62. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:52 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:57. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:53 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:58. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:53 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:59. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:54 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:60. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:55 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:61. In some embodiments, the anti-tau antibody comprises a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:56 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:62. [Table 6-1] [Table 6-2] [Table 7-1]

Table 7-2

[0086] In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to a sequence selected from SEQ ID NOs: 63-67. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to a sequence selected from SEQ ID NOs: 68-73. The nucleic acid sequences of the heavy chains of the anti-tau antibodies described herein are listed in Table 8, and the nucleic acid sequences of the light chains of the anti-tau antibodies described herein are listed in Table 9. The nucleic acid sequences listed in Tables 8 and 9 may be used in the process of in vitro production of the antibodies described herein. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 63. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 64. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 65. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO: 66. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:67. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:68. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:69. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:70. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:71. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:72. In some embodiments, the anti-tau antibody comprises a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:73. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:63 and a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:68. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:64 and a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:69.In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:64 and a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:70. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:65 and a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:71. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:66 and a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:72. In some embodiments, the anti-tau antibody comprises a heavy chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:67 and a light chain encoded by a nucleic acid comprising a sequence identical to SEQ ID NO:73. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 9-1] [Table 9-2] [Table 9-3]

[0087] Methods of the Disclosure Disclosed herein is a method for detecting phosphorylated tau in a sample from an individual using the antibody described herein.In some embodiments, the phosphorylated tau is selected from the group consisting of pTau-212, pTau-217, pTau-231, pTau-214 and pTau-220.In some embodiments, the method for detecting phosphorylated tau in a sample from an individual using the antibody described herein comprises improved specificity and sensitivity.

[0088] Described herein is a method for detecting phosphorylated tau in a sample from an individual, comprising performing an assay on the sample using an antibody or antibody fragment that binds to phosphorylated tau. Described herein is a method for detecting phosphorylated tau in a sample from an individual, comprising performing an immunoassay on the sample using an antibody or antibody fragment that binds to phosphorylated tau. In some embodiments, the phosphorylated tau is selected from the group consisting of pTau-212, pTau-217, pTau-231, pTau-214, pTau-220 and pTau-181. In some embodiments, the phosphorylated tau is selected from the group consisting of 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. In some embodiments, the phosphorylated tau is pTau-181, pTau-217 and pTau-231.

[0089] Further described herein are methods for detecting phosphorylated tau in a sample from an individual, comprising performing an assay on the sample using an antibody or antibody fragment that binds to multiple 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.

[0090] Described herein is a method for detecting phosphorylated tau in a sample from an individual, the method detecting pTau-217 and pTau-231 in a sample selected from the group consisting of plasma and serum samples. In some embodiments, the method detects pTau-212 and pTau-217 in a sample selected from the group consisting of plasma and serum samples. In some embodiments, the method detects pTau-212 and pTau-231 in a sample selected from the group consisting of plasma and serum samples. In some embodiments, the method detects pTau-212, pTau-217 and pTau-231 in a sample selected from the group consisting of plasma and serum samples.

[0091] The methods described herein may include performing an assay on a sample, the sample being selected from the group consisting of a plasma sample and a serum sample. In some examples, the sample is a blood sample. In some examples, 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 from a finger prick. The sample may be obtained by a health care provider or by 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.

[0092] In some embodiments, the method for determining the level of a biomarker selected from the group consisting of Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranin, SNAP-25, synaptotagmin, alpha-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof is further described herein. In some examples, the biomarker is Aβ42. In some examples, the biomarker is Aβ40. In some examples, the biomarker is Aβ42 and Aβ40. In some examples, the biomarker is APOE. In some examples, the biomarker is selected from the group consisting of APOE2, APOE3, and APOE4. In some examples, the biomarker is APOE4.

[0093] In some embodiments, the method for detecting phosphorylated tau in a sample comprises an immunoassay or ligand assay using an antibody or antibody fragment described herein. In some cases, the assay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), colorimetric immunoassay, homogeneous immunoassay, non-optical immunoassay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescence resonance energy transfer (FRET) immunoassay, time-resolved fluorescent immunoassay, lateral flow immunoassay, microspot immunoassay, surface plasmon resonance assay, ligand assay, coagulation assay, chromatography assay, and immunocapture coupled with mass spectrometry. In some cases, the assay comprises an immunoassay. In some cases, the assay is selected from the group consisting of Western blot, enzyme-linked immunosorbent assay (ELISA), and chromatography. In some cases, the immunoassay is single-plexed. In some cases, the immunoassay is multiplexed.

[0094] The method described herein may include a plurality of immunoassays using the antibody or antibody fragment described herein. In some cases, the plurality of immunoassays are the same immunoassay (e.g., four or more ELISA assays). When the plurality of immunoassays are the same immunoassay, each of the plurality of immunoassays can detect different phosphorylated tau. When the plurality of immunoassays are the same immunoassay, each of the plurality of immunoassays can be carried out in the same reaction chamber or in different reaction chambers. The reaction chamber can be any suitable space for carrying out an immunoassay. Examples of reaction chambers include, but are not limited to, wells in a microplate, Eppendorf tubes, or droplets.

[0095] 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.

[0096] In some cases, the assay comprises a non-immunoassay. In some cases, the assay is selected from the group consisting of 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.

[0097] The method described herein using the antibody described herein can be used to confirm Alzheimer's disease in an individual based on the detection of phosphorylated tau.In some embodiments, Alzheimer's disease in an individual is confirmed when pTau-212, pTau-217, pTau-231, pTau-214, pTau-220, or a combination thereof is detected in a sample from the individual.

[0098] The method described herein using the antibody described herein can be used for individual prognosis of developing Alzheimer's disease based on detecting phosphorylated tau.In some embodiments, the individual prognosis of developing Alzheimer's disease is determined when pTau-212, pTau-217, pTau-231, pTau-214, pTau-220, or a combination thereof is detected in the sample from the individual.

[0099] The methods described herein using the antibodies described herein may be used to accurately and specifically establish Alzheimer's Disease (AD) in an individual compared to a disease or disorder selected from the group consisting of non-Alzheimer's Disease (AD) neurodegenerative disease, Aβ-negative non-AD neurodegenerative disease, Aβ-positive non-AD neurodegenerative disease, behavioral 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 conditions, and combinations thereof, or neurologically and cognitively unimpaired controls. In some embodiments, the methods described herein using the antibodies described herein include improved accuracy or specificity of at least or about 70%, 80%, 90%, 95%, 99% or more in establishing AD compared to a disease or disorder or a neurologically and cognitively unimpaired state.

[0100] The methods described herein using the antibodies described herein can be used to accurately and specifically establish Alzheimer's disease (AD) in an individual compared to neuropathological testing or clinical diagnosis. In some embodiments, the methods described herein using the antibodies described herein include improved accuracy or specificity of at least or about 70%, 80%, 90%, 95%, 99% or more in establishing AD compared to neuropathological testing or clinical diagnosis. In some embodiments, the neuropathological testing or clinical diagnosis includes neurological testing, psychiatric testing, or brain imaging (e.g., MRI, CT, or PET scan).

[0101] The methods described herein using the antibodies described herein may be capable of detecting phosphorylated tau in a sample with a low detection limit. In some embodiments, the methods described herein using the antibodies described herein are capable of detecting phosphorylated tau in a sample with a detection limit of at least about 1.5 picograms per milliliter (pg / mL). In some embodiments, the methods described herein using the antibodies described herein are capable of detecting phosphorylated tau in a sample with a detection limit of at least about 5 picograms per milliliter (pg / mL). In some embodiments, the methods described herein using the antibodies described herein are capable of detecting phosphorylated tau in a sample with a detection limit within the range of 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. Tau antibody production

[0102] In some embodiments, the antibodies or antibody fragments described herein are produced using any method known in the art to be useful for the synthesis of antibodies or antibody fragments, particularly by chemical synthesis or recombinant expression, and are preferably produced by recombinant expression techniques.

[0103] In some examples, antibodies or binding fragments thereof are recombinantly expressed and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesis of overlapping oligonucleotides containing portions of the antibody encoding sequence, annealing and ligation of these oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0104] Alternatively, nucleic acid molecules encoding antibodies are generated, as appropriate, from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for particular gene sequences.

[0105] In some examples, the antibody or binding thereof is optionally generated by immunizing an animal, e.g., a mouse, to produce polyclonal antibodies, e.g., 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 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), or more preferably, by generating monoclonal antibodies. Alternatively, clones encoding at least the Fab portion of the antibody can be obtained, as desired, by screening a Fab expression library for clones of Fab fragments that bind a specific antigen (e.g., as described in Huse et al., 1989, Science 246:1275-1281) or by screening an antibody library (see, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0106] In some embodiments, techniques developed for the production of "chimeric antibodies" (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) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as molecules having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

[0107] In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. 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 adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge to produce a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also used, if desired (Skerra et al., 1988, Science 242:1038-1041).

[0108] In some embodiments, an expression vector containing the antibody nucleotide sequence or the antibody nucleotide sequence is transferred into a host cell by conventional techniques (e.g., electroporation, liposomal transfection and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0109] In some embodiments, various host-expression vector systems are used to express the antibodies or binding fragments thereof described herein. Such host-expression systems refer to vehicles in which antibody coding sequences are produced and subsequently purified, but also to cells that express the antibody or binding fragments thereof in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include microorganisms, such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody or binding fragment coding sequence; recombinant viral expression vectors (e.g., cauliflower) containing the antibody or binding fragment coding sequence; These include, but are not limited to, plant cell lines infected with the Tobacco Mosaic Virus (CaMV) and Tobacco Mosaic Virus (TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody or binding fragment thereof coding sequence; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0110] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines stably expressing antibodies are engineered as needed. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, the engineered cells are then grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are then cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing antibodies or binding fragments thereof.

[0111] In some examples, several selection systems are used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, which are used in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance has also been used as the basis of selection for 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 & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the 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).Methods generally known in the art of recombinant DNA technology that may be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0112] In some cases, the expression level of the antibody is 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)). If the marker in the antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture increases the copy number of the marker gene. Since the amplified region is related to the nucleotide sequence of the antibody, production of the antibody also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0113] In some examples, any method known in the art for purification of antibodies is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purification of proteins. Expression vector

[0114] In some embodiments, the vector includes any suitable vector derived from either eukaryotic or prokaryotic sources. In some cases, the vector is obtained from bacteria (e.g., E. coli), insects, yeast (e.g., Pichia pastoris), algae or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, 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.

[0115] 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, a FLAG vector, e.g., pPolh-FLAG1 or pPolh-MAT 2, or a MAT vector, e.g., pPolh-MAT1 or pPolh-MAT2.

[0116] In some cases, the yeast vector includes a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 15 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® destination vector, a pAO815 Pichia vector, a pFLD1 Pichi pastoris vector, a pGAPZA, B and C Pichia pastoris vector, a pPIC3.5K Pichia vector, a pPIC6 A, B and C Pichia vector, a pPIC9K Pichia vector, a pTEF1 / Zeo, a pYES2 yeast vector, a pYES2 / CT yeast vector, a pYES2 / NT A, B and C yeast vector, or a pYES3 / CT yeast vector.

[0117] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0118] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors can 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. Mammalian stable expression vectors can 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.

[0119] In some cases, cell-free systems are mixtures of cytoplasmic and / or nuclear components from cells, and are used for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize either prokaryotic or eukaryotic cell components. Sometimes, nucleic acid synthesis is obtained 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 Extract system, E.coli T7 S30 system, or PURExpress®.

[0120] host cell In some embodiments, the host cell includes any suitable cell, such as a naturally occurring cell or a genetically modified cell. In some examples, the host cell is a production host cell. In some examples, the host cell is a eukaryotic cell. In other examples, the host cell is a prokaryotic cell. In some cases, the eukaryotic cell includes a fungus (e.g., a yeast cell), an animal cell, or a plant cell. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include gram-positive bacteria or gram-negative bacteria. Sometimes, the gram-negative bacteria are anaerobic, rod-shaped, or both.

[0121] In some cases, the gram-positive bacteria include Actinomycetes, Firmicutes, or Tenericutes. In some cases, the gram-negative bacteria include Aquifex, Deinococcus-Thermus, Fibrobacter-Chlorobium / Bacteroidetes (FCB group), Fusobacterium, Gemmatimonas, Nitrospira, Planctomyces-Verrucomicrobium / Chlamydia (PVC group), Proteobacteria, Spirochaetes, or Synergistes. Other bacteria can be Acidobacterium, Chloroflexus, Chrysiogenes, Cyanobacteria, Deferibacter, Dictyoglomus, Thermodesulfobacteria, or Thermotoga. The bacterial cell can be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0122] 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™.

[0123] In some cases, the animal cells include cells from vertebrates or invertebrates. In some cases, the animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, the fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0124] Fungi include Ascomycota, such as yeasts, molds, filamentous fungi, Basidiomycota, or Zygomycota. In some cases, yeasts include Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycetes (true yeast, e.g., Saccharomyces cerevisiae (baker's yeast)) or Taphrina (e.g., Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycota includes Agaricota (e.g., Tremella) or Puccinia (e.g., Microbotrytium).

[0125] Exemplary yeasts or filamentous fungi include, for example, the genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or molds include, for example, the species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0126] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains, such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strains, such as INVSc1.

[0127] In some cases, the additional animal cell comprises a cell obtained from a mollusc, an arthropod, an annelid, or a sponge.In some cases, the additional animal cell is a mammalian cell, such as a primate, an ape, a horse, a cow, a pig, a dog, a cat, or a rodent.In some cases, the rodent comprises a mouse, a rat, a hamster, a gerbil, a hamster, a chinchilla, a fancy rat, or a guinea pig.

[0128] Exemplary mammalian host cells include 293A cell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO cells. CHOK1, Expi293F(TM) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-In(TM)-3T3 cell line, Flp-In(TM)-BHK cell line, Flp-In (trademark)-CHO cell line, Flp-In(TM)-CV-1 cell line, Flp-In(TM)-Jurkat cell line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293 These include, but are not limited to, MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line and T-REx™-HeLa cell line.

[0129] In some instances, the mammalian host cell is a stable cell line, or a cell line that has incorporated the genetic material of interest into its genome and has the ability to express the product of the genetic material after many generations of cell division. In some instances, the mammalian host cell is a transient cell line, or a cell line that has not incorporated the genetic material of interest into its genome and does not have the ability to express the product of the genetic material after many generations of cell division.

[0130] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells and expresSF+® cells. In some examples, plant cells include cells derived from algae. Exemplary insect cell lines include, but are not limited to, strains derived from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0131] Numbered embodiments Numbered embodiment 1 includes a method for detecting phosphorylated tau in a sample from an individual, comprising performing an immunoassay on the sample using an antibody or antibody fragment comprising a variable domain heavy chain region (VH) and a variable domain light chain region (VL), wherein the VH comprises an amino acid sequence at least about 90% identical to a sequence set forth in any one of SEQ ID NOs: 30-34, and the VL comprises an amino acid sequence at least about 90% identical to a sequence set forth in any one of SEQ ID NOs: 35-40. Numbered embodiment 2 includes the method of numbered embodiment 1, wherein the phosphorylated tau is selected from the group consisting of pTau-181, pTau-212, pTau-217, pTau-231, pTau-214 and pTau-220. Numbered embodiment 3 includes the methods of numbered embodiments 1 to 2, wherein the phosphorylated tau is pTau-217. Numbered embodiment 4 includes the methods of numbered embodiments 1 to 2, wherein the phosphorylated tau is pTau-231. Numbered embodiment 5 includes the method of numbered embodiment 2, detecting pTau-217 and pTau-231. Numbered embodiment 6 includes the method of numbered embodiment 2, wherein pTau-212 and pTau-217 are detected. Numbered embodiment 7 includes the method of numbered embodiment 2, wherein pTau-212 and pTau-231 are detected. Numbered embodiment 8 includes the method of numbered embodiment 2, wherein pTau-181 and pTau-217 are detected. Numbered embodiment 9 includes the method of numbered embodiment 2, wherein pTau-181 and pTau-231 are detected. Numbered embodiment 10 includes the method of numbered embodiment 2, wherein pTau-181, pTau-217 and pTau-231 are detected. Numbered embodiment 11 includes the method of numbered embodiment 2, wherein pTau-212, pTau-217 and pTau-231 are detected. Numbered embodiment 12 includes the method of numbered embodiment 5, wherein pTau-217 and pTau-231 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 13 includes the method of numbered embodiment 6, wherein pTau-212 and pTau-217 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 14 includes the method of numbered embodiment 7, wherein pTau-212 and pTau-231 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 15 includes the method of numbered embodiment 11, wherein pTau-181 and pTau-217 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 16 includes the method of numbered embodiment 11, wherein pTau-181 and pTau-231 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 17 includes the method of numbered embodiment 11, wherein pTau-181, pTau-217 and pTau-231 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 18 includes the method of numbered embodiment 11, wherein pTau-212, pTau-217 and pTau-231 are detected in a sample selected from the group consisting of a plasma sample and a serum sample. Numbered embodiment 19 includes the methods of numbered embodiments 1 to 18, wherein VH comprises an amino acid sequence according to any one of SEQ ID NOs: 30-34. Numbered embodiment 20 includes the methods of numbered embodiments 1 to 19, wherein the VL comprises an amino acid sequence according to any one of SEQ ID NOs: 35-40. Numbered embodiment 21 includes the methods of numbered embodiments 1 to 20, wherein VH comprises an amino acid sequence according to any one of SEQ ID NOs: 30-34, and VL comprises an amino acid sequence according to any one of SEQ ID NOs: 35-40. Numbered embodiment 22 includes the methods of numbered embodiments 1 to 21, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO:30, and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO:35. Numbered embodiment 23 includes the methods of numbered embodiments 1 to 21, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO:31 and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO:36. Numbered embodiment 24 includes the methods of numbered embodiments 1 to 21, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO:31 and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO:37. Numbered embodiment 25 includes the methods of numbered embodiments 1 to 21, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO:32 and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO:38. Numbered embodiment 26 includes the methods of numbered embodiments 1 to 21, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO:33 and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO:39. Numbered embodiment 27 includes the methods of numbered embodiments 1 to 21, wherein the VH comprises an amino acid sequence at least about 90% identical to SEQ ID NO:34, and the VL comprises an amino acid sequence at least about 90% identical to SEQ ID NO:40. Numbered embodiment 28 includes the methods of numbered embodiments 1 to 27, wherein the antibody or antibody fragment comprises an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 41-51. Numbered embodiment 29 includes the methods of numbered embodiments 1 to 28, further comprising performing an assay on the sample to determine a level of a biomarker selected from the group consisting of Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranin, SNAP-25, synaptotagmin, alpha-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. Numbered embodiment 30 includes the methods of numbered embodiments 1 to 29, wherein the sample is selected from the group consisting of a blood sample, a plasma sample, a serum sample, and a cerebrospinal fluid (CSF) sample. Numbered embodiment 31 includes the methods of numbered embodiments 1 to 30, further comprising establishing Alzheimer's disease in the individual based on the detection of phosphorylated tau. Numbered embodiment 32 includes the methods of numbered embodiments 1 to 31, further comprising establishing a prognosis for the individual for developing Alzheimer's disease based on the detection of phosphorylated tau. Numbered embodiment 33 includes the method of numbered embodiment 32, further determining the individual's age, genotype, or expression of the biomarker. Numbered embodiment 34 includes the method of numbered embodiment 33, wherein the biomarker is selected from the group consisting of Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranin, SNAP-25, synaptotagmin, alpha-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. Numbered embodiment 35 includes the methods of numbered embodiments 1 to 34, having at least about 80% specificity for detecting phosphorylated tau. Numbered embodiment 36 includes the methods of numbered embodiments 1 to 34, having at least about 85% specificity for detecting phosphorylated tau. Numbered embodiment 37 includes the methods of numbered embodiments 1 to 34 having at least about 90% specificity for detecting phosphorylated tau. Numbered embodiment 38 includes the methods of numbered embodiments 1 to 37, having at least about 80% sensitivity for detecting phosphorylated tau. Numbered embodiment 39 includes the methods of numbered embodiments 1 to 37, having at least about 85% sensitivity for detecting phosphorylated tau. Numbered embodiment 40 includes the methods of numbered embodiments 1 to 37 having at least about 90% sensitivity for detecting phosphorylated tau. Numbered embodiment 41 includes the methods of numbered embodiments 1 to 40, wherein the method is capable of detecting phosphorylated tau in a sample with a detection limit of at least about 1.0 picograms per milliliter (pg / mL). Numbered embodiment 42 includes the methods of numbered embodiments 1 to 40, wherein the method is capable of detecting phosphorylated tau in a sample with a detection limit of at least about 1.5 picograms per milliliter (pg / mL). Numbered embodiment 43 includes the methods of numbered embodiments 1 to 40, wherein the method is capable of detecting phosphorylated tau in a sample with a detection limit of at least about 5 picograms per milliliter (pg / mL).

[0132] Numbered embodiment 44 includes an anti-tau antibody comprising i) a heavy chain comprising a variable heavy (VH) domain and ii) a light chain comprising a variable light (VL) domain, wherein the VH domain comprises an HCDR1 sequence comprising a sequence selected from SEQ ID NOs: 1-5, an HCDR2 sequence comprising a sequence selected from SEQ ID NOs: 6-9, and an HCDR3 sequence comprising a sequence selected from SEQ ID NOs: 10-13, and the VL domain comprises an LCDR1 sequence comprising a sequence selected from SEQ ID NOs: 14-19, an LCDR2 sequence comprising a sequence selected from SEQ ID NOs: 20-23, and an LCDR3 sequence comprising a sequence selected from SEQ ID NOs: 24-29. Numbered embodiment 45 includes the anti-tau antibody of numbered 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. Numbered embodiment 46 includes the anti-tau antibody of numbered embodiment 44, in which 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. Numbered embodiment 47 includes the anti-tau antibody of numbered embodiment 44, in which 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. Numbered embodiment 48 includes the anti-tau antibody of numbered embodiment 44, in which 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. Numbered embodiment 49 includes the anti-tau antibody of numbered embodiment 44, in which 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. Numbered embodiment 50 includes the anti-tau antibody of numbered embodiment 44, in which 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. Numbered embodiment 51 includes the anti-tau antibody of numbered embodiment 44, wherein the VH domain comprises at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 30-34. Numbered embodiment 52 includes the anti-tau antibody of numbered embodiment 44, wherein the VL domain comprises at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 35-40.

[0133] Numbered embodiment 53 includes the anti-tau antibody of numbered embodiments 44 to 52, which is a chimeric antibody or an antigen-binding fragment thereof. Numbered embodiment 54 includes the anti-tau antibody of numbered embodiments 44 to 53, comprising an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, a sc diabody, a sc diabody-CH3, a triple body, a mini-antibody, a minibody, a TriBi minibody, a scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a Fab', F(ab')2, F(ab')3, F(ab')2-scFv2, a scFv, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a sc diabody-Fc, a diabody-Fc, a tandem scFv-Fc, or an intrabody. Numbered embodiment 55 includes the anti-tau antibody of numbered embodiments 44 to 54, which is an IgG1 antibody. Numbered embodiment 56 includes the anti-tau antibody of numbered embodiments 44 to 55, which is an IgG2 antibody. Numbered embodiment 57 includes the anti-tau antibody of numbered embodiments 44 to 56, which is an IgG4 antibody. Numbered embodiment 58 includes the anti-tau antibody of numbered embodiments 44 to 57, wherein the light chain is a kappa chain. Numbered embodiment 59 includes the anti-tau antibody of numbered embodiments 44 to 58, having a binding affinity for human tau of about 100 pM to about 3 nM. Numbered embodiment 60 includes the anti-tau antibody of numbered embodiments 44 to 59, comprising a VH domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 52-56. Numbered embodiment 61 includes the anti-tau antibodies of numbered embodiments 44 to 60, comprising a VL domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to a sequence selected from SEQ ID NOs: 57-62. Numbered embodiment 62 includes the anti-tau antibody of numbered embodiments 44 to 61, comprising a VH domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 52-56, and a VL domain encoded by a nucleic acid comprising at least 80%, at least 85%, at least 90%, at least 95% sequence identity to a sequence selected from SEQ ID NOs: 57-62. Numbered embodiment 63 includes the anti-tau antibodies of numbered embodiments 44 to 62, comprising a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NOs: 52-56. Numbered embodiment 64 includes the anti-tau antibodies of numbered embodiments 44 to 63, comprising a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NOs: 57-62. Numbered embodiment 65 includes the anti-tau antibodies of numbered embodiments 44 to 64, comprising a VH domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NOs: 52-56 and a VL domain encoded by a nucleic acid comprising a sequence identical to SEQ ID NOs: 57-62. EXAMPLES

[0134] The following examples are provided for the purpose of illustrating various embodiments of the present invention, and are not meant to limit the present invention in any manner. The examples, together with the methods described herein, are currently representative of preferred embodiments, are illustrative, and are not to be construed as limitations on the scope of the present invention. Variations therein and other uses encompassed within the spirit of the present invention as defined by the scope of the claims will occur to those skilled in the art.

[0135] Example 1 Tau antibody screening

[0136] Tau antibodies detecting phosphorylated tau were assayed with Simoa® bead assays using either a 2-step or 3-step protocol assay according to the manufacturer's instructions, see Figure 1.

[0137] The antibodies tested were Antibody 1, Antibody 2, Antibody 3, Antibody 4, Antibody 5 and Antibody 6. The capture results can be seen in Figures 2A-2D. Figure 2A is data from a 2-step protocol assay where all capture antibodies were tested against a tau-12 detector (detects tau at the N-terminus). The data demonstrates that all captures had similar results with improved sensitivity seen with Antibody 6. Figure 2B is data from a 2-step protocol assay where all capture antibodies were tested against a HT7-BT2 detector (detects tau in the central domain region). Both biotinylated antibodies were used. The data shows a roughly 10-fold increase in background compared to the tau-12 detector. No signal was detected at 1000 pg / mL for any capture. Figure 2C is data from a 3-step protocol assay where all capture antibodies were tested against a tau-12 detector. The data demonstrates reduced sensitivity compared to the 2-step protocol and improved sensitivity seen with Antibody 6. FIG. 2D shows data from a 3-step protocol assay in which all capture antibodies were tested against the HT7-BT2 detector. Both biotinylated antibodies were used. The data show a 10-fold increase in background compared to the Tau-12 detector. Based on these results, the 2-step protocol was then further optimized for sensitivity.

[0138] Example 2 Pharmacokinetics of Tau Antibodies

[0139] The antibodies were tested for their pharmacokinetic profile.

[0140] Antigen information for the antibodies can be found in Table 10. [Table 10]

[0141] Antibodies were generated and purified. Antibodies were assayed using a standard indirect ELISA protocol. Briefly, peptide antigens corresponding to SEQ ID NOs: 74-81 were diluted to 1ug / ml in PBS and plated on Greiner Bio One Microlon 96-well plates. Peptide antigens were produced 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 are indicated by (pT) for phosphorylated threonine or (pS) for phosphorylated serine. After blocking with 1% BSA in PBS pH 7.4, antibodies were serially diluted 1:4 at an initial concentration of 1ug / ml. After incubation, unbound antibody was washed away with 1xTBST and HRP-labeled goat anti-rabbit secondary antibody was applied according to the manufacturer's instructions. Subsequently, unbound secondary antibody was washed away with 1xTBST and 3,3'5,5'-tetramethylbenzidine (TMB) was applied for 5 minutes at room temperature and the plate was read at 650 nm. The data can be seen in Figure 3, which shows the screening data of different monoclonal antibodies against varying peptide concentrations.

[0142] Example 3 Tau antibodies for immunohistochemistry

[0143] The tau antibodies described herein were tested in immunohistochemistry assays.

[0144] Briefly, all antibodies were optimized using a range of concentrations (0.01-3.00ug / ml) and stained using a Leica Bond RX automated IHC platform: ER1 antigen retrieval (sodium citrate, pH6), 20 minutes at 100°C; primary antibody, 15 minutes at RT; IVD grade Leica Polymer Refine HRP detection, 8 minutes at room temperature; DAB chromogen, 10 minutes at room temperature, and finally, hematoxylin counterstain, 5 minutes at room temperature. Antibodies that passed basic IHC staining were advanced to undergo IHC staining after alkaline phosphatase (AP) treatment (200U / ml, 60 minutes at 37°C). Vehicle only controls (buffer without AP) were also used. Positive antigen control tissues were FFPE normal human cerebral cortex, and cerebral cortex from an Alzheimer's patient. Negative antigen control tissues were FFPE normal human liver, skeletal muscle, and cardiac muscle. All tissues were sequenced into tissue microarrays to simplify the IHC staining process. A negative reagent (detection system only) control was used and shown to be negative. Benchmark antibodies stained with the test antibodies were rabbit monoclonal against tau [EPR22524-95] (ab254256, Abcam plc) and rabbit monoclonal against tau (phospho-S214) [EPR1884(2)] (ab170892, Abcam plc).

[0145] The benchmark antibodies demonstrated that the antibodies showed negative staining in negative control tissues and positive staining in positive control tissues (data not shown). Data for the tau antibodies can be seen in Figures 4A-4G (Antibody 6), Figures 5A-5G (Antibody 5) and Figures 6A-6G (Antibody 2). Antibody 5 and Antibody 6 demonstrated similar data to the benchmark antibodies in that negative staining was observed in negative control tissues including normal heart, normal liver, normal skeletal muscle and normal cerebral cortex, and positive staining was observed in positive control tissues including Alzheimer's cerebral cortex. Antibodies 1-4 did not show similar data to the benchmark antibodies.

[0146] Example 4 Detection of tau peptides using tau antibodies

[0147] The tau antibodies described herein that detect phosphorylated tau were tested in an ELISA assay. The tau antibodies were first tested for pTau217 reactivity by indirect ELISA. Figure 7 shows a diagram of the indirect ELISA assay format used. Briefly, streptavidin beads were bound to a plate, and a biotinylated peptide was added to the plate under conditions that allow biotin-streptavidin binding. The biotinylated peptide was a synthetic peptide that contained a portion of tau and had a threonine residue phosphorylated at position 217 (pT217). This was the target peptide. After binding of the synthetic peptide to the plate by the formation of a biotin-streptavidin complex, a tau antibody was added to the plate under conditions that allow antibody-target peptide binding. After binding, the plate was washed to remove any unbound antibody, and then a secondary antibody or tracer antibody against the species from which the tau antibody was derived (either 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, and then the plate was added with TMB ELISA peroxidase chromogenic substrate (3,3',5,5'-tetramethylbenzidine) to visualize antibody reactivity in indirect ELISA experiments. Antibody sample binding was quantified using an ELISA microplate reader.

[0148] As shown in Figure 7, five antibodies were tested for their ability to detect phosphorylated tau 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 (pTau217). PT3 corresponds to a mouse anti-phospho(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. All five antibodies were assayed for reactivity against pTau217 in two separate ELISA instruments at the following concentrations: 10 per plate; -2 , 10 -1 , 10 -0 , 10 1 , 10 2 , 10 3 and 10 4 Both IBA493 mAb and PT3 demonstrated robust concentration-dependent levels of reactivity to pTau217 as seen in the Bio-pt655 (phospho-T217) and Bio-pt660 (phospho-T217) graphs. Antibody 2 was administered at 10 4 Antibody 5 demonstrated more modest concentration-dependent levels of reactivity to pTau217, evident in ng / mL. Antibody 5 and Antibody 6 demonstrated no reactivity to pTau217 in these assays. The graph in FIG. 7 showing the results of this ELISA assay using the five test antibodies against phosphatase-treated pTau demonstrated the specificity of antibodies IBA493 mAb, PT3 and Antibody 2 in detecting phosphorylated tau.

[0149] The tau antibodies described herein that detect phosphorylated tau were tested in a Simoa®-based assay. Figures 8-24 show the results of a Simoa® assay designed for sensitive testing of tau reactivity to the antibodies described herein. In some embodiments, a Simoa®-based assay can be approximately 1000 times more sensitive in detecting a given analyte when compared to detection of the same analyte in an indirect ELISA assay. This increased sensitivity of a Simoa®-based assay allows for the development and use of biomarkers that were previously unable to generate a detectable signal using traditional assays, e.g., indirect ELISA. The increased sensitivity of a Simoa®-based assay compared to a conventional immunoassay, e.g., indirect ELISA, is due to the fact that the Simoa® method is capable of detecting a single target molecule, whereas a conventional immunoassay typically requires large reaction volumes and millions of antibody-conjugated enzymes reacted with millions of fluorophores or chromogenic substrates before an optical signal can be detected. For Simoa®-based assays, the average enzyme / bead (AEB) represents the raw signal output.

[0150] In FIG. 8, antibody 2 capable of detecting p-tau217, as described herein, was used in a Simoa®-based assay to detect the level of analyte per plasma sample from an individual. The signal-to-noise (S / N) ratio was determined by Simoa® for each sample and plotted on a graph. 120 plasma samples were obtained and assayed from an individual. The graphed S / N ratios showed that all samples tested, except one, produced signals within the expected concentration range. When the plasma samples were diluted 1:3 and then assayed again, only three of the 120 samples produced results below the blank control measurements, and only five samples recorded S / N measurements of 1.5, which was determined to be the limit of detection (LOD). In FIG. 8, calculations were performed with each of the 120 plasma samples assayed to determine the coefficient of variation (CV%) for each sample, and the results were graphed against the measured concentrations. Ten of the 120 samples yielded a CV% greater than 20, and from this analysis, the estimated analytical lower limit of quantification (LLOQ) was determined to be 0.08 pg / mL. This LLOQ value represents the lowest amount of analyte (tau phosphorylated at T217) that could be quantitatively determined with an acceptable level of precision. These results in Figure 8 demonstrated the sensitivity of the Simoa® method to detect tau phosphorylated at T217 using Antibody 2.

[0151] In Figure 9, a calibration curve was generated for the Simoa® p Tau-217 assay and graphed using 68 CSF samples and 120 plasma samples separated onto four plates using Antibody 2 and four plates using ADx Neuroscience antibody ADx204 [AEB vs log(CAL) pg / mL]. In another graph from this assay run on a separate instrument, AEB was plotted on a logarithmic scale [AEB vs log(CAL) pg / mL] to demonstrate that a fit of the data can allow for accurate analyte quantification calculations when measuring samples.

[0152] In Figure 10, 38 paired CSF and EDTA plasma samples were measured using the Simoa® pTau-217 assay using antibody 2. This assay is also called ALZpath Dx. The results are graphed and the samples are shown with their clinical diagnosis (non-AD, indeterminate or AD). These results and their statistical analysis showed a strong correlation between CSF pTau levels and plasma pTau levels when measured using the Simoa® pTau-217 assay using antibody 2 (R value of approximately 0.7 and P value for two-tailed T-test <0.0001 between non-AD and AD).

[0153] In Figure 11, 42 CSF samples were measured using the Simoa® pTau-217 assay using Antibody 2 and the Simoa® pTau-181 assay using the pTau-181 antibody from Quanterix® (Quanterix® Corp., product number 103714) and plotted against each other. This demonstrated that the Simoa® pTau-217 assay using Antibody 2 showed the expected association with an analyte involved in AD (pTau-181) detected in CSF. Statistical analysis showed an R value of approximately 0.8 and a P value of <0.0001 for a two-tailed T-test.

[0154] In Figure 12, 42 CSF samples were measured using the Simoa® pTau-217 assay using Antibody 2 and the Simoa® pTau assay using the Innotest pTau-181 antibody and plotted against each other. This demonstrated that the Simoa® pTau-217 assay using Antibody 2 showed the expected association with an analyte involved in AD (pTau) detected in CSF. Statistical analysis showed an R value of approximately 0.77 and a P value of <0.0001 for a two-tailed T-test.

[0155] In Figure 13, 42 CSF samples were measured using the Simoa® HD-X assay using Antibody 2 as the capture antibody, ADx204 antibody as the detector, and peptides as calibrators. This demonstrated that the Simoa® assay using known AD biomarkers showed the expected association. Statistical analysis showed an R value of approximately 0.9 and a P value of <0.0001 for a two-tailed T-test.

[0156] In FIG. 14, CSF and plasma samples were measured using the Simoa® pTau-217 assay using Antibody 2 and graphed in separate graphs. A clinical diagnosis of AD or a control without an AD diagnosis was used as the classifier for each sample. Analysis of the graphed results showed significant differences between samples from individuals with a clinical AD diagnosis versus controls for both CSF and plasma samples. Area under the curve (AUC) calculations were 0.94 for CSF samples and 0.86 for plasma samples. These results showed that the Simoa® pTau-217 assay using Antibody 2 was able to distinguish AD cases in CSF and plasma.

[0157] In Figure 15, four EDTA plasma samples with high pTau levels (denoted QC_L1, QC_L2, QC_M and QC_H), which serve as quality controls, were measured in duplicate using the Simoa® pTau-217 assay using Antibody 2 and pTau levels were calculated. Results from replicate runs are plotted to demonstrate the precision and reproducibility of the Simoa® pTau-217 assay using Antibody 2.

[0158] In Figure 16, the control sample and additional measured samples from Figure 15 were measured using the Simoa® pTau-217 assay using Antibody 2 and plotted in two separate experiments to generate a precision profile. The precision profile is based on the measured sample concentrations and the inter-run CV% of the four QC samples. From this experiment, the functional LLOQ of pTau-217 in this assay was determined to be 0.26 pg / mL.

[0159] In FIG. 17, parallelism was evaluated with the Simoa® pTau-217 assay using antibody 2. The determination of parallelism is also important in that it indicates whether the signal is specific. Parallelism determines whether actual samples containing high endogenous analyte concentrations provide the same degree of detection in the assay in the standard curve after dilution. This may indicate differences in antibody binding affinity to the endogenous analyte and the standard or calibration analyte. This may ensure that the recombinant standard parallels the natural recognition of the endogenous analyte. In this experiment, four plasma samples, each from a different donor, with a relatively high concentration of detected pTau-217, and the spiked dilution buffer (sample 5), were diluted by a factor of 2 in five steps, starting with a dilution of 3×. The concentration fell below the LLOD for all four plasma samples from a dilution factor of 12× onwards. In a graph of log(measured pg / mL) vs. log[dilution factor (DF)], the plasma measurements vs. spiked measurements demonstrated linearity in detection along the various dilutions. Three of the four plasma samples were determined to fall within the acceptable range of parallelism, with sample 4 falling just outside the acceptable range. The acceptable range of parallelism is <15%. These results demonstrated that the Simoa® pTau-217 assay using antibody 2 on plasma samples produced consistent and accurate calculations of pTau-217 levels across a range of concentrations, thus demonstrating its utility as a biomarker assay.

[0160] In Figures 18-19, dilution linearity using the Simoa® pTau-217 assay using antibody 2 was performed to demonstrate that samples with spike concentrations at approximately the upper limit of quantification (ULOQ) could be diluted to concentrations within the working range while still producing reliable assay results. In Figure 18, three spiked samples (s1, s2, and s3) and a calibration sample were assayed and plotted as log(measured pg / mL) vs. log(DF) to determine dilution linearity. In Figure 19, three spiked samples (s1, s2, and s3) and a calibration sample were assayed and plotted as log(measured pg / mL) vs. log(DF) to determine dilution linearity, with the highest spike points excluded from s1, s2, and s3 as they were outside the calibration range of 50 pg / mL.

[0161] In Figure 20, Simoa® pTau-217 assay using antibody 2 was used to evaluate samples obtained from the forgetfulness clinic cohort. Plasma samples were measured and graphed for calculated pTau-217 concentration. Clinical diagnosis of AD was used as the classifier. AUC was calculated at 0.916, which indicates the success of Simoa® pTau-217 assay using antibody 2 in distinguishing AD+ from AD- in this cohort. Figure 20 also plots the receiver operating characteristic (ROC) curve to show the diagnostic ability of this binary classifier (AD+ or AD-) system, since it is possible to vary the discrimination threshold between the classifiers.

[0162] In Figures 21-22, the clinical performance of the Simoa® p Tau-217 assay using Antibody 2 was compared to the Simoa® p Tau-181 assay using Antibody P-Tau 181 - Quanterix®. In Figure 21, Antibody 2 was able to distinguish between assayed plasma samples obtained from AD dementia individuals vs. controls (1.3e for Antibody 2). -12In FIG. 22, the commercially available P-tau181-Quanterix® Simoa® assay (Quanterix® Corp., product number 103714) was also able to distinguish assayed plasma samples obtained from AD dementia individuals versus controls (9.6e -08 P values ​​for the individual samples from which the data from Figures 21-22 were derived are listed in Figure 22.

[0163] In Figure 23, precision plots were generated for the Simoa® assay using P-Tau217 Antibody 2 and P-Tau181 - Quanterix® Antibody (Quanterix® Corp., Product No. 103714). The calculated LLODs were 0.55 pg / mL and 0.24 pg / mL, respectively. Concentrations were not back-calculated and therefore LLOD values ​​were not back-calculated.

[0164] In FIG. 24, ROC curves were plotted for P-tau 181 - Quanterix® antibody (Quanterix® Corp., product number 103714) and P-tau 217 antibody 2. Analysis of the data showed that P-tau 217 antibody 2 showed superior sensitivity and specificity in distinguishing AD dementia from controls compared to the Simoa® assay using P-tau 181 - Quanterix® antibody. The diagnostic accuracy of antibody 2 for AD dementia in this Simoa® method is 92.5% when tested on plasma samples. The diagnostic specificity of antibody 2 for AD dementia in this Simoa® method is 85% when tested on plasma samples.

[0165] In Figure 25, a schematic diagram of a tau polypeptide is shown showing the relative positions of various protein domains and the positions of threonine residues that can be assayed for phosphorylation status using the methods disclosed herein. The position of pT217 within the P2 domain of tau is shown. pT181 is within the P1 domain, and pT231 is near the border between the P2 and R1 domains.

[0166] In Figure 26, various tau antibodies were assayed using an indirect ELISA to graph the degree of reactivity to tau fragments with non-phosphorylated T217 (Bio-pt654) and full-length tau (tau441). IBA493 mAB and PT3 showed concentration-dependent reactivity to both Bio-pt654 and tau441. Antibodies 2, 5 and 6 described herein showed no reactivity to either Bio-pt654 or tau441 in this assay, demonstrating the accuracy and specificity of antibodies 2, 5 and 6 in detecting pTau-217.

[0167] In FIG. 27, various tau antibodies were assayed using indirect ELISA to graph the degree of reactivity to tau fragments with phosphorylated T181 (Bio-pt126) and phosphorylated T231 (Bio-pt146). IBA493 mAB and antibody 2 described herein showed concentration-dependent reactivity to Bio-pt126. IBA493 mAB was the only antibody tested that showed concentration-dependent reactivity to Bio-pt146. This demonstrates that IBA493 mAB, PT3, and antibody 2 described herein were each distinguishable based on which analyte each antibody interacted with via indirect ELISA. IBA493 mAB interacts with pTau-217, non-phospho T217, full-length tau, pTau-181, and pTau-231. PT3 interacts with pTau-217, non-phospho T217, and full-length tau. Antibody 2 interacts with pTau-217 and pTau-181. It was also shown that the interaction of IBA493 mAB with non-phospho T217 was much lower than that of PT3 with non-phospho T217.

[0168] In FIG. 28, a diagram of an assay using Antibody 2 to detect capture of specific tau peptides is shown. In this assay, Antibody 2 is bound to a plate and sample wells from the plate are subjected to various biotinylated peptides under conditions conducive to the formation of specific antibody-ligand interactions. The samples are then washed to remove excess unbound biotinylated peptides. Peroxidase-conjugated streptavidin beads are then added to the samples to form biotin-streptavidin complexes on the peptide-bound antibodies. TMB is then added to the samples. redSubstrate is added and samples are measured for colorimetric color development using an ELISA plate reader. Results are graphed and show that Adx-pt655 produced specific dose-dependent reactivity among the various pTau-217, pTau-231 and pTau-181 peptides tested. These results show the specificity of antibody 2 for a specific feature of pTau (i.e., tau phosphorylated at threonine 217). Antibody 5 and antibody 6, tested by indirect ELISA under the same conditions using the same tau peptide, did not produce specific dose-dependent reactivity to the tested tau peptide.

[0169] Various p-tau-217 antibodies corresponding to antibody 1, antibody 2, antibody 3, antibody 4, antibody 5 and antibody 5 described herein were also evaluated as capture antibodies on the Mesoscale Discovery technology platform, either directly coated on plates or on streptavidin-coated plates. This system uses non-radioactive electrochemiluminescent labels, thereby conferring significant advantages over traditional ELISA assays. These advantages include lower background signal, improved sensitivity and dynamic range of detection.

[0170] In Figure 29, Western blots were used to evaluate the binding of various antibodies to brain lysate samples from AD patients and control subjects. In the five Western blots shown, samples are loaded according to the same sample key as shown in Figure 29. Protein ladders were run in lanes 1 and 10. Phosphatase-treated p-tau, loaded at 0.05ug, was run in lane 2. Full-length tau (tau 411), loaded at 0.05ug, was run in lane 3. Lanes 4-6 contain samples from different control subjects at a dilution factor of 5. Lanes 7-9 contain samples from different AD subjects at a dilution factor of 5. These results showed that both IBA394 mAb and PT3 bound to and immunoprecipitated different length isoforms of tau in control and AD samples, and immunoprecipitated significantly more tau in AD samples, but did not interact with synthetic full-length tau or phosphatase-treated p-tau. Antibody 2 (30H2L10) bound to and immunoprecipitated different length isoforms of tau in AD samples but did not immunoprecipitate significant amounts of tau in samples from control individuals. Antibodies 5 and 6 did not produce a detectable Western blot signal in this assay.

[0171] While preferred embodiments of the present 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. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be used in carrying out the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

**Claim 1**: A method for detecting phosphorylated tau in a sample derived from an individual, comprising the step of performing an immunoassay on the sample using an antibody or antibody fragment comprising a variable domain heavy chain region (VH) and a variable domain light chain region (VL), wherein the VH comprises an amino acid sequence that is at least about 90% identical to the sequence shown in any one of SEQ ID NOs: 30 to 34, and the VL comprises an amino acid sequence that is at least about 90% identical to the sequence shown in any one of SEQ ID NOs: 35 to 40; wherein the VH comprises an HCDR1 sequence, an HCDR2 sequence, and an HCDR3 sequence, and the VL comprises an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, (a) 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; (b) 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; (c) 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; (d) 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; (e) 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; or (f) a method 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. **Claim 2**: The method according to claim 1, 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. **Claim 3**: The method according to claim 1, wherein the VH comprises SEQ ID NO: 31 and the VL comprises SEQ ID NO:

36. **Claim 4**: The method according to claim 1, wherein the heavy chain comprises SEQ ID NO: 43 and the light chain comprises SEQ ID NO:

44. **Claim 5**: The method according to claim 1, 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. **Claim 6**: The method according to claim 1, wherein the VH comprises SEQ ID NO: 33 and the VL comprises SEQ ID NO:

39. **Claim 7**: The method according to claim 1, wherein the heavy chain comprises SEQ ID NO: 48 and the light chain comprises SEQ ID NO:

49. **Claim 8**: The method according to claim 1, 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. **Claim 9**: The method according to claim 1, wherein the VH comprises SEQ ID NO: 34 and the VL comprises SEQ ID NO:

40. **Claim 10**: The method according to claim 1, wherein the heavy chain comprises SEQ ID NO: 50 and the light chain comprises SEQ ID NO:

51. **Claim 11**: The method according to claim 1, wherein the phosphorylated tau is selected from the group consisting of p-tau-181, p-tau-212, p-tau-217, p-tau-231, p-tau-214, and p-tau-220. The method according to claim 1, wherein the antibody or antibody fragment is a monoclonal antibody, or a chimeric antibody or antibody fragment thereof. The method according to claim 1, wherein the antibody or antibody fragment is a rabbit monoclonal antibody. The method according to claim 1, wherein the antibody or antibody fragment comprises an IgG1 constant region. The method according to claim 1, wherein the sample is selected from the group consisting of a blood sample, a plasma sample, a serum sample, and a cerebrospinal fluid (CSF) sample. The method according to claim 1, further comprising detecting the antibody or antibody fragment bound to phosphorylated tau, thereby detecting phosphorylated tau in the sample; wherein the detected phosphorylated tau is for use in demonstrating Alzheimer's disease in the individual based on the detected phosphorylated tau, or for use in demonstrating a prognosis prediction of the individual for the onset of Alzheimer's disease. The method according to claim 1, further comprising determining the age, genotype, or biomarker expression of the individual. The method according to claim 1, further comprising determining the age, genotype, or biomarker expression of the individual, wherein the biomarker is selected from the group consisting of Aβ42, Aβ40, Aβ38, BACE1, hFABP, TREM2, YKL-40, IP-10, neurogranin, SNAP-25, synaptotagmin, alpha-synuclein, TDP-43, ferritin, VILIP-1, NfL, GFAP, and combinations thereof. The method according to claim 1, wherein the method has at least about 85% specificity or at least about 85% sensitivity for discriminating Alzheimer's disease in the individual from a normal subject based on the detected phosphorylated tau. The method according to claim 1, wherein the method is performed in vitro.

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