Methods for using antibodies that recognize tau

Abstract

The present invention provides a method for treating tauopathies (such as Alzheimer's disease) using an antibody that binds to human tau. The antibody inhibits or slows the progression of tau-related pathology and associated symptoms. In one aspect, the present invention provides a method for reducing cellular tau internalization in a subject, comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces cellular tau internalization, the antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 149,359, filed February 14, 2021, which is incorporated by reference in its entirety for all purposes. This application is related to U.S. Provisional Application No. 16 / 808,209, filed March 3, 2020, which claims priority to U.S. Provisional Application No. 62 / 813,126, filed March 3, 2019, U.S. Provisional Application No. 62 / 813,137, filed March 3, 2019, and U.S. Provisional Application No. 62 / 838,159, filed April 24, 2019, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing Reference This application contains an electronic sequence listing in file named 574677SEQLST.TXT, created on Feb. 11, 2022, and containing 168,698 bytes, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0003] Tau is a well-known human protein that can exist in phosphorylated forms (see, e.g., Goedert, Proc. Natl. Acad. Sci. USA 85:4051-4055(1988); Goedert, EMBO J. 8:393-399(1989); Lee, Neuron 2:1615-1624(1989); Goedert, Neuron 3:519-526(1989); Andreadis, Biochemistry 31:10626-10633(1992)). Tau has been reported to play a role in stabilizing microtubules, particularly those in the central nervous system. It has been reported that t-tau (total tau, i.e., phosphorylated and non-phosphorylated forms), as well as phospho-tau (p-tau, i.e., phosphorylated tau), are released by the brain in response to neuronal injury and neurodegeneration and appear at elevated levels in the CSF of Alzheimer's patients compared to the general population (Jack et al., Lancet Neurol 9: 119-28 (2010)).

[0004] Tau is the main component of neurofibrillary tangles, which, together with plaques, are a hallmark of Alzheimer's disease. These tangles consist of abnormal fibrils measuring 10 nm in diameter that are in the form of paired coiled helices with a regular period of 80 nm. Tau in neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated), with phosphate groups attached to specific sites on the molecule. In Alzheimer's disease, significant involvement of neurofibrillary tangles is found in neurons of layer II of the entorhinal cortex, the CA1 and subiculum regions of the hippocampus, the amygdala, and in deeper layers of the neocortex (layers III, V, and superficial layer VI). Hyperphosphorylated tau has also been reported to interfere with microtubule assembly, which may promote the collapse of neuronal networks. Tau inclusions are part of the defining neuropathology of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and Pick's disease. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Goedert, Proc. Natl. Acad. Sci. USA 85:4051-4055(1988) [Non-Patent Document 2] Goedert, EMBO J. 8:393-399 (1989) [Non-Patent Document 3] Lee, Neuron 2:1615-1624(1989) [Non-Patent Document 4] Goedert, Neuron 3:519-526(1989) [Non-Patent Document 5] Andreadis, Biochemistry 31:10626-10633(1992) [Non-Patent Document 6] Jack et al., Lancet Neurol 9: 119-28 (2010) Summary of the Invention

[0006] In one aspect, the invention provides a method of reducing cellular internalization of tau in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces cellular internalization of tau, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0007] In another aspect, the invention provides a method of reducing tau-induced toxicity in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces tau-induced toxicity, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0008] In another aspect, the invention provides a method of reducing or delaying the onset of a behavioral disorder in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that results in a reduction or delay in the onset of the behavioral disorder, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0009] In another aspect, the invention provides a method of reducing the level of a marker of tau pathology in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces the level of a marker of tau pathology, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0010] In another aspect, the invention provides a method of reducing the development of tau pathology in a subject, the method comprising administering to a subject in need thereof a tau pathology-reducing amount of an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0011] In some methods, the subject has a pathological hallmark of Alzheimer's disease. In some methods, the subject has Alzheimer's disease.

[0012] In some methods, the CDR-L2 of the antibody or antigen-binding fragment comprises SEQ ID NO: 13. In some methods, the CDR-L2 of the antibody or antigen-binding fragment comprises SEQ ID NO: 168.

[0013] In some methods, the heavy chain variable region of the antibody or antigen-binding fragment comprises the mature heavy chain variable region of SEQ ID NO: 18, and the light chain variable region of the antibody or antigen-binding fragment comprises the mature light chain variable region of SEQ ID NO: 122. In some methods, the antibody or antigen-binding fragment is a humanized version of a murine antibody characterized by the mature heavy chain variable region of SEQ ID NO: 7 and the mature light chain variable region of SEQ ID NO: 11.

[0014] In some methods, the antibody comprises a light chain comprising a mature light chain variable region fused to a light chain constant region, and a heavy chain comprising a mature heavy chain variable region fused to a heavy chain constant region.

[0015] In some methods, the heavy chain constant region of the antibody comprises the amino acid sequence of SEQ ID NO: 176, with or without the C-terminal lysine. In some methods, the mature heavy chain variable region fused to the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 178, with or without the C-terminal lysine.

[0016] In some methods, the antibody further comprises a signal peptide fused to the mature heavy chain variable region and / or the mature light chain variable region. In some methods, the heavy chain comprises the amino acid sequence of SEQ ID NO: 180, with or without the C-terminal lysine.

[0017] In some methods, the light chain constant region of the antibody comprises the amino acid sequence of SEQ ID NO: 177. In some methods, the mature light chain variable region fused to the light chain constant region comprises the amino acid sequence of SEQ ID NO: 179. In some methods, the light chain comprises the amino acid sequence of SEQ ID NO: 181.

[0005] In some methods, the heavy chain comprises the amino acid sequence of SEQ ID NO: 178, with or without the C-terminal lysine, and the light chain comprises the amino acid sequence of SEQ ID NO: 179. In some methods, the heavy chain comprises the amino acid sequence of SEQ ID NO: 180, with or without the C-terminal lysine, and the light chain comprises the amino acid sequence of SEQ ID NO: 181.

[0018] In some methods, the antibody comprises at least one mutation in the constant region. In some methods, the antibody comprises at least one mutation in the constant region that reduces complement binding or activation by the constant region or reduces binding to an Fcγ receptor compared to a native human heavy chain constant region. In some methods, the antibody comprises a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 according to EU numbering, or comprises alanines at positions 318, 320, and 322. [Brief description of the drawings]

[0019] [Figure 1] The results of a tau internalization assay for mouse 3D6 and hu3D6VHv1bA11 / L2-DIM4 are shown.

[0020] [Diagram 2]A and B show that mouse 3D6 disrupts tau seeding in an in vivo disease model of Alzheimer's disease.

[0021] [Diagram 3] We show that treatment with mouse 3D6 reduces pathological tau and ameliorates behavioral deficits in a transgenic tau model.

[0022] [Figure 4] 1 shows that mouse 3D6 protects mouse primary cortical neurons from tau-induced toxicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] A brief description of the sequence SEQ ID NO:1 represents the amino acid sequence of a human tau isoform (Swiss-Prot P10636-8).

[0024] SEQ ID NO:2 represents the amino acid sequence of a human tau isoform (Swiss-Prot P10636-7).

[0025] SEQ ID NO:3 represents the amino acid sequence of a human tau isoform (Swiss-Prot P10636-6), (4R0N human tau).

[0026] SEQ ID NO: 4 represents the amino acid sequence of a human tau isoform (Swiss-Prot P10636-5).

[0027] SEQ ID NO:5 represents the amino acid sequence of a human tau isoform (Swiss-Prot P10636-4).

[0028] SEQ ID NO:6 represents the amino acid sequence of a human tau isoform (Swiss-Prot P10636-2).

[0029] SEQ ID NO:7 represents the amino acid sequence of the heavy chain variable region of the murine 3D6 antibody.

[0030] SEQ ID NO:8 represents the amino acid sequence of the Kabat / Chothia composite CDR-H1 of the murine 3D6 antibody.

[0031] SEQ ID NO: 9 represents the amino acid sequence of the Kabat CDR-H2 of the murine 3D6 antibody.

[0032] SEQ ID NO: 10 represents the amino acid sequence of the Kabat CDR-H3 of the murine 3D6 antibody.

[0033] SEQ ID NO: 11 represents the amino acid sequence of the light chain variable region of the murine 3D6 antibody and the murine 6A10 antibody.

[0034] SEQ ID NO: 12 represents the amino acid sequence of the Kabat CDR-L1 of the murine 3D6 antibody and the murine 6A10 antibody.

[0035] SEQ ID NO: 13 represents the amino acid sequence of the Kabat CDR-L2 of the murine 3D6 antibody and the murine 6A10 antibody.

[0036] SEQ ID NO: 14 represents the amino acid sequence of the Kabat CDR-L3 of the murine 3D6 antibody and the murine 6A10 antibody.

[0037] SEQ ID NO: 15 represents the amino acid sequence of the heavy chain variable region hu3D6VHv1 of the humanized 3D6 antibody.

[0038] SEQ ID NO: 16 represents the amino acid sequence of the heavy chain variable region hu3D6VHv2 of the humanized 3D6 antibody.

[0039] SEQ ID NO: 17 represents the amino acid sequence of the heavy chain variable region hu3D6VHv1b of the humanized 3D6 antibody.

[0040] SEQ ID NO: 18 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv1bA11.

[0041] SEQ ID NO: 19 represents the amino acid sequence of the heavy chain variable region hu3D6VHv5 of the humanized 3D6 antibody.

[0042] SEQ ID NO: 20 represents the amino acid sequence of the light chain variable region hu3D6VLv1 of the humanized 3D6 antibody.

[0043] SEQ ID NO: 21 represents the amino acid sequence of the light chain variable region hu3D6VLv2 of the humanized 3D6 antibody.

[0044] SEQ ID NO: 22 represents the amino acid sequence of the light chain variable region hu3D6VLv3 of the humanized 3D6 antibody.

[0045] SEQ ID NO: 23 represents the amino acid sequence of the light chain variable region of the humanized 3D6 antibody, hu3D6VLv4.

[0046] SEQ ID NO:24 represents the amino acid sequence of the heavy chain variable acceptor Acc.#BAC01986.1.

[0047] SEQ ID NO: 25 is the heavy chain variable acceptor Acc.#IMGT#IGHV1-69-2 * This represents the amino acid sequence of 01.

[0048] SEQ ID NO: 26 is the heavy chain variable acceptor Acc.#IMGT#IGKJ1 * This represents the amino acid sequence of 01.

[0049] SEQ ID NO: 27 is the light chain variable acceptor Acc.#IMGT#IGKV2-30 * This represents the amino acid sequence of 02.

[0050] SEQ ID NO: 28 is the light chain variable acceptor Acc.#IMGT#IGKJ2 * This represents the amino acid sequence of 01.

[0051] SEQ ID NO:29 represents the amino acid sequence of the light chain variable acceptor Acc.#AAZ09048.1.

[0052] SEQ ID NO:30 represents the nucleic acid sequence encoding the heavy chain variable region of the murine 3D6 antibody.

[0053] SEQ ID NO:31 represents the nucleic acid sequence encoding the light chain variable region of the murine 3D6 antibody.

[0054] SEQ ID NO: 32 represents the amino acid sequence of the Kabat CDR-H1 of the murine 3D6 antibody.

[0055] SEQ ID NO: 33 represents the amino acid sequence of the Chothia CDR-H1 of the murine 3D6 antibody.

[0056] SEQ ID NO: 34 represents the amino acid sequence of the Chothia CDR-H2 of the murine 3D6 antibody.

[0057] SEQ ID NO: 35 represents the amino acid sequence of AbM CDR-H2 of the murine 3D6 antibody.

[0058] SEQ ID NO: 36 represents the amino acid sequence of Contact CDR-L1 of mouse 3D6 antibody.

[0059] SEQ ID NO: 37 represents the amino acid sequence of Contact CDR-L2 of mouse 3D6 antibody.

[0060] SEQ ID NO: 38 represents the amino acid sequence of Contact CDR-L3 of mouse 3D6 antibody.

[0061] SEQ ID NO: 39 represents the amino acid sequence of Contact CDR-H1 of mouse 3D6 antibody.

[0062] SEQ ID NO: 40 represents the amino acid sequence of Contact CDR-H2 of mouse 3D6 antibody.

[0063] SEQ ID NO: 41 represents the amino acid sequence of Contact CDR-H3 of mouse 3D6 antibody.

[0064] SEQ ID NO: 42 represents the amino acid sequence of an alternative Kabat-Chothia composite CDR-H1 of the humanized 3D6 antibody (such as that found in hu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3, hu3D6VHv1e, and hu3D6VHv1f).

[0065] SEQ ID NO: 43 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHv5 and hu3D6VHv1bA11B6H3).

[0066] SEQ ID NO: 44 represents the consensus amino acid sequence among the heavy chain variable regions (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) of murine 3D6 and selected humanized 3D6 antibodies (represented as "Majority" in Figure 2 of PCT / IB2017 / 052544).

[0067] SEQ ID NO: 45 shows the consensus amino acid sequence between the light chain variable region of mouse 3D6 and the light chain variable region of selected humanized 3D6 antibodies (represented as "Majority" in Figure 3 of PCT / IB2017 / 052544).

[0068] SEQ ID NO: 46 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv1bA11B6G2.

[0069] SEQ ID NO: 47 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv1bA11B6H3.

[0070] SEQ ID NO: 48 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv1c.

[0071] SEQ ID NO: 49 represents the amino acid sequence of the heavy chain variable region hu3D6VHv1d of the humanized 3D6 antibody.

[0072] SEQ ID NO: 50 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv1e.

[0073] SEQ ID NO: 51 represents the amino acid sequence of the heavy chain variable region hu3D6VHv1f of the humanized 3D6 antibody.

[0074] SEQ ID NO: 52 represents the amino acid sequence of the heavy chain variable region hu3D6VHv3 of the humanized 3D6 antibody.

[0075] SEQ ID NO: 53 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv3b.

[0076] SEQ ID NO: 54 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv3c.

[0077] SEQ ID NO: 55 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv4.

[0078] SEQ ID NO: 56 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv4b.

[0079] SEQ ID NO: 57 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHv4c.

[0080] SEQ ID NO:58 represents the amino acid sequence of an alternative Kabat-Chothia composite CDR-H1 of the humanized 3D6 antibody (such as found in hu3D6VH1c).

[0081] SEQ ID NO:59 represents the amino acid sequence of an alternative Kabat-Chothia composite CDR-H1 of the humanized 3D6 antibody (such as found in hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c).

[0082] SEQ ID NO:60 represents the amino acid sequence of an alternative Kabat-Chothia composite CDR-H1 of the humanized 3D6 antibody (such as found in hu3D6VHv3b and hu3D6VHv4b).

[0083] SEQ ID NO:61 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHv1bA11B6G2).

[0084] SEQ ID NO:62 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as that found in hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b).

[0085] SEQ ID NO: 63 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c).

[0086] SEQ ID NO:64 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHv1e).

[0087] SEQ ID NO:65 represents the amino acid sequence of an alternative Kabat CDR-H3 of the humanized 3D6 antibody (such as found in hu3D6VHv1f).

[0088] SEQ ID NO: 66 represents the amino acid sequence of the heavy chain variable region of the murine 6A10 antibody.

[0089] SEQ ID NO: 67 represents the amino acid sequence of the combined Kabat / Chothia CDR-H1 of the murine 6A10 antibody.

[0090] SEQ ID NO: 68 represents the amino acid sequence of the Kabat CDR-H2 of the murine 6A10 antibody.

[0091] SEQ ID NO: 69 represents the amino acid sequence of the Kabat CDR-H3 of the murine 6A10 antibody.

[0092] SEQ ID NO: 70 represents the amino acid sequence of the VH region of a murine antibody (pdb code 1CR9) used as a structural template for heavy chain humanization.

[0093] SEQ ID NO: 71 represents the consensus amino acid sequence among the heavy chain variable regions of selected humanized 3D6 antibodies (VHv1, VHv1b, VHv1bA11, VHv1bA11B6G2, VHv1bA11B6H3, VHv1c, VHv1d, VHv1e, VHv1f, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) (represented as "Majority" in Figures 4A and 4B of PCT / IB2017 / 052544).

[0094] SEQ ID NO: 72 represents the amino acid sequence of the heavy chain of the chimeric 3D6 antibody.

[0095] SEQ ID NO: 73 represents the amino acid sequence of the light chain of the chimeric 3D6 antibody.

[0096] SEQ ID NO: 74 represents the amino acid sequence of the heavy chain variable structure model Acc.#5MYX-VH_mSt.

[0097] SEQ ID NO: 75 represents the amino acid sequence of the heavy chain variable acceptor Acc.#2RCS-VH_huFrwk.

[0098] SEQ ID NO: 76 represents the amino acid sequence of the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody.

[0099] SEQ ID NO: 77 represents the amino acid sequence of the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody.

[0100] SEQ ID NO: 78 represents the amino acid sequence of the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody.

[0101] SEQ ID NO: 79 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb4.

[0102] SEQ ID NO: 80 represents the amino acid sequence of the heavy chain variable region hu3D6VHvb5 of the humanized 3D6 antibody.

[0103] SEQ ID NO: 81 represents the amino acid sequence of the light chain variable structure model Acc.#5MYX-VL_mSt.

[0104] SEQ ID NO: 82 represents the amino acid sequence of the light chain variable acceptor Acc.#ARX71335-VL_huFrwk.

[0105] SEQ ID NO: 83 represents the amino acid sequence of the light chain variable region hu3D6VLvb1 of the humanized 3D6 antibody.

[0106] SEQ ID NO: 84 represents the amino acid sequence of the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb2.

[0107] SEQ ID NO: 85 represents the amino acid sequence of the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb3.

[0108] SEQ ID NO:86 represents the amino acid sequence of an alternative Kabat-Chothia composite CDR-H1 of the humanized 3D6 antibody (such as found in hu3D6VHvb4 and hu3D6VHvb5).

[0109] SEQ ID NO:87 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHvb3 and hu3D6VHvb4).

[0110] SEQ ID NO:88 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHvb5).

[0111] SEQ ID NO:89 represents the amino acid sequence of an alternative Kabat CDR-L1 of the humanized 3D6 antibody (such as found in hu3D6VLvb3).

[0112] SEQ ID NO: 90 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb6.

[0113] SEQ ID NO: 91 represents the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb7.

[0114] SEQ ID NO: 92 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in hu3D6VHvb6 and hu3D6VHvb7).

[0115] SEQ ID NO: 93 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54D.

[0116] SEQ ID NO: 94 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54G.

[0117] SEQ ID NO: 95 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L45N.

[0118] SEQ ID NO: 96 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54E.

[0119] SEQ ID NO: 97 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50E.

[0120] SEQ ID NO: 98 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54Q.

[0121] SEQ ID NO: 99 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50D.

[0122] SEQ ID NO: 100 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54K.

[0123] SEQ ID NO: 101 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54R.

[0124] SEQ ID NO: 102 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54T.

[0125] SEQ ID NO: 103 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50G.

[0126] SEQ ID NO: 104 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant I48G.

[0127] SEQ ID NO: 105 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant I48D.

[0128] SEQ ID NO: 106 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47G.

[0129] SEQ ID NO: 107 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant Y49E.

[0130] SEQ ID NO: 108 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54V.

[0131] SEQ ID NO: 109 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54S.

[0132] SEQ ID NO: 110 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant S52G.

[0133] SEQ ID NO: 111 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47N.

[0134] SEQ ID NO: 112 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47D.

[0135] SEQ ID NO: 113 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47E.

[0136] SEQ ID NO: 114 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47P.

[0137] SEQ ID NO: 115 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47T.

[0138] SEQ ID NO: 116 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47S.

[0139] SEQ ID NO: 117 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47A.

[0140] SEQ ID NO: 118 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50V.

[0141] SEQ ID NO: 119 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R.

[0142] SEQ ID NO: 120 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G.

[0143] SEQ ID NO: 121 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54G.

[0144] SEQ ID NO: 122 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R.

[0145] SEQ ID NO: 123 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54T.

[0146] SEQ ID NO: 124 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D.

[0147] SEQ ID NO: 125 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54R.

[0148] SEQ ID NO: 126 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54G.

[0149] SEQ ID NO: 127 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54D.

[0150] SEQ ID NO: 128 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G.

[0151] SEQ ID NO: 129 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D.

[0152] SEQ ID NO: 130 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54T.

[0153] SEQ ID NO: 131 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G.

[0154] SEQ ID NO: 132 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54G.

[0155] SEQ ID NO: 133 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54R.

[0156] SEQ ID NO: 134 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54G.

[0157] SEQ ID NO: 135 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54R.

[0158] SEQ ID NO: 136 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R_G100Q.

[0159] SEQ ID NO: 137 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G_G100Q.

[0160] SEQ ID NO: 138 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R_G100Q.

[0161] SEQ ID NO: 139 represents the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D_G100Q.

[0162] SEQ ID NO: 140 represents the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50D_L54G_G100Q.

[0163] SEQ ID NO: 141 represents the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50D_L54R_G100Q.

[0164] SEQ ID NO: 142 represents the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50V_L54D_G100Q.

[0165] SEQ ID NO: 143 represents the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q.

[0166] SEQ ID NO: 144 represents the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant G100Q.

[0167] SEQ ID NO: 145 represents the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L54E.

[0168] SEQ ID NO: 146 represents the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant D60E (also known as h3D6VHvb8).

[0169] SEQ ID NO: 147 represents the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant L82cV.

[0170] SEQ ID NO: 148 represents the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant D60E_L80M_Q81E_L82cV_T83R (also known as h3D6VHvb9).

[0171] SEQ ID NO: 149 represents the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (such as found in h3D6VHvb8 and h3D6VHvb9).

[0172] SEQ ID NO: 150 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54D and hu3D6VLv2 L37Q_L54D).

[0173] SEQ ID NO: 151 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54G and hu3D6VLv2 L37Q_L54G).

[0174] SEQ ID NO: 152 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54N).

[0175] SEQ ID NO: 153 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54E and hu3D6VLv2 L37Q_L54E).

[0176] SEQ ID NO: 154 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L50E).

[0177] SEQ ID NO: 155 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54Q).

[0178] SEQ ID NO: 156 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L50D and hu3D6VLv2 L37Q_L50D).

[0179] SEQ ID NO: 157 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54K).

[0180] SEQ ID NO: 158 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54R and hu3D6VLv2 L37Q_L54R).

[0181] SEQ ID NO: 159 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54T and hu3D6VLv2 L37Q_L54T).

[0182] SEQ ID NO: 160 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L50G and hu3D6VLv2 L37Q_L50G).

[0183] SEQ ID NO: 161 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54V).

[0184] SEQ ID NO: 162 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L54S).

[0185] SEQ ID NO: 163 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 S52G and hu3D6VLv2 L37Q_S52G).

[0186] SEQ ID NO: 164 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L50V).

[0187] SEQ ID NO: 165 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50G_L54R and hu3D6VLv2 L37Q_L50G_L54R_G100Q).

[0188] SEQ ID NO: 166 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50G_L54G and hu3D6VLv2 L37Q_L50G_L54G_G100Q).

[0189] SEQ ID NO: 167 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_S52G_L54G).

[0190] SEQ ID NO: 168 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_S52G_L54R and hu3D6VLv2 L37Q_S52G_L54R_G100Q).

[0191] SEQ ID NO: 169 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_S52G_L54T).

[0192] SEQ ID NO: 170 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_S52G_L54D and hu3D6VLv2 L37Q_S52G_L54D_G100Q).

[0193] SEQ ID NO: 171 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50D_L54G and hu3D6VLv2 L37Q_L50D_L54G_G100Q).

[0194] SEQ ID NO: 172 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50D_L54R and hu3D6VLv2 L37Q_L50D_L54R_G100Q).

[0195] SEQ ID NO: 173 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50E_L54G).

[0196] SEQ ID NO: 174 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50E_L54R).

[0197] SEQ ID NO: 175 represents the amino acid sequence of an alternative Kabat CDR-L2 of the humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50V_L54D_G100Q).

[0198] SEQ ID NO: 176 represents the amino acid sequence of the heavy chain constant region (IgG1: allotype G1m17,1).

[0199] SEQ ID NO: 177 represents the amino acid sequence of the light chain constant region (kappa).

[0200] SEQ ID NO: 178 represents the amino acid sequence of the mature heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype).

[0201] SEQ ID NO: 179 represents the amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa).

[0202] SEQ ID NO: 180 represents the amino acid sequence of the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with the bovine alpha-lactalbumin signal peptide at the N-terminus.

[0203] SEQ ID NO: 181 represents the amino acid sequence of the light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0204] SEQ ID NO: 182 represents the nucleotide sequence encoding the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with the N-terminal bovine alpha-lactalbumin signal peptide.

[0205] SEQ ID NO: 183 represents the nucleotide sequence encoding the light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0206] SEQ ID NO: 184 represents the amino acid sequence of tau microtubule-binding repeat region 1 (amino acid residues 255 to 271 of SEQ ID NO: 1).

[0207] SEQ ID NO: 185 represents the amino acid sequence of tau microtubule-binding repeat region 2 (amino acid residues 286 to 302 of SEQ ID NO: 1).

[0208] SEQ ID NO: 186 represents the amino acid sequence of tau microtubule-binding repeat region 3 (amino acid residues 317 to 333 of SEQ ID NO: 1).

[0209] SEQ ID NO: 187 represents the amino acid sequence of tau microtubule-binding repeat region 4 (amino acid residues 349 to 365 of SEQ ID NO: 1).

[0210] SEQ ID NO: 188 represents the amino acid sequence of the tau core motif in MBTR1 to which 3D6 binds.

[0211] SEQ ID NO: 189 represents the amino acid sequence of the tau sequence N-terminal to the tau core motif in MBTR1 to which 3D6 binds.

[0212] SEQ ID NO: 190 represents the amino acid sequence of the tau sequence C-terminal to the tau core motif in MBTR1 to which 3D6 binds.

[0213] SEQ ID NO: 191 represents the amino acid sequence of the epitope of 3D6.

[0214] SEQ ID NO: 192 represents the amino acid sequence of the tau core motif in MBTR2 to which 3D6 binds.

[0215] SEQ ID NO: 193 represents the amino acid sequence of the tau core motif in MBTR3 to which 3D6 binds.

[0216] SEQ ID NO: 194 represents the amino acid sequence of the tau core motif in MBTR4 to which 3D6 binds.

[0217] definition Monoclonal antibodies or other biological entities are usually provided in isolated form. This means that the antibody or other biological entity usually contains interfering proteins and other contaminants resulting from its production or purification with a purity of at least 50 (w / w)%, but does not exclude the possibility that the monoclonal antibody may be combined with an excess of a pharma- ceutical acceptable carrier or other vehicle intended to facilitate its use. In some cases, the monoclonal antibody is at least 60 (w / w)%, 70 (w / w)%, 80 (w / w)%, 90 (w / w)%, 95 (w / w)%, or 99 (w / w)% pure of interfering proteins and contaminants resulting from its production or purification. In many cases, an isolated monoclonal antibody or other biological entity is the major macromolecular species remaining after its purification.

[0218] Specific binding of an antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 M -1 Specific binding refers to the affinity and / or avidity of an antibody. Specific binding is detectably significant in magnitude and is comparable to non-specific binding occurring to at least one unrelated target. Specific binding can be the result of the formation of bonds between specific functional groups or specific spatial matches (e.g., lock and key types), whereas non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily imply that an antibody binds to one and only one target.

[0219] The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, light chain mature variable region refers to the light chain variable region without the light chain signal peptide. The carboxy terminus of each chain defines a constant region primarily responsible for effector function.

[0220] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).

[0221] An immunoglobulin light or heavy chain variable region (also referred to herein as a "light chain variable domain" ("VL domain") or a "heavy chain variable domain" ("VH domain"), respectively) consists of a "framework" region interrupted by three "complementarity determining regions" or "CDRs". The framework regions act to align the CDRs for specific binding to an epitope of an antigen. The CDRs contain the amino acid residues of an antibody that primarily contribute to antigen binding. From the amino-terminus to the carboxyl-terminus, the VL and VH domains comprise the following framework regions (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively; CDR1, 2, and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. Where the present application discloses a VL sequence with R as the C-terminal residue, R may alternatively be considered as the N-terminal residue of the light chain constant region, and thus, it should be understood that the present application also discloses a VL sequence without a C-terminal R.

[0222] The amino acid assignments for each VL and VH domain follow one of the conventional CDR definitions, including the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the Chothia-Kabat CDR combination, in which CDR-H1 is a combination of the Chothia and Kabat CDRs; and the Oxford The AbM definition used by antibody modeling software from Molecular; and the contact definition from Martin et al. (bioinfo.org.uk / abs) (see Table 1). Kabat provides a widely used numbering scheme (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number. When an antibody is said to contain a CDR according to a particular definition of CDR (e.g. Kabat), this definition specifies the minimum number of CDR residues present in the antibody (i.e. Kabat CDRs). This does not exclude that other residues that are within the alternative conventional CDR definitions but outside the scope of the specified definition may be present as well. For example, an antibody that contains a CDR as defined by Kabat includes, among other possibilities, an antibody in which the CDR contains Kabat CDR residues and no other CDR residues, and an antibody in which the CDR H1 is a composite Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues and no further CDR residues according to other definitions. [Table 1]

[0223] The term "antibody" includes intact antibodies and binding fragments thereof, typically consisting of separate heavy and light chains, Fab, Fab', F(ab') 2 Fragments, including F(ab)c, Dabs, Nanobodies, and Fv, compete with the intact antibody from which they are derived for specific binding to a target. Fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific and / or humanized antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). In some bispecific antibodies, the two different heavy / light chain pairs include a humanized 3D6 heavy / light chain pair and a heavy / light chain pair specific for a different epitope on tau other than the one bound by 3D6.

[0224] In some bispecific antibodies, one heavy / light chain pair is a humanized 3D6 antibody, as further disclosed below, and the other heavy / light chain pair is derived from an antibody that binds to a receptor expressed at the blood-brain barrier, such as the insulin receptor, insulin-like growth factor (IGF) receptor, leptin receptor, or lipoprotein receptor, or transferrin receptor (Friden et al., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such bispecific antibodies can be delivered through the blood-brain barrier by receptor-mediated transcytosis. Brain uptake of bispecific antibodies can be further enhanced by engineering the bispecific antibody to reduce its affinity for the blood-brain barrier receptor. The reduced affinity for the receptor resulted in a broader distribution in the brain (see, e.g., Atwal et al., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

[0225] Also exemplary bispecific antibodies include: (1) dual variable domain antibodies (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) Tandbab, a fusion of two single-chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each of the target antigens; (3) flexibodies, a combination of diabody and scFv resulting in a multivalent molecule; (4) so-called "dock and dock antibodies" based on the "D / D domain" of protein kinase A, which when applied to Fabs, can result in a trivalent bispecific binding protein consisting of two identical Fab fragments bound to different Fab fragments. or (5) so-called Scorpion molecules, which comprise, for example, two scFvs fused to either end of a human Fc region. Examples of platforms useful for the preparation of bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor), or DuoBody (based on the exchange of Fab arms, Genmab).

[0226] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are usually retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding (conformational epitopes) are usually lost after treatment with denaturing solvents. An epitope comprises at least three, usually at least five or eight to ten amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0227] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the property of one antibody competing with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography of the antibody bound to its antigen to identify the exact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0228] Competition between antibodies is determined by an assay in which the antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by competitive assays (competitor antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody due to steric hindrance that occurs.

[0229] The term "pharmaceutically acceptable" means the carrier, diluent, excipient or auxiliary must be compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0230] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0231] An individual is at high risk for a disease if the subject has at least one known risk factor (genetic factors, biochemical factors, familial, and situational exposures) that places the individual with the risk factor at a statistically significantly higher risk of developing the disease than individuals without the risk factor.

[0232] The term "biological sample" refers to a sample of biological material available in or from a biological source, such as a human or mammalian subject. Such samples may be organs, organelles, tissues, tissue sections, bodily fluids, peripheral blood, plasma, serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample may also encompass any material derived by processing the sample. Derived materials may include cells or their passages. Processing of biological samples may include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.

[0233] The term "control sample" refers to a biological sample that is not known or suspected to contain a region affected by a tau-related disease, or at least not known or suspected to contain a disease region of a given type. The control sample may be obtained from an individual not affected by a tau-related disease. Alternatively, the control sample may be obtained from a patient affected by a tau-related disease. Such a biological sample may be obtained at the same time as the biological sample suspected to contain a tau-related disease, or in a different context. Both the biological sample and the control sample may be obtained from the same tissue. Preferably, the control sample is made of an essentially or grossly normal and healthy region, and may be used for comparison with the biological sample suspected to contain a region affected by a tau-related disease. Preferably, the tissue of the control sample is of the same type as the tissue of the biological sample. Preferably, the cells affected by a tau-related disease suspected to be in the biological sample originate from the same cell type (e.g., neuron or glia) as the cell type in the control sample.

[0234] The term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, disease, disorder, pathology, disorder, condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

[0235] The term "symptom" refers to subjective evidence of disease, such as a change in gait, as perceived by the subject. "Sign" refers to objective evidence of disease as observed by a physician.

[0236] The term "positive response to a treatment" refers to a more favorable individual patient response or the average response of a population of patients compared to the average response of a control population that does not receive the treatment.

[0237] For the purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions constitute the exchange of a member of one of these classes for another.

[0238] Percentage sequence identity is determined with maximally aligned antibody sequences according to the Kabat numbering convention. After alignment, when a region of an antibody of interest (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in the subject and reference antibodies, divided by the total number of aligned positions of the two regions, not accounting for gaps, multiplied by 100 to convert to a percentage.

[0239] A composition or method "comprising or including" one or more recited elements may include other elements not specifically recited. For example, a composition "comprising" an antibody may include the antibody alone or the antibody in combination with other components. It will be understood that where the disclosure refers to a feature comprising particular elements, the disclosure also alternatively refers to a feature consisting essentially of or consisting of those particular elements.

[0240] The representation of a range of values ​​includes every integer within or defining that range, and every subrange defined by the integers within that range.

[0241] Unless otherwise clear from the context, the term "about" encompasses definite values, such as values ​​within the standard error (eg, SEM) of measurement of the stated value.

[0242] Statistical significance means p≦0.05.

[0243] The singular forms of articles "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0244] Detailed Description I. General Provisions The present invention provides methods for treating tauopathies (such as Alzheimer's disease) using antibodies that bind to human tau.

[0245] II.Target molecule Unless otherwise clear from the context, reference to tau refers to the native human form of tau, including all isoforms, whether or not post-translational modifications (e.g., phosphorylation, glycosylation, or acetylation) are present. There are six major isoforms (splice variants) of tau present in the human brain. The longest of these variants has 441 amino acids, the first met residue of which is truncated. Residues are numbered according to the 441 isoform. Thus, for example, reference to phosphorylation at position 404 refers to position 404 in the 441 isoform, or the corresponding position in any other isoform when maximally aligned with the 441 isoform. The amino acid sequences and Swiss-Prot numbers of the isoforms are set forth below. [ka] [ka]

[0246] Reference to tau includes known naturally occurring variations and permutations thereof, about 30 of which are listed in the Swiss-Prot database, as well as mutations associated with tau pathologies, such as dementia, Pick's disease, supranuclear palsy, etc. (see, e.g., the Swiss-Prot database and Poorkaj, et al. Ann Neurol. 43:815-825 (1998)). Some examples of tau mutations, numbered by the 441 isoforms, are a lysine to threonine mutation at amino acid residue 257 (K257T); an isoleucine to valine mutation at amino acid position 260 (I260V); a glycine to valine mutation at amino acid position 272 (G272V); an asparagine to lysine mutation at amino acid position 279 (N279K); an asparagine to histidine mutation at amino acid position 296 (N296H); a proline to serine mutation at amino acid position 301 (P301S); a proline to leucine mutation at amino acid position 301 (P301L). ); a glycine to valine mutation at amino acid position 303 (G303V); a serine to asparagine mutation at position 305 (S305N); a glycine to serine mutation at amino acid position 335 (G335S); a valine to methionine mutation at position 337 (V337M); a glutamic acid to valine mutation at position 342 (E342V); a lysine to isoleucine mutation at amino acid position 369 (K3691); a glycine to arginine mutation at amino acid position 389 (G389R); and an arginine to tryptophan mutation at amino acid position 406 (R406W).

[0247] Tau may be phosphorylated at one or more amino acid residues, including tyrosines at amino acid positions 18, 29, 97, 310, and 394, serines at amino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293, 324, 356, 396, 400, 404, 409, 412, 413, and 422; and threonines at amino acid positions 175, 181, 205, 212, 217, 231, and 403. Unless otherwise clear from the context, reference to Tau or a fragment thereof includes the naturally occurring human amino acid sequence, including isoforms, mutants, and allelic variants thereof.

[0248] III. Antibodies A. Binding Specificity and Functional Properties The present invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within KXXSXXNX(K / H)H (SEQ ID NO:191). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 259-268 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 290-299 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 321-330 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies bind to a peptide comprising, consisting essentially of, or consisting of amino acid residues 353-362 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies bind to two, three, or all four of these peptides. Some antibodies specifically bind to an epitope within residues 199-213 (corresponding to residues 257-271 of SEQ ID NO:1) of the 383 amino acid 4RON human tau protein (SEQ ID NO:3). Some antibodies specifically bind to an epitope within residues 262-276 (corresponding to residues 320-334 of SEQ ID NO:1) of the 383 amino acid 4RON human tau protein (SEQ ID NO:3). Some antibodies of the invention specifically bind to a peptide consisting of residues 257-271 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies of the invention specifically bind to a peptide consisting of residues 320-334 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies of the invention specifically bind to a peptide consisting of residues 259-268 of the 441 amino acid tau protein (SEQ ID NO:1) (i.e., KIGSTENLKH (SEQ ID NO:188)). Some antibodies of the present invention specifically bind to a peptide consisting of residues 290-299 (i.e., KCGSKDNIKH (SEQ ID NO:192)) of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies of the present invention specifically bind to a peptide consisting of residues 321-330 (i.e., KCGSLGNIHH (SEQ ID NO:193)) of the 441 amino acid tau protein (SEQ ID NO:1).Some antibodies of the invention specifically bind to a peptide consisting of residues 353-362 (i.e., KIGSLDNITH (SEQ ID NO:194)) of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies of the invention specifically bind to a peptide consisting of the consensus motif KXXSXXNX(K / H)H (SEQ ID NO:191). Some antibodies bind to an epitope consisting of residues 259, 262, 265, 267, 268, residues 290, 293, 296, 298, 299, residues 321, 324, 327, 329, 330, or residues 353, 356, 359, 362 of the 441 amino acid tau protein (SEQ ID NO:1). Some antibodies bind to tau regardless of phosphorylation state. Some antibodies bind to epitopes that do not include residues that are subject to phosphorylation. These antibodies may be obtained by immunization with tau polypeptides purified from natural sources or expressed recombinantly. Antibodies can be screened for binding to tau in its unphosphorylated form and in its form in which one or more phosphorylation-susceptible residues are phosphorylated. Such antibodies preferably bind to phosphorylated tau with indistinguishable affinity or at least within 1.5-fold, 2-fold, or 3-fold affinity compared to unphosphorylated tau (i.e., are "pan-specific"). 3D6 is an example of a pan-specific monoclonal antibody. The present invention also provides antibodies that bind to the same epitope as any of the above antibodies (e.g., the epitope of 3D6). Antibodies that compete with any of the above antibodies for binding to tau (e.g., compete with 3D6) are also included.

[0249] The above antibodies may be produced de novo by immunizing with a peptide comprising, consisting essentially of, or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or with a peptide comprising, consisting essentially of, or consisting of residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO:1, or with a full-length tau polypeptide or a fragment thereof comprising such residues and screening for specific binding to peptides comprising such residues. Preferably, such peptides are conjugated to a heterologous conjugate molecule that helps elicit an antibody response against the peptide. Conjugation may be direct or via a spacer peptide or amino acid. Cysteine ​​is used as a spacer amino acid because its free SH group facilitates attachment of the carrier molecule. Polyglycine linkers (e.g., 2-6 glycines) with or without cysteine ​​residues between the glycine and the peptide may also be used. The carrier molecule acts to provide T cell epitopes that aid in eliciting an antibody response to the peptide. Several carriers are commonly used, notably keyhole limpet hemocyanin (KLH), ovalbumin, and bovine serum albumin (BSA). Peptide spacers may be added to the peptide immunogen as part of solid phase peptide synthesis. Carriers are usually added by chemical cross-linking.Some examples of chemical cross-linking agents that can be used include cross-N-maleimido-6-aminocaproyl ester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see, e.g., Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1988; Sinigaglia et al., Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47 (1993); Hammer et al., Cell 74:197-203 (1993); Falk K. et al., Immunogenetics, 39:230-242 (1994); International Patent Publication No. 98 / 23635; and Southwood et al. J. Immunology, 160:3363-3373). (1998)). Carriers and spacers, when present, can be attached to either end of the immunogen.

[0250] Peptides, including optional spacers and carriers, may be used to immunize laboratory animals or B cells, as described in more detail below. Hybridoma supernatants may be tested for the ability to bind to one or more peptides comprising, consisting essentially of, or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or for the ability to bind to one or more peptides comprising, consisting essentially of, or consisting of residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO:1, and / or for the ability to bind phosphorylated and non-phosphorylated forms of tau (such as, for example, the full-length isoform of tau in which position 404 is phosphorylated). The peptides may be conjugated to a carrier or other tag to facilitate screening assays. In this case, the carrier or tag is selectively different from the combination of spacer and carrier molecule used in the immunization to exclude antibodies specific for the spacer or carrier rather than the tau peptide. Any of the isoforms can be used.

[0251] The present invention provides a monoclonal antibody that binds to an epitope within tau. The antibody designated 3D6 is an example of such a murine antibody. Unless otherwise clear from the context, reference to 3D6 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. The antibody has been deposited under [DEPOSIT NUMBER]. The antibody specifically binds to an epitope designated KXXSXXNX(K / H)H (SEQ ID NO: 191). The antibody specifically binds within amino acid residues 199-213 and / or 262-276 (corresponding to amino acid residues 257-271 and / or 320-334 of SEQ ID NO: 1, respectively) of the 383 amino acid 4R0N human tau protein (SEQ ID NO: 3). The antibody specifically binds to amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1, and any combination of two, any combination of three, or all four thereof. The antibody is further characterized by its ability to bind to both phosphorylated and non-phosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded / aggregated forms of tau. Humanized antibody hu3D6VHv1bA11 / L2-DIM4 binds equally to phosphorylated and non-phosphorylated tau, binds to all splice isoforms of tau, and binds to neurofibrillary tangles and degenerating neurites in tissue sections from each of the six Alzheimer's disease donor samples tested. The antibody designated 6A10 is an example of such a murine antibody. Unless otherwise clear from the context, reference to 6A10 should be understood as a reference to any of the murine, chimeric, veneered, and humanized forms of this antibody. The Kabat / Chothia composite CDRs of the heavy chain of 6A10 are set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the Kabat CDRs of the light chain of 6A10 are set forth in SEQ ID NOs: 12, 13, and 14, respectively. Murine 6A10 shares 82.1% VH sequence identity and 100% VL sequence identity with the murine 3D6 VH and VL chains, respectively.

[0252] Some antibodies of the invention bind to the same or overlapping epitope as the antibody designated 3D6. The sequences of the heavy and light chain mature variable regions of this antibody are designated SEQ ID NOs: 7 and 11, respectively. Other antibodies with such binding specificity can be produced by immunizing mice with tau or a portion thereof comprising, consisting essentially of, or consisting of the desired epitope (e.g., residues 199-213 and / or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 and / or 320-334 of SEQ ID NO:1, respectively), or, for example, residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1, any combination of two thereof, any combination of three thereof, or all four), and screening the resulting antibodies for binding to tau, optionally in competition with an antibody having the variable region of mouse 3D6 (IgG1 kappa). A fragment of tau containing the desired epitope may be coupled to a carrier that helps elicit an antibody response against the fragment and / or combined with an adjuvant that helps elicit the response. Such antibodies may be screened for differential binding to tau or its fragments compared to mutants of the specified residues. Screening against the mutants allows for the identification of antibodies whose binding is inhibited by mutagenesis of specific residues, perhaps sharing the functional properties of the other exemplified antibodies, by more precisely defining the binding specificity. The mutations may be systematic replacement substitutions with alanine (or serine, if alanine is already present), one residue at a time or at more widely spaced intervals, across the target or across that section where the epitope is known to remain. If the same set of mutations significantly reduces the binding of the two antibodies, then the two antibodies bind to the same epitope.

[0253] Antibodies having the binding specificity of a selected murine antibody (e.g., 3D6) can also be produced using a variant of the phage display method. See Winter, International Patent Publication No. 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, the heavy or light chain variable region of a selected murine antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Phages (e.g., at least 10 8 , preferably at least 10 9 M -1 ) are selected. The heavy chain variable region from this phage then serves as starting material for constructing a further phage library, in which each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable region may be obtained from a library of rearranged human variable light chain regions. Again, phage that show strong specific binding to tau or a fragment thereof are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

[0254] The Kabat / Chothia composite CDRs of the heavy chain of 3D6 are designated SEQ ID NOs:8, 9, and 10, respectively, and the Kabat / Chothia composite CDRs of the light chain of 3D6 are designated SEQ ID NOs:12, 13, and 14, respectively.

[0255] Table 2 shows the 3D6 CDRs defined by Kabat, Chothia, Chothia and Kabat composite (also referred to herein as the "Kabat / Chothia composite"), AbM, and Contact. [Table 2]

[0256] Other antibodies can be obtained by mutagenesis of cDNAs encoding the heavy and light chains of exemplary antibodies such as 3D6. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 3D6 in the amino acid sequence of the mature heavy and / or light chain variable regions and that maintain their functional properties and / or differ from each antibody by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions are also included in the present invention. Also included are monoclonal antibodies that have at least one or all six CDRs, as defined by Kabat, either conventionally defined, that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of 3D6.

[0257] The invention also provides antibodies having some or all (e.g., 3, 4, 5, and 6) CDRs, completely or substantially, from 3D6. Such antibodies may comprise a heavy chain variable region having at least two, usually three, CDRs, completely or substantially from the heavy chain variable region of 3D6, and / or a light chain variable region having at least two, usually three, CDRs, completely or substantially from the light chain variable region of 3D6. The antibodies may comprise both heavy and light chains. The CDRs are substantially derived from the corresponding 3D6 CDRs when they contain no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that CDR-H2 (as defined by Kabat) may have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to 3D6 in the amino acid sequence of the mature heavy chain variable region and / or mature light chain variable region, and may retain its functional properties and / or may differ from 3D6 by a small number of functionally minor amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0258] Some antibodies identified by such assays may bind to monomeric, misfolded, aggregated, phosphorylated, or nonphosphorylated forms of tau, or other forms. Similarly, some antibodies are immunoreactive with nonpathological and pathological forms and conformations of tau.

[0259] B. Non-human antibodies Production of other non-human antibodies, e.g., mouse, guinea pig, primate, rabbit, or rat antibodies, against tau or a fragment thereof (e.g., amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO:1) can be achieved, for example, by immunizing the animal with tau or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988), incorporated by reference for all purposes. Such immunogens can be obtained from natural sources, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused to or otherwise complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used, as described below. Complete Freund's adjuvant, followed by incomplete adjuvant, is preferred for immunization of laboratory animals. Rabbits or guinea pigs are usually used for the production of polyclonal antibodies. Mice are usually used for the production of monoclonal antibodies. Antibodies are screened for specific binding to tau or an epitope within tau (e.g., an epitope comprising one or more of amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or an epitope comprising one or more of amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1).Such screening may be accomplished by determining binding of the antibody to a collection of tau variants (such as tau variants comprising amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or tau variants comprising amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO:1, or tau variants comprising mutations within such residues) and determining which tau variants bind to the antibody. Binding may be assessed, for example, by Western blot, FACS, or ELISA.

[0260] C. Humanized Antibodies A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted into the sequences of a human "acceptor" antibody (see, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 (Queen); U.S. Pat. Nos. 5,225,539 (Winter); 6,407,213 (Carter); 5,859,205 (Adair); and 6,881,557 (Foote)). The sequences of the acceptor antibody can be, for example, the sequences of a mature human antibody, a composite of such sequences, a consensus sequence of human antibody sequences, or a sequence of a germline region. Thus, a humanized antibody is an antibody having at least three, four, five, or all of the CDRs, completely or substantially, derived from the donor antibody, and variable region framework sequences and constant regions, if present, completely or substantially, derived from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs entirely or substantially from the heavy chain of a donor antibody, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, substantially from the human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs entirely or substantially from the donor antibody light chain, and a light chain variable region framework sequence and a light chain constant region, if present, substantially from the human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. The CDRs of a humanized antibody are derived from substantially the corresponding CDRs in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (defined by any conventional definition, but preferably defined by Kabat) are identical between each CDR. The framework sequence of a variable region of an antibody chain or a constant region of an antibody chain is substantially derived from a framework sequence of a human variable region or a human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues as defined by Kabat are identical.For a humanized antibody to be classified as humanized under the 2014 World Health Organization (WHO) International Nonproprietary Name (INN) definition, the antibody must have at least 85% identity with the human germline antibody sequence (i.e., prior to somatic mutations). A mixed antibody is one in which one antibody chain (e.g., the heavy chain) matches the threshold, but the other chain (e.g., the light chain) does not. If neither chain matches the threshold, even though the variable framework regions in both chains are substantially human with some mouse back mutations, the antibody is classified as chimeric. See Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1, 1-9, DOI: 10.1080 / 19420862.2015.1114320. See also "WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review)" (Internet) 2014. (Available at http: / / www. who.int / medicines / services / inn / BioRev2014.pdf) (hereby incorporated by reference). For the avoidance of doubt, the term "humanized" as used herein is not intended to be limited to the 2014 WHO INN definition of a humanized antibody. Some of the humanized antibodies provided herein have at least 85% sequence identity with human germline sequences, and some of the humanized antibodies provided herein have less than 85% sequence identity with human germline sequences. Some of the heavy chains of the humanized antibodies provided herein have about 60-100% sequence identity with human germline sequences, e.g., within the range of about 60%-69%, 70%-79%, 80%-84%, or 85%-89% sequence identity.Some heavy chains fall below the 2014 WHO INN definition, e.g., have about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identity to human germline sequences, and other heavy chains meet the 2014 WHO INN definition, have about 85%, 86%, 87%, 88%, 89% or more sequence identity to human germline sequences. Some of the light chains of the humanized antibodies provided herein have about 60%-100% sequence identity to human germline sequences, e.g., within the range of about 80%-84% or 85%-89% sequence identity. Some light chains fall below the 2014 WHO INN definition, e.g., have about 81%, 82%, 83%, or 84% sequence identity to human germline sequences, while other light chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89%, or more sequence identity to human germline sequences. Some humanized antibodies provided herein that are "chimeric" under the 2014 WHO INN definition have a heavy chain that has less than 85% identity to human germline sequences paired with a light chain that has less than 85% identity to human germline sequences. For example, some humanized antibodies provided herein that have a heavy chain that has less than 85% identity to human germline sequences paired with a light chain that has less than 85% identity to human germline sequences, or vice versa, are "mixed" under the 2014 WHO INN definition. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized" and have a heavy chain with at least 85% sequence identity to the human germline sequence paired with a light chain with at least 85% sequence identity to the human germline sequence. Additional humanized antibodies of the invention meet the 2014 WHO INN definition of "mixed."

[0261] Humanized antibodies often incorporate all six CDRs (as defined by any conventional definition, but preferably as defined by Kabat) from a murine antibody, although they can also be made with fewer than all of the CDRs from a murine antibody (e.g., at least three, four, or five CDRs) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, J. Immunol., 164:1432-1441, 2000).

[0262] In some antibodies, only a portion of the CDRs, called SDRs, i.e., a subset of CDR residues required for binding, are required to retain binding of the humanized antibody. CDR residues that do not contact the antigen and are not in the SDRs can be identified from regions of the Kabat CDRs other than the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and / or experience, or based on previous testing as described in Gonzales et al., Mol. Immunol. 41: 863, 2004 (e.g., residues H60-H65 of CDR H2 are often not required). In such humanized antibodies, where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid residue that occupies that position can be the amino acid that occupies the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of substitutions of acceptor donor amino acids in such included CDRs reflects a balance of competing considerations. Such substitutions are potentially advantageous in reducing the number of mouse amino acids in the humanized antibody and thus reducing potential immunogenicity, and / or to meet the WHO INN definition of "humanization". However, substitutions may also result in changes in affinity, and significant reductions in affinity are preferably avoided. The position of substitution in the CDR and the amino acid to be substituted may also be empirically selected.

[0263] The sequence of the human acceptor antibody may optionally be selected from the many known human antibodies to provide a high degree of sequence identity (e.g., 65-85% identity) between the framework of the human acceptor sequence variable region and the framework of the corresponding variable region of the donor antibody chain.

[0264] Some humanized and chimeric antibodies have the same (within experimental error) or improved functional properties (e.g., binding affinity to human tau, inhibition of internalization of tau into neurons) (which may be assayed as described in the Examples of U.S. Publication No. 2020 / 0369755A1) compared to the mouse antibody from which they are derived. For example, some humanized and chimeric antibodies have binding affinities within a factor of 3, 2, or 1 of the mouse antibody from which they are derived, or affinities that are indistinguishable within experimental error. Some humanized and chimeric antibodies inhibit internalization of tau into neurons (which may be assayed as described in the Examples of U.S. Publication No. 2020 / 0369755A1) within 3-fold, 2-fold, or 1-fold of the mouse antibody from which they are derived, or inhibit it as well (within experimental error) as the mouse antibody from which they are derived. Some humanized antibodies have reduced immunogenicity, increased affinity, increased thermostability, and / or improved expression compared to previously reported humanized forms of the 3D6 antibody (see WO2017 / 191560 and U.S. Publication No. 2020 / 0369755A1). hu3D6VHv1bA11 / L2-DIM4 has improved affinity over the parent hu3D6VHv1bA11 / hu3D6VLv2, as supported by on-rates, off-rates, and Kd numbers. hu3D6VHv1bA11 / L2-DIM4 has increased thermostability and potency over the parent hu3D6VHv1bA11 / hu3D6VLv2 (see U.S. Publication No. 2020 / 0369755A1). Some antibodies of the invention bind with high affinity to specific tau isoforms as measured by surface plasmon resonance. For example, hu3D6VHv1bA11 / L2-DIM4 has K of 154 pM and 206 pM for 3R2N-tau (Swiss-protID: P10636-5) and 4R2N-tau (Swiss-protID: P10636-8), respectively. D Combine with.

[0265] An example of an acceptor sequence for the heavy chain is the human mature heavy chain variable region of humanized 48G7 Fab (PDB accession code 2RCS-VH_huFrwk (SEQ ID NO: 75)). The variable domains of 3D6 and 48G7 Fab are also identical in length of the CDR-H1 and CDR-H2 loops. An example of an acceptor sequence for the heavy chain is the human mature heavy chain variable region IMGT#IGHV1-69-2 * 01 (SEQ ID NO: 25). IMGT#IGHV1-69-2 * 01 (SEQ ID NO: 25) shares the canonical form of CDR-H1 and CDR-H2 of the mouse 3D6 heavy chain. IMGT#IGHV1-69-2 * 01 (SEQ ID NO: 25) belongs to human heavy chain subgroup 1. An example of an acceptor sequence for the light chain is the human mature light chain variable region (PDB accession code human antibody ARX71335VL (SEQ ID NO: 82)). The variable light domain of 3D6 and the variable light domain of the ARX71335 antibody are also identical in length for the CDR-L1, CDR-L2, and CDR-L3 loops. An example of an acceptor sequence for the light chain is the human mature light chain variable region (IMGT#IGKV2-30 * 02 (SEQ ID NO: 27). IMGT#IGKV2-30 * 02 (SEQ ID NO: 27) has the same canonical class as mouse 3D6 for CDR-L1, CDR-L2, and CDR-L3. * 02 (SEQ ID NO:27) belongs to human kappa subgroup 2.

[0266] If more than one human acceptor antibody sequence is selected, a composite or hybrid of these acceptors can be used, and the amino acids used at different positions in the humanized light and heavy chain variable regions can be taken from any of the human acceptor antibody sequences used. For example, to humanize the 3D6 mature heavy chain variable region, IMGT#IGHV1-69-2 *The human mature heavy chain variable regions of 3D6-01 (SEQ ID NO: 25) and PDB accession code#2RCS-VH_huFrwk (SEQ ID NO: 75) were used as acceptor sequences. An example of a position that differs in these two acceptors is position H17 (T or S). A humanized version of the 3D6 heavy chain variable region may contain either amino acid at this position. For example, to humanize the 3D6 mature light chain variable region, the human mature light chain variable region (IMGT#IGKV2-30 * 02 (SEQ ID NO: 27) and PDB code #ARX71335-VL_huFrwk (SEQ ID NO: 82) were used as acceptor sequences. One example of a position that differs in these two acceptors is position L100 (Q or A). Humanized versions of the 3D6 light chain variable region can contain either amino acid at this position.

[0267] Particular amino acids from the human variable region framework residues may be selected for substitution based on their possible effect on the CDR conformation and / or binding to antigen, which may be examined by modeling, testing the characteristics of the amino acid at a particular position, or by experimental observation of the effect of substitution or mutagenesis of a particular amino acid.

[0268] For example, if an amino acid differs between a residue in the mouse variable region framework and a residue in the selected human variable region framework, the amino acid in the human framework may be (1) Direct non-covalent binding to the antigen, (2) adjacent to or within the CDR regions, as defined by Chothia but not by Kabat; (3) interact with the CDR region in some other way (e.g., within about 6 Å of the CDR region) (e.g., identified by modeling the light or heavy chain on the resolved structure of a homologous known immunoglobulin chain); or (4) Residues involved in the VL-VH interface Where it is reasonably predicted that this will be the case, amino acids from the equivalent framework from the mouse antibody may be substituted.

[0269] In one embodiment, humanized sequences are generated using QuikChange site-directed mutagenesis, a two-step PCR protocol that allows for the introduction of multiple mutations, deletions, and insertions [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682].

[0270] Framework residues of classes (1)-(3) as defined by Queen et al., U.S. Patent No. 5,530,101, are sometimes alternatively referred to as canonical and vernier residues. Framework residues that help define the conformation of the CDR loops are sometimes referred to as canonical residues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton & Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues that help define the conformation of the antigen-binding loops and play a role in fine-tuning the fit of the antibody to the antigen are sometimes referred to as veneer residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).

[0271] Other framework residues that are candidates for substitution are those that create potential glycosylation sites. Further candidates for substitution are amino acids in the acceptor human framework that are rare in human immunoglobulins at that position. These amino acids can be replaced with amino acids from the equivalent position in the mouse donor antibody or from the equivalent position in a more typical human immunoglobulin.

[0272] Another framework residue that is a candidate for substitution is the N-terminal glutamine residue (Q), which can be replaced with glutamic acid (E) to minimize the possibility of conversion to pyroglutamic acid [Y. Diana Liu, et al., 2011, J. Biol. Chem., 286: 11211-11217]. Conversion of glutamic acid (E) to pyroglutamic acid (pE) is slower than conversion from glutamine (Q). Due to the loss of a primary amine in the conversion of glutamine to pE, the antibody becomes more acidic. Incomplete conversion leads to antibody heterogeneity, which can be observed as multiple peaks using charge-based analytical methods. Differences in heterogeneity may represent loss of process control.

[0273] An example of a humanized antibody is the humanized form of murine 3D6, designated Hu3D6.

[0274] The murine antibody 3D6 comprises a mature heavy chain variable region and a mature light chain variable region having amino acid sequences comprising SEQ ID NO: 7 and SEQ ID NO: 11, respectively. The present invention provides humanized forms of the murine 3D6 antibody, which include ten exemplary humanized heavy chain mature variable regions (hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6VHvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)), as well as 56 exemplary humanized light chain mature variable regions (hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), hu3D6VLvb3 (SEQ ID NO: 85), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), hu3D6VLv2 L50D (SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).

[0275] Figures 2 and 3 of US 2020 / 0369755A1 show the alignment of the heavy and light chain variable regions, respectively, for mouse 3D6 and various humanized antibodies. Figures 9A and 9B of US 2020 / 0369755A1 show the alignment of the heavy chain variable region of mouse 3D6 with the heavy chain variable regions of various humanized antibodies. Figures 10A, 10B, 10C, and 10D of US 2020 / 0369755A1 show the alignment of the light chain variable region of hu3D6VLv2 with the light chain variable regions of various humanized antibodies.

[0276] The following 35 variable region framework positions were considered as candidates for substitution in the 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions, as further detailed in the Examples of U.S. Publication No. 2020 / 0369755A1, for reasons such as possible effects on CDR conformation and / or binding to antigen, interactions between heavy and light chains, mediating interactions with the constant region, being sites of desirable or undesirable post-translational modifications, being rare residues at that position in human variable region sequences and therefore potentially immunogenic, potentially causing aggregation, and other reasons: L7 (T7S), L10 (T10S), L15 (I15L), L17 (Q17E), L37 (L37Q), L45 (K45R), L5 (K5S), L6 (K6S), L7 (K7S), L8 (K8S), L9 (K9S), L10 (K9S), L15 (I15L), L17 (Q17E), L37 (L37Q), L45 (K45R), L5 (K9S), L6 (K9S), L7 (K9S), L8 (K9 ... 47(L47G, L47N, L47D, L47E, L47P, L47T, L47S, or L47A), L48(I48G or I48D), L49(Y49E), L83(L83V), L86(H86Y), L100(A100Q), L106(L106I), H1(Q1E), H5(Q5V), H11(L11V), H17(S17T), H20(L20I), H23(T23 K), H38(K38R), H42(E42G), H43(Q43K), H66(K66R), H67(A67V), H75(S75T), H76(N76D), H80(L80M), H81(Q81E), H82c(L82cV), H83(T83R), H91(Y91F), H93(A93S), H94(S94T), H108(T108L), and H109(L109V). The following nine variable region CDR positions were considered as candidates for substitution in the 56 exemplified human mature light chain variable regions and the 10 exemplified human mature heavy chain variable regions, as further detailed in the Examples of U.S. Publication No. 2020 / 0369755A1: L24 (K24R), L50 (L50E, L50D, L50G, or L50V), L52 (S52G), L54 (L54D, L54G, L54N, L54E, L54Q, L54K, L54R, L54T, L54V, or L54S), H28 (N28T), H54 (N54D), H56 (D56E), H58 (V58I), and H60 (D60E). In some humanized 3D6 antibodies, the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:87.In some humanized 3D6 antibodies, the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 149. In some humanized 3D6 antibodies, the Kabat-Chothia composite CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87. In some humanized 3D6 antibodies, the Kabat-Chothia composite CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 88. In some humanized 3D6 antibodies, the Kabat-Chothia composite CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 92. In some humanized 3D6 antibodies, the Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89. In some humanized 3D6 antibodies, the Kabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.

[0277] Here, as elsewhere, the first mentioned residue is the residue of a humanized antibody formed by grafting a Kabat CDR, or in the case of CDR-H1, a composite Chothia-Kabat CDR, onto a human acceptor framework, and the second mentioned residue is the residue proposed to replace that residue. Thus, within the variable region frameworks the first mentioned residue is human and within the CDRs the first mentioned residue is murine.

[0278] The exemplified antibodies include any permutation or combination of the exemplified mature heavy chain variable regions and mature light chain variable regions: VHvb1 / VLvb1, VHvb1 / VLvb2, VHvb1 / VLvb3, VHvb2 / VLvb1, VHvb2 / VLvb2, VHvb2 / VLvb3, VHvb3 / VLvb1, VHvb3 / VLvb2, VHvb3 / VLvb3, VHvb4 / VLvb1, VHvb4 / VLvb2, VHvb4 / VLvb3, VHvb5 / VLvb1, VHvb5 / VLvb2, VHvb5 / VLvb 3, including VHvb6 / VLvb1, VHvb6 / VLvb2, VHvb6 / VLvb3, VHvb7 / VLvb1, VHvb7 / VLvb2, VHvb7 / VLvb3. Exemplary antibodies include the following mature heavy chain variable regions: hu3D6VHvb1 (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO:77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO:146), hu3D6VHv1bA11 L82cV (SEQ ID NO:147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)) and humanized 3D6VL light chain variable region (hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), hu3D6VLvb3 (SEQ ID NO: 85), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), hu3D6VLv2 L50D (SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144)).

[0279] Exemplary antibodies include the following mature heavy chain variable regions: hu3D6VHvb1 (SEQ ID NO:76), hu3D6VHvb2 (SEQ ID NO:77), hu3D6VHvb3 (SEQ ID NO:78), hu3D6VHvb4 (SEQ ID NO:79), hu3D6Hvb5 (SEQ ID NO:80), hu3D6VHvb6 (SEQ ID NO:90), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHvb7 (SEQ ID NO:91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO:146), hu3D6VHv1bA11 L82cV (SEQ ID NO:147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)) and any of the humanized 3D6VL light chain variable regions (hu3D6VLv1 (SEQ ID NO: 20), hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv3 (SEQ ID NO: 22), and hu3D6VLv4 (SEQ ID NO: 22)). Exemplary antibodies include the following exemplary mature light chain variable regions: hu3D6VLvb1 (SEQ ID NO:83), hu3D6VLvb2 (SEQ ID NO:84), hu3D6VLvb3 (SEQ ID NO:85), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2 L54G (SEQ ID NO:94), hu3D6VLv2 L54N (SEQ ID NO:95), hu3D6VLv2 L54E (SEQ ID NO:96), hu3D6VLv2 L50E (SEQ ID NO:97), hu3D6VLv2 L54Q (SEQ ID NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO:100), hu3D6VLv2 L54R (SEQ ID NO:101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2G100Q (SEQ ID NO: 144)) and humanized 3D6 heavy chain variable region (hu3D6VHv1 (SEQ ID NO: 15), hu3D6VHv2 (SEQ ID NO: 16), hu3D6VHv1b (SEQ ID NO: 17), hu3D6VHv1bA11 (SEQ ID NO: 18), hu3D6VHv5 (SEQ ID NO: 19), hu3D6VHv1bA11B6G2 (SEQ ID NO: 46), hu3D6VHv1bA11B6H3 (SEQ ID NO: 47), hu3D6VHv1c (SEQ ID NO: 48), Any permutation or combination of hu3D6VHv1d (sequence number 49), hu3D6VHv1e (sequence number 50), hu3D6VHv1f (sequence number 51), hu3D6VHv3 (sequence number 52), hu3D6VHv3b (sequence number 53), hu3D6VHv3c (sequence number 54), hu3D6VHv4 (sequence number 55), hu3D6VHv4b (sequence number 56), and hu3D6VHv4c (sequence number 57)).

[0280] The invention provides an antibody in which the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54R (L2-DIM4, SEQ ID NO: 122). The invention provides an antibody in which the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54T (L2-DIM5, SEQ ID NO: 123). The invention provides an antibody in which the humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54R (L2-DIM4, SEQ ID NO: 122). The invention provides an antibody in which the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54G (L2-DIM3, SEQ ID NO: 121). The invention provides an antibody in which the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 S52G (L2-DIM9, SEQ ID NO: 110). The invention provides an antibody in which the humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with the humanized light chain variable region hu3D6VLv2 L54G (L2-DIM7, SEQ ID NO: 94). The present invention provides an antibody that combines the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) with the humanized light chain variable region hu3D6VLv2 L50G (L2-DIM22, SEQ ID NO: 103).

[0281] The present invention provides an antibody in which any one of the exemplary humanized heavy chain variable regions is combined with a human heavy chain constant region. An example of a human heavy chain constant region is provided as SEQ ID NO: 176 (IgG1:allotype G1m17,1). For example, SEQ ID NO: 178 represents the amino acid sequence of the mature heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype). For example, SEQ ID NO: 180 represents the amino acid sequence of the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with a bovine alpha-lactalbumin signal peptide at the N-terminus. The present invention provides an antibody in which any one of the exemplary humanized light chain variable regions is combined with a light chain constant region. An example of a light chain constant region is provided as SEQ ID NO: 177 (kappa). For example, SEQ ID NO: 179 represents the amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4kappa). For example, SEQ ID NO: 181 represents the amino acid sequence of the light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4kappa) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0282] The present invention relates to hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), or hu3D6VHv1bA11 hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), hu3D6VLv2 L50D (SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), or hu3D6VLv2Variants of the 3D6 humanized antibody are provided, in which the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity to G100Q (SEQ ID NO: 144). In some such antibodies, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty-one, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, thirty-nine, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty-one, forty-one, forty-two, forty-three, or all fourteen of the back mutations or other mutations in SEQ ID NOs: 76 to 80, SEQ ID NOs: 90 to 91, SEQ ID NOs: 146 to 148, SEQ ID NOs: 83 to 85, and SEQ ID NOs: 93 to 145.

[0283] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H93 is occupied by S and H94 is occupied by T. In some humanized 3D6 antibodies, positions H93 and H94 are occupied by S and T, respectively.

[0284] In some humanized 3D6 antibodies, position H91 in the VH region is occupied by F.

[0285] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by E, H5 is occupied by V, H11 is occupied by V, H20 is occupied by I, H23 is occupied by K, H38 is occupied by R, H42 is occupied by G, H43 is occupied by K, H66 is occupied by R, H75 is occupied by T, H76 is occupied by D, H81 is occupied by E, H108 is occupied by L, and H109 is occupied by V. In some humanized 3D6 antibodies, positions H1, H5, H11, H20, H23, H38, H42, H43, H66, H75, H76, H81, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, L, and V, respectively.

[0286] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H17 is occupied by T, H80 is occupied by M, and H83 is occupied by R. In some humanized 3D6 antibodies, positions H17, H80, and H83 in the VH region are occupied by T, M, and R, respectively.

[0287] In some humanized 3D6 antibodies, position H58 in the VH region is occupied by I.

[0288] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H28 is occupied by T and H67 is occupied by V. In some humanized 3D6 antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.

[0289] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H54 is occupied by D and H56 is occupied by E. In some humanized 3D6 antibodies, positions H54 and H56 in the VH region are occupied by D and E, respectively.

[0290] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H17 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, H54 is occupied by N or D, and H5 is occupied by N or D. 6 is occupied by D or E, H58 is occupied by V or I, H66 is occupied by K or R, H67 is occupied by A or V, H75 is occupied by S or T, H76 is occupied by N or D, H80 is occupied by L or M, H81 is occupied by Q or E, H83 is occupied by T or R, H91 is occupied by F or Y, H93 is occupied by S, H94 is occupied by T, H108 is occupied by T or L, and H109 is occupied by L or V.

[0291] In some humanized 3D6 antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively, as in huVHvb1. In some humanized 3D6 antibodies, positions H1, H5, H11, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T, L, and V, respectively, as in huVHvb2. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, R, G, K, I, R, T, D, M, E, R, S, T, L, and V, respectively, as found in huVHvb3. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, as found in huVHvb4. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, as found in huVHvb5.In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, F, S, T, L, and V, respectively, as found in huVHvb6. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, S, T, L, and V, respectively, as found in huVHvb7.

[0292] In some humanized 3D6 antibodies, position H60 is occupied by E, as in hu3D6VHv1bA11 D60E (h3D6VHvb8). In some humanized 3D6 antibodies, position H82C is occupied by V, as in hu3D6VHv1bA11 L82cV. In some humanized 3D6 antibodies, positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, as in hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9).

[0293] The heavy chain variable region of any of the above reference antibodies may be modified to further reduce immunogenicity, for example, in some of the humanized antibodies, position H80 is occupied by M and / or H82c is occupied by V.

[0294] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L7 is occupied by S, L10 is occupied by S, L15 is occupied by L, L83 is occupied by V, L86 is occupied by Y, and L106 is occupied by I. In some humanized 3D6 antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.

[0295] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L7 is T or S, L10 is T or S, L15 is I or L, L17 is Q or E, L24 is K or R, L37 is L or Q, L45 is K or R, L83 is L or V, L86 is H or Y, L100 is A or Q, and L106 is L or I.

[0296] In some humanized 3D6 antibodies, positions L7, L10, L15, L83, L86, and L106 in the VL region are occupied by S, S, L, V, Y, and I, respectively, as in huVLvb2. In some humanized 3D6 antibodies, positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100, and L106 in the VL region are occupied by S, S, L, E, R, Q, R, V, Y, Q, and I, respectively, as in huVLvb3.

[0297] The light chain variable region of any of the above reference antibodies may be modified to further reduce immunogenicity. For example, in some of the humanized antibodies, position L47 is occupied by G, N, D, E, P, T, S, or A, position L48 is occupied by G or D, position L49 is occupied by E, position L50 is occupied by E, D, G, or V, position L52 is occupied by G, and / or position L54 is occupied by D, G, N, E, Q, K, R, T, V, or S. The heavy chain variable region of any of the above reference antibodies may be modified to further reduce immunogenicity. For example, in some of the humanized antibodies, position H80 is occupied by M, and / or H82c is occupied by V.

[0298] In some humanized 3D6 antibodies, position L54 is occupied by D, as in hu3D6VLv2 L54D. In some humanized 3D6 antibodies, position L54 is occupied by G, as in hu3D6VLv2 L54G. In some humanized 3D6 antibodies, position L54 is occupied by N, as in hu3D6VLv2 L54N. In some humanized 3D6 antibodies, position L54 is occupied by E, as in hu3D6VLv2 L54E. In some humanized 3D6 antibodies, position L50 is occupied by E, as in hu3D6VLv2 L50E. In some humanized 3D6 antibodies, position L54 is occupied by Q, as in hu3D6VLv2 L54Q. In some humanized 3D6 antibodies, position L50 is occupied by D, as in hu3D6VLv2 L50D. In some humanized 3D6 antibodies, position L54 is occupied by K, as in hu3D6VLv2 L54K. In some humanized 3D6 antibodies, position L54 is occupied by R, as in hu3D6VLv2 L54R. In some humanized 3D6 antibodies, position L54 is occupied by T, as in hu3D6VLv2 L54T. In some humanized 3D6 antibodies, position L50 is occupied by G, as in hu3D6VLv2 L50G. In some humanized 3D6 antibodies, position L48 is occupied by G, as in hu3D6VLv2 I48G. In some humanized 3D6 antibodies, position L48 is occupied by D, as in hu3D6VLv2 I48D. In some humanized 3D6 antibodies, position L47 is occupied by G, as in hu3D6VLv2 L47G. In some humanized 3D6 antibodies, position L49 is occupied by E, as in hu3D6VLv2 Y49E. In some humanized 3D6 antibodies, position L54 is occupied by V, as in hu3D6VLv2 L54V. In some humanized 3D6 antibodies, position L54 is occupied by S, as in hu3D6VLv2 L54S.In some humanized 3D6 antibodies, position L52 is occupied by G, as in hu3D6VLv2 S52G. In some humanized 3D6 antibodies, position L47 is occupied by N, as in hu3D6VLv2 L47N. In some humanized 3D6 antibodies, position L47 is occupied by D, as in hu3D6VLv2 L47D. In some humanized 3D6 antibodies, position L47 is occupied by E, as in hu3D6VLv2 L47E. In some humanized 3D6 antibodies, position L47 is occupied by P, as in hu3D6VLv2 L47P. In some humanized 3D6 antibodies, position L47 is occupied by T, as in hu3D6VLv2 L47T. In some humanized 3D6 antibodies, position L47 is occupied by S, such as in hu3D6VLv2 L47S. In some humanized 3D6 antibodies, position L47 is occupied by A, such as in hu3D6VLv2 L47A. In some humanized 3D6 antibodies, position L50 is occupied by V, such as in hu3D6VLv2 L50V.

[0299] In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and R, respectively, as in hu3D6VLv2 L37Q_L50G_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and G, respectively, as in hu3D6VLv2 L37Q_L50G_L54G. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and G, respectively, as in hu3D6VLv2 L37Q_S52G_L54G. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and R, respectively, as in hu3D6VLv2 L37Q_S52G_L54R. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and T, respectively, as in hu3D6VLv2 L37Q_S52G_L54T. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively, as in hu3D6VLv2 L37Q_S52G_L54D.

[0300] In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and R, respectively, as in hu3D6VLv2 L37Q_L54R. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q_L54G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q_L54D. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q_L50G. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q_L50D. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and T, respectively, as in hu3D6VLv2 L37Q_L54T. In some humanized 3D6 antibodies, positions L37 and L52 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q_S52G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and E, respectively, as in hu3D6VLv2 L37Q_L54E.

[0301] In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, D, and G, respectively, as in hu3D6VLv2 L37Q_L50D_L54G. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, D, and R, respectively, as in hu3D6VLv2 L37Q_L50D_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, E, and G, respectively, as in hu3D6VLv2 L37Q_L50E_L54G. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, E, and R, respectively, as in hu3D6VLv2 L37Q_L50E_L54R.

[0302] In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_S52G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, D, and Q, respectively, as in hu3D6VLv2 L37Q_S52G_L54D_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, G, and Q, respectively, as in hu3D6VLv2 L37Q_L50D_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, R, and Q, respectively, as in hu3D6VLv2 L37Q_L50D_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, V, D, and Q, respectively, as in hu3D6VLv2 L37Q_L50V_L54D_G100Q.

[0303] In some humanized 3D6 antibodies, position L37 is occupied by Q, such as in hu3D6VLv2 L37Q. In some humanized 3D6 antibodies, position L100 is occupied by Q, such as in hu3D6VLv2 G100Q.

[0304] Some humanized 3D6 antibodies comprise a mature heavy chain variable region comprising CDR H1, CDR H2, and CDR H3 comprising SEQ ID NOs: 8, 9, and 10, respectively (with the proviso that position H28 may be occupied by N or T, H54 may be occupied by N or D, H56 may be occupied by D or E, position H58 may be occupied by V or I, and position H60 may be occupied by D or E), and CDR L1, CDR L2, and CDR L3 comprising SEQ ID NOs: 12, 13, and 14, respectively. and a mature light chain variable region comprising L3, with the proviso that position L24 may be occupied by K or R, position L50 may be occupied by L, E, D, G, or V, position L52 may be occupied by S or G, and position L54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S, and at least one of the following positions is occupied by the specified amino acid: H1 is occupied by Q, H5 is occupied by Q, H11 is occupied by L, H20 is occupied by L, H23 is occupied by T, H38 is occupied by K, H75 is occupied by S, H56 is occupied by E, and H58 is occupied by I. H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L83 is occupied by L, L86 is occupied by H, L100 is occupied by Q, and L106 is occupied by L.

[0305] Some humanized 3D6 antibodies comprise the three light chain CDRs and the three heavy chain CDRs of monoclonal antibody 3D6, where 3D6 is a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7, and a light chain variable region having an amino acid sequence comprising SEQ ID NO:11, with the proviso that: position H27 may be occupied by F or Y; position H28 may be occupied by N or T; position H29 may be occupied by I or F; position H30 may be occupied by K or T; position H51 may be occupied by I or V; position H54 may be occupied by N or D; position H60 may be occupied by D, A, or E; position H61 may be occupied by P or E; and position H102 may be occupied by F or Y; Position L50 may be occupied by L, E, D, G, or V, position L52 may be occupied by S or G, position L54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S, and at least one of the following positions is occupied by the specified amino acid: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L100 is occupied by Q, H60 is occupied by E, and H82c is occupied by V.

[0306] In some humanized 3D6 antibodies, the variable heavy chain has 85% or more identity to the human sequence. In some humanized 3D6 antibodies, the variable light chain has 85% or more identity to the human sequence. In some humanized 3D6 antibodies, the variable heavy chain and the variable light chain each have 85% or more identity to the human germline sequence. In some humanized 3D6 antibodies, the three heavy chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 8, 9, and 10, respectively) and the three light chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 12, 13, and 14); except that position H28 is occupied by N or T, position H54 is occupied by N or D, position H56 is occupied by D or E, position H58 is occupied by V or I, position H60 is occupied by D or E, position L24 is occupied by K or R, position L50 is occupied by L, E, D, G, or V, position L52 is occupied by S or G, and position L54 is occupied by L, D, G, N, E, Q, K, R, T, V, or S. In some humanized 3D6 antibodies, the Kabat / Chothia composite CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86. In some humanized 3D6 antibodies, the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 92, or SEQ ID NO: 149. In some humanized 3D6 antibodies, the Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89. In some humanized 3D6 antibodies, the Kabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.

[0307] The CDR regions of such humanized antibodies may be identical or substantially identical to the CDR regions of 3D6. The CDR regions may be defined by any conventional definition (e.g., Chothia or a combination of Chothia and Kabat), but are preferably as defined by Kabat.

[0308] Variable region framework positions are according to Kabat numbering unless otherwise stated. Other such variants typically differ in sequence from the illustrated Hu3D6 heavy and light chains by a small number of substitutions, deletions, or insertions (e.g., usually no more than 1, 2, 3, 5, 10, or 15). Such differences are typical in the frameworks, but may also occur in the CDRs.

[0309] A further possibility for variation of humanized 3D6 variants is further back mutations in the variable region framework. Many of the framework residues that do not contact the CDRs of the humanized mAb can accommodate substitutions of amino acids from the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and many potential CDR contact residues are also amenable to substitution. Furthermore, amino acids in the CDRs can also be changed, for example with residues found at the corresponding positions of the human acceptor sequence used to provide the variable region framework. Furthermore, a different human acceptor sequence can be used, for example in the heavy and / or light chain. When a different acceptor sequence is used, one or more of the back mutations recommended above may not occur, since the corresponding donor and acceptor residues are already the same without back mutations.

[0310] Preferably, the substitutions or back mutations in the humanized 3D6 variants (whether conservative or not) do not substantially affect the binding affinity or potency of the humanized mAb, ie, its properties to bind to tau.

[0311] The humanized 3D6 antibodies are further characterized by their property of binding to phosphorylated and non-phosphorylated tau, as well as misfolded / aggregated forms of tau.

[0312] D. Chimeric Antibodies and Veneering Forms The present invention further provides chimeric and veneered forms of non-human antibodies, particularly the 3D6 antibody of the examples.

[0313] A chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the mouse antibody and are approximately 2 / 3 of the human sequence. In one embodiment, the chimeric 3D6 antibody has the heavy chain amino acid sequence of SEQ ID NO:72 and the light chain amino acid sequence of SEQ ID NO:73.

[0314] A veneered antibody is a type of humanized antibody that retains some, usually all, of the CDRs of a non-human antibody and some of the non-human variable region framework residues, but replaces other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions in a human antibody sequence. The result is an antibody in which the CDRs are completely or substantially from a non-human antibody and the variable region framework of the non-human antibody has been made more human-like by the substitutions. Veneered forms of the 3D6 antibody are encompassed by the present invention.

[0315] E. Human antibodies Human antibodies against tau or fragments thereof (e.g., amino acid residues 199-213 and / or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 and / or 320-334 of SEQ ID NO:1, respectively), or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1, or any combination of two, any combination of three, or all four thereof) are provided by various techniques described below. Some human antibodies are selected by competitive binding experiments or by the phage display method of Winter, supra, or other methods to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the Examples. Human antibodies may be screened for a particular epitope specificity by using only a fragment of tau as the target antigen (such as a tau fragment comprising only amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or a tau fragment comprising only amino acid residues 259-268, or 290-299, or 321-330, or 353-362 of SEQ ID NO:1), and / or by screening antibodies against a collection of tau variants (such as tau variants comprising various mutations within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or tau variants comprising various mutations within amino acid residues 259-268, or 290-299, or 321-330, or 353-362 of SEQ ID NO:1).

[0316] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); U.S. Patent No. 4,634,664 to Oestberg; and U.S. Patent No. 4,634,666 to Engleman et al., the use of transgenic mice containing human immunoglobulin genes (see, e.g., Longberg et al., International Patent Publication No. WO 93 / 12227 (1993); U.S. Patent No. 5,877,397; U.S. Patent No. 5,874,299; U.S. Patent No. 5,814,318; U.S. Patent No. 5,789,650; U.S. Patent No. 5,770,429; U.S. Patent No. 5,661,016; U.S. Patent No. 5,633,425; U.S. Patent No. 5,625,126; U.S. Patent No. 5,569,825; U.S. Patent No. 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996); and Kucherlapati, WO 91 / 10741 (1991)), phage display methods (see, e.g., Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047; U.S. Pat. Nos. 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and 5,565,332); and the methods described in WO 2008 / 081008 (e.g., immortalizing memory B cells isolated from a human, e.g., with EBV, screening for the desired property, and cloning and expressing the recombinant form).

[0317] F. Selection of Constant Regions The heavy and light chain variable regions of a chimeric, veneered, or humanized antibody may be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is desired. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, whereas human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa. Numbering conventions for constant regions include EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991), IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol., 29, 185-203 (2005), and IMGT exon numbering (Lefranc, supra).

[0318] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, such as the lysine at the C-terminus of the heavy chain, may be deleted or derivatized in some or all of these molecules. Substitutions may be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, for example, U.S. Patent No. 5,624,821 to W inter et al.; U.S. Patent No. 5,834,597 to Tso et al.; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006) or to increase half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions to increase antibody half-life include Gln at position 250 and / or Leu at position 428 (EU numbering is used in this paragraph for constant regions). Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce the affinity of Fcγ receptors, particularly FcγRI receptors (see, e.g., U.S. Patent No. 6,624,821). Alanine substitutions at positions 234, 235, and 237 of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. Optionally, positions 234, 236, and / or 237 of human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, e.g., U.S. Patent No. 5,624,821). Some antibodies use mutations at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 according to EU numbering of human IgG1. In some antibodies, mutations are used at one or more of positions 318, 320, and 322 according to EU numbering of human IgG1. In some antibodies, positions 234 and / or 235 are substituted with alanine and / or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4.

[0319] Antibodies can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as single-chain antibodies in which the heavy and light chain mature variable domains are linked via a spacer.

[0320] Human constant regions exhibit allotypic and isoallotypic variations between different individuals, i.e., constant regions may differ in different individuals at one or more polymorphic locations. Isoallotypes differ from allotypes in that serum recognizes isoallotypes that bind to non-polymorphic regions of one or more other isotypes. Thus, for example, another heavy chain constant region is the constant region of IgG1 G1m3, with or without a C-terminal lysine. Reference to a human constant region includes constant regions with either natural allotypes or permutations of residues that occupy the positions of natural allotypes. An example of a heavy chain constant region is SEQ ID NO: 176 (with or without a C-terminal lysine), and an example of a light chain constant region is SEQ ID NO: 177.

[0321] G. Expression of Recombinant Antibodies Many methods are known for producing chimeric and humanized antibodies using antibody expressing cell lines (e.g., hybridomas). For example, the immunoglobulin variable regions of antibodies can be cloned and sequenced using well-known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are used with the VH recognizing leader peptide containing the translation initiation codon as the 5' primer and the 3' primer specific for the g2b constant region. Exemplary primers are described in U.S. Patent Publication No. 2005 / 0009150 by Schenk et al. (hereinafter "Schenk"). Sequences from multiple independently derived clones can be compared to ensure that no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing the VH fragment obtained by 5' RACE RT-PCR and 3' g2b specific primers.

[0322] The light chain variable VL region can be cloned in a similar manner. In one approach, a consensus primer set is designed for the amplification of the VL region using a 5' primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3' primer specific to the Ck region downstream of the VJ joining region. In the second approach, a 5'RACE RT-PCR method is used to clone the cDNA encoding the VL. Exemplary primers are described in Schenk, supra. The cloned sequence is then combined with sequences encoding the constant region of human (or other non-human species).

[0323] In one approach, the heavy and light chain variable regions are reengineered to encode the respective VDJ junction or splice donor sequence downstream of the VJ junction and cloned into mammalian expression vectors, such as pCMV-hγ1 for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human γ1 and Ck constant regions as exon fragments downstream of the inserted variable region cassettes. After sequence verification, the heavy and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned medium is harvested 48 hours after transfection and assayed for antibody production by Western blot analysis or antigen binding by ELISA. The chimeric antibodies are humanized as described above.

[0324] Chimeric, veneered, humanized, and human antibodies are usually produced by recombinant expression. Recombinant polynucleotide constructs contain expression control sequences operably linked to the coding sequences of the antibody chains, usually including naturally associated control elements or heterologous expression control elements, such as promoters. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vectors are incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and for the recovery and purification of cross-reacting antibodies.

[0325] These expression vectors are usually replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, such as ampicillin-resistance or hygromycin-resistance, to permit detection of those cells transformed with the desired DNA sequences.

[0326] E. coli is one of the prokaryotic hosts useful for the expression of antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is a yeast host that includes suitable vectors with expression control sequences, origins of replication, termination sequences, etc., as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

[0327] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing myelomas including Sp2 / 0 and NS0. The cells can be non-human. Expression vectors for these cells can include expression control sequences, such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0328] Alternatively, antibody coding sequences can be incorporated as a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. Nos. 5,741,957; 5,304,489; and 5,849,992). Suitable transgenes include light and / or heavy chain coding sequences operably linked to a promoter and enhancer derived from a mammary gland specific gene such as casein or beta-lactoglobulin.

[0329] The vector containing the DNA segment of interest can be transferred into the host cell by a method that depends on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For the generation of transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be integrated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs) and the nucleus of the cell transferred to the enucleated oocyte.

[0330] After introducing the vectors encoding the heavy and light chains of the antibody into the cell culture, the pool of cells can be screened for growth productivity and product quality in serum-free medium. The top producing cell pool can then be subjected to FACS-based single cell cloning to generate monoclonal lines. Specific productivities of more than 50 pg or more than 100 pg per cell per day, corresponding to product titers of more than 7.5 g / culture, can be used. The antibodies produced by the single cell clones can also be tested with binding assays such as turbidity, filtration properties, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and ELISA or Biascore. The selected clones can then be deposited in multiple vials and stored frozen for further use.

[0331] After expression, antibodies can be purified by standard methods in the art, including Protein A capture, HPLC purification, column chromatography, gel electrophoresis, etc. (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0332] Methods for commercial production of antibodies may be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single cell cloning, cell banking, use of selection markers for copy number amplification, CHO terminators, or improved protein titer (see, e.g., U.S. Pat. Nos. 5,786,464; 6,114,148; 6,063,598; 7,569,339; International Patent Publication Nos. 2004 / 050884; 2008 / 012142; 2008 / 012142; 2005 / 019442; 2008 / 107388; 2009 / 027471; and U.S. Pat. No. 5,888,809).

[0333] H. Antibody Screening Assays Antibodies can be initially screened for the intended binding specificity as described above. Similarly, active immunogens can be screened for the property of inducing antibodies with that binding specificity. In this case, the active immunogen is used to immunize laboratory animals and the resulting sera tested for the appropriate binding specificity.

[0334] Antibodies with desired binding specificity can then be tested in cell and animal models. The cells used for this screening are preferentially neuronal cells. A cell model of tau pathology has been reported in which neuroblastoma cells are transfected with the four repeat domains of tau, optionally with mutations associated with tau pathology (e.g. delta K280, see Khlistunova, Current Alzheimer Research 4, 544-546 (2007)). In another model, tau is induced in neuroblastoma N2a cell line by adding doxycycline. This cell model allows testing the toxicity of tau to cells in soluble or aggregated state, the appearance of tau aggregates after switching on tau gene expression, the dissolution of tau aggregates after switching off gene expression again, and the effectiveness of antibodies in inhibiting the formation of tau aggregates or their disaggregation.

[0335] Antibodies or active immunogens can also be screened in transgenic animal models of tau-related diseases. Such transgenic animals can contain a tau transgene (e.g., any of the human isoforms) and, optionally, a human APP transgene, such as a kinase that phosphorylates tau, ApoE, presenilin, or alpha-synuclein. Such transgenic animals are treated to develop at least one sign or symptom of a tau-related disease.

[0336] An exemplary transgenic animal is the K3 strain of mice (Itner et al., Proc. Natl. Acad. Sci. USA 105(41):15997-6002 (2008)). These mice have a K369I mutation (which is associated with Pick's disease) and a human tau transgene with a Thy1.2 promoter. This model shows a rapid course of neurodegeneration, motor impairment, and degeneration of afferent fibers and cerebellar granule cells. Another exemplary animal is the JNPL3 strain of mice. These mice have a P301L mutation (which is associated with frontotemporal dementia) and a human tau transgene with a Thy1.2 promoter (Taconic, Germantown, NY, Lewis, et al., Nat Genet. 25:402-405 (2000)). These mice have a slower course of neurodegeneration. These mice develop neurofibrillary tangles in several brain regions and spinal cord (incorporated herein by reference in its entirety). This is an excellent model for studying the consequences of tangle development and for screening therapies that may inhibit the production of these aggregates. Another advantage of these animals is the relatively early onset of pathology. In homozygous strains, behavioral abnormalities associated with tau pathology can be observed at least as early as 3 months, but the animals remain relatively healthy until at least 8 months of age. In other words, at 8 months of age, the animals are able to walk around, feed themselves, and perform behavioral tasks well enough to allow monitoring of therapeutic efficacy. Effective immunization of these mice with AI wI KLH-PHF-1 for 6-13 months resulted in titers of approximately 1,000 and showed fewer neurofibrillary tangles, less pSer422, and reduced weight loss compared to untreated control mice (ice).

[0337] The activity of the antibody or effective agent can be evaluated by various criteria, including reduction of the amount of total or phosphorylated tau, reduction of other pathological features, such as amyloid deposits of Aβ, and inhibition or delay of behavioral disorders. Active immunogens can also be tested for induction of antibodies in serum. Passive and active immunogens can be tested for passage of antibodies to the brain through the blood-brain barrier of transgenic animals. Antibodies or antibody-inducing fragments can also be tested in non-human primates that naturally or through induction develop symptoms of diseases characterized by tau. Tests with antibodies or effective agents are usually performed with controls in which parallel experiments are performed except that the antibody or effective agent is not present (e.g., replaced by vehicle). Reduction, delay, or inhibition of signs or symptoms of the disease caused by the antibody or effective agent under test can then be evaluated compared to the control.

[0338] I. Methods of Using the Antibodies of the Invention The antibodies or antigen-binding fragments thereof described herein may inhibit or reduce internalization of tau by cells, inhibit or reduce tau-induced toxicity, reduce or delay the onset of behavioral disorders, inhibit or reduce the levels of markers of tau pathology, or inhibit or reduce the onset of tau pathology.

[0339] Also provided herein is a method of reducing cellular internalization of tau in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces cellular internalization of tau, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0340] In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces tau internalization by cells by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% (e.g., compared to the level of tau internalization in the subject prior to administration, or compared to the level of tau internalization in a subject to which the antibody or antigen-binding fragment thereof is not administered). In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces tau internalization by cells by about 10% to about 99%, about 20% to about 90%, about 30% to about 80%, about 40% to 80%, or about 50% to 75% (e.g., compared to the level of tau internalization in a subject prior to administration, or compared to the level of tau internalization in a subject to which the antibody or antigen-binding fragment thereof has not been administered). In some embodiments, administration results in a reduction (e.g., compared to the level of tau internalization in the subject prior to administration, or compared to the level of tau internalization in a subject to which the antibody or antigen-binding fragment thereof was not administered) of about 10% to about 99% (e.g., about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 50 ... % to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 99%, about 20% to about 95%,About 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to About 30%, about 20% to about 25%, about 25% to about 99%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, About 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 99%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to About 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, About 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to About 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, About 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to About 60%, about 50% to about 55%, about 55% to about 99%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 60% to about 95%,About 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to A decrease of about 80%, about 70% to about 75%, about 75% to about 99%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% occurs.

[0341] Also provided herein is a method of reducing tau-induced toxicity in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces tau-induced toxicity, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0342] In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces tau-induced toxicity by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% (e.g., compared to the level of tau-induced toxicity in the subject prior to administration, or compared to the level of tau-induced toxicity in a subject to which the antibody or antigen-binding fragment thereof was not administered). In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces tau-induced toxicity by about 10% to about 99%, about 20% to about 90%, about 30% to about 80%, about 40% to 80%, or about 50% to 75% (e.g., compared to the level of tau-induced toxicity in the subject prior to administration, or compared to the level of tau-induced toxicity in a subject to which the antibody or antigen-binding fragment thereof was not administered). In some embodiments, administration results in about a 10% to about 99% reduction (e.g., about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 50%, about 10% to about 50%, about 10% to about 50%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 90%, about 10% to about 90%, about 10% to about 90%, about 10% to about 90%, about 10% to about 10 ... % to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 99%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%,About 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 99%, about 25% to about 95%, about 25% to about 90%, about 25% to About 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 99%, About 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to About 35%, about 35% to about 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, About 35% to about 40%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to About 45%, about 45% to about 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 99%, About 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 99%, about 55% to about 95%, about 55% to about 90%, about 55% to About 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%,About 60% to about 65%, about 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 99%, A decrease of about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% occurs.

[0343] Also provided herein is a method of reducing or delaying the onset of a behavioral disorder in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that results in a reduction or delay in the onset of the behavioral disorder, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0344] In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces or delays the onset of the behavioral disorder by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% (e.g., compared to the level of the behavioral disorder in the subject prior to administration or compared to the level of the behavioral disorder in a subject to which the antibody or antigen-binding fragment thereof is not administered). In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces or delays the onset of the behavioral disorder by about 10% to about 99%, about 20% to about 90%, about 30% to about 80%, about 40% to 80%, or about 50% to 75% (e.g., compared to the level of the behavioral disorder in the subject prior to administration, or compared to the level of the behavioral disorder in a subject to which the antibody or antigen-binding fragment thereof is not administered). In some embodiments, administration results in about a 10% to about 99% reduction (e.g., about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 1 0% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 99%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%,About 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 99%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to About 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 99%, about 30% to about 95%, About 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to About 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, About 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to About 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 99%, about 50% to about 95%, About 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 99%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to About 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%,About 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 99%, about 75% to A decrease of about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% occurs.

[0345] Also provided herein is a method of reducing the level of a marker of tau pathology in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces the level of the marker of tau pathology, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0346] In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces the level of a marker of tau pathology by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% (e.g., compared to the level of the marker of tau pathology in the subject prior to administration, or compared to the level of the marker of tau pathology in a subject to which the antibody or antigen-binding fragment thereof is not administered). In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces the level of a marker of tau pathology by about 10% to about 99%, about 20% to about 90%, about 30% to about 80%, about 40% to 80%, or about 50% to 75% (e.g., compared to the level of the marker of tau pathology in the subject prior to administration, or compared to the level of the marker of tau pathology in a subject to which the antibody or antigen-binding fragment thereof is not administered). In some embodiments, administration results in about a 10% to about 99% reduction (e.g., about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 50%, about 10% to about 55%, about 10% to about 50%, about 10% to about 50%, about 10% to about 65%, about 10% to about 60%, about 10% to about 75%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 90%, about 10% to about 15 ... 0% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 99%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%,About 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 99%, about 25% to About 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, About 25% to about 30%, about 30% to about 99%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to About 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, About 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to About 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, About 45% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 99%, about 55% to About 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%,About 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, A decrease of about 75% to about 99%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% occurs.

[0347] Also provided herein is a method of reducing the development of tau pathology in a subject, the method comprising administering to a subject in need thereof an amount of an antibody or antigen-binding fragment thereof that reduces the development of tau pathology, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising an LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.

[0348] In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces the onset of tau pathology by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% (e.g., compared to the level of onset of tau pathology in the subject prior to administration, or compared to the level of onset of tau pathology in a subject to which the antibody or antigen-binding fragment thereof is not administered). In some embodiments, administration of an antibody or antigen-binding fragment thereof described herein reduces the onset of tau pathology by about 10% to about 99%, about 20% to about 90%, about 30% to about 80%, about 40% to 80%, or about 50% to 75% (e.g., compared to the level of onset of tau pathology in the subject prior to administration, or compared to the level of onset of tau pathology in a subject to which the antibody or antigen-binding fragment thereof was not administered). In some embodiments, administration results in about a 10% to about 99% reduction (e.g., about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%) in the level of onset of tau pathology in the subject prior to administration, or in a subject to which the antibody or antigen-binding fragment thereof was not administered. , about 10% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% ~ about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 99%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%,About 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 99%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to About 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 99%, about 30% to about 95%, About 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to About 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, About 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to About 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 99%, about 50% to about 95%, About 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 99%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to About 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%,About 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 99%, about 75% to A decrease of about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 85% to about 99%, about 85% to about 95%, about 85% to about 90%, about 90% to about 99%, about 90% to about 95%, or about 95% to about 99% occurs.

[0349] IV. Patients Suitable for Treatment The presence of neurofibrillary tangles has been found in several diseases, including Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, and progressive supranuclear palsy (PSP). The present regime may also be used to treat or prevent any of these diseases. Due to the broad association between neurological diseases and pathologies and tau, the present regime can be used to treat or prevent any subject that exhibits high levels of tau or phosphorylated tau (e.g., in CSF) compared to the average value of individuals without neurological disease. The present regime can also be used to treat or prevent neurological disease in individuals with tau mutations associated with neurological disease. The present method is particularly suitable for treating or preventing Alzheimer's disease, particularly in patients.

[0350] Patients suitable for treatment include individuals at risk for the disease but who do not show symptoms, and those who currently show symptoms. Patients at risk for the disease include those with a known genetic risk for the disease. Such individuals include those who have relatives who have already experienced the disease, and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk include mutations in tau, such as those mentioned above, and mutations in other genes associated with neurological diseases. For example, alleles of ApoE in heterozygous and even homozygous forms are associated with risk of Alzheimer's disease. Other markers of risk for Alzheimer's disease include mutations in the APP gene, particularly at positions 717 and 670 and 671, called the Hardy and Swedish mutations, respectively, mutations in the presenilin genes, PS1 and PS2, family history of AD, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by PET imaging, characteristic dementia, and the presence of the risk factors mentioned above. In addition, many diagnostic tests are available to identify individuals with AD. These include measuring the levels of CSF tau or phosphotau and Aβ42. Elevated tau or phosphotau and decreased Aβ42 levels indicate the presence of AD. Some mutations have been linked to Parkinson's disease. There are mutations in other genes associated with Parkinson's disease, such as Ala30Pro or Ala53, or leucine-rich repeat kinase, PARK8. Individuals may also be diagnosed with any of the above-mentioned neurological disorders by DSM IV TR criteria.

[0351] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, 30). However, it is usually not necessary to begin treatment until the patient reaches 40, 50, 60, or 70 years of age. Treatment usually entails multiple administrations over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response decreases, booster administration is indicated. In the case of potential Down's syndrome patients, treatment can begin before birth by administering therapeutic agents to the mother or soon after birth.

[0352] V. Nucleic acids The present invention further provides nucleic acids encoding any of the above-mentioned heavy and light chains (e.g., SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 76-80, SEQ ID NO: 90-91, SEQ ID NO: 146-148, SEQ ID NO: 83-85, SEQ ID NO: 93-145, and SEQ ID NO: 178-181). An example of a nucleic acid encoding a heavy chain of the present invention is SEQ ID NO: 182, and an example of a nucleic acid encoding a light chain of the present invention is SEQ ID NO: 183. Optionally, such nucleic acids may further encode a signal peptide and be expressed with the signal peptide linked to the variable region. The coding sequence of the nucleic acid may be operably linked to control sequences for ensuring expression of the coding sequence, such as a promoter, an enhancer, a ribosome binding site, a transcription termination signal, and the like. The control sequences may include a promoter, e.g., a prokaryotic promoter or a eukaryotic promoter. The nucleic acids encoding the heavy or light chains may be codon-optimized for expression in a host cell. The nucleic acids encoding the heavy and light chains may encode selectable genes. The nucleic acids encoding the heavy and light chains may occur in isolated form or may be cloned into one or more vectors. The nucleic acids can be synthesized, for example, by solid-state synthesis of overlapping oligonucleotides or by PCR. The nucleic acids encoding the heavy and light chains can be joined as one contiguous nucleic acid, for example in an expression vector, or can be separate, for example each cloned into its own expression vector.

[0353] VI. Conjugated Antibodies Conjugated antibodies that specifically bind to antigens such as tau are useful for detecting the presence of tau in patients with Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial to monitor and evaluate the efficacy of therapeutic agents used to treat patients diagnosed with progressive supranuclear palsy (PSP); inhibiting or reducing tau aggregation; inhibiting or reducing tau fibril formation; reducing or removing tau deposits; stabilizing a non-toxic conformation of tau; or to treat Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease The antibodies are useful for treating or preventing chronic pulmonary disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). For example, such antibodies may be conjugated to other therapeutic moieties, other proteins, other antibodies, and / or other detectable labels. See International Patent Publication No. WO 03 / 057838; U.S. Patent No. 8,455,622.Such a therapeutic moiety may be any agent that may be used to treat, combat, ameliorate, prevent, or ameliorate an undesirable condition or disease in a patient, such as Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP).

[0354] The conjugated therapeutic moiety may comprise a cytotoxic agent, a cytostatic agent, a neurotrophic agent, a neuroprotective agent, a radiotherapeutic agent, an immunomodulatory agent, or any biologically effective agent that promotes or enhances the activity of the antibody. A cytotoxic agent may be any agent that is toxic to cells. A cytostatic agent may be any agent that inhibits cell proliferation. A neurotrophic agent may be any agent, including chemical or proteinaceous agents, that promotes the maintenance, proliferation, or differentiation of neurons. A neuroprotective agent may be an agent, including chemical or proteinaceous agents, that protects neurons from acute insults or degenerative processes. An immunomodulatory agent may be any agent that stimulates or inhibits the development or maintenance of an immune response. A radiotherapeutic agent may be any molecule or compound that emits radiation. When such a therapeutic moiety is coupled to an antibody specific for tau, such as the antibodies described herein, the coupled therapeutic moiety has a more specific affinity for cells affected by a tau-related disease compared to normal cells. As a result, administration of the conjugated antibody directly targets cancer cells while minimizing damage to surrounding normal, healthy tissue. This can be particularly useful for therapeutic moieties that are too toxic to administer alone. Furthermore, smaller amounts of the therapeutic moiety can be used.

[0355] Some such antibodies can be modified to act as immunotoxins. See, e.g., U.S. Pat. No. 5,194,594. For example, ricin, a plant-derived cytotoxin, can be coupled to antibodies using the bifunctional reagents S-acetylmercaptosuccinic anhydride and succinimidyl 3-(2-pyridyldithio)propionate. See Pietersz et al., Cancer Res. 48(16):4469-4476 (1998). This coupling results in loss of ricin's B-chain binding activity without compromising the toxic potential of ricin's A-chain or the activity of the antibody. Similarly, saporin, an inhibitor of ribosome assembly, can be coupled to antibodies via a disulfide bond between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

[0356] Some such antibodies may be conjugated to a radioisotope, such as yttrium. 90 (90Y), Indium 111 (111In), 131 I, 99 mTc, radioactive silver-111, radioactive silver-199, and bismuth 213 . Attachment of radioisotopes to antibodies can be performed using conventional bifunctional chelates. For attachment of radioactive silver-111 and radioactive silver-199, sulfur-based bonds can be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Attachment of silver radioisotopes can include reducing immunoglobulins with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used, which reacts with the isotope to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0357] Some such antibodies may be conjugated to other therapeutic moieties. Such therapeutic moieties may be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective. For example, the antibodies may be conjugated to a toxic chemotherapeutic agent, such as maytansine, geldanamycin, a tubulin inhibitor, such as a tubulin binding agent (e.g., auristatin), or a minor groove binding agent, such as calicheamicin. Other representative therapeutic moieties include agents known to be useful in the treatment, management, or amelioration of Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP).

[0358] Antibodies can also be coupled to other proteins. For example, antibodies can be coupled to fynomers. Fynomers are small molecule binding proteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They can be stable and soluble, and they can lack cysteine ​​residues and disulfide bonds. Fynomers can be engineered to bind target molecules with the same affinity and specificity as antibodies. They are suitable for the creation of antibody-based multispecific fusion proteins. For example, fynomers can be fused to the N-terminus and / or C-terminus of antibodies to create bispecific and trispecific FynomAbs with different structures. Fynomers can be selected using fynomer libraries through screening techniques using FACS, Biacor, and cell-based assays that allow efficient selection of fynomers with optimal properties. Examples of finomers are disclosed in Grabulovski et al., J. Biol. Chem. 282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner et al., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013); and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

[0359] The antibodies disclosed herein may also be coupled or conjugated to one or more other antibodies (e.g., to form an antibody heteroconjugate). Such other antibodies may bind to a different epitope within tau or may bind to a different tau antigen.

[0360] The antibody may also be coupled to a detectable label. Such an antibody may be used to diagnose and / or evaluate the efficacy of treatments for, for example, Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP). Such antibodies are particularly useful for making such determinations in a subject having or susceptible to Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP), or a suitable biological sample obtained from such a subject.Exemplary detectable labels which can be coupled or bound to an antibody include various enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radioactive silver-111, radioactive silver-199, bismuth. 213 , iodine ( 131 I, 125 I, 123 I, 121 I), Carbon ( 14 C), sulfur ( 5 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In, 111 In), Technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), Molybdenum ( 99 Mo), Xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr,32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117 tin; positron-emitting metals in various positron emission tomography applications; non-radioactive paramagnetic metal ions; and metals radiolabeled or conjugated to certain radioisotopes.

[0361] The attachment of radioisotopes to antibodies can be carried out using conventional bifunctional chelates. For radioactive silver-111 and radioactive silver-199, sulfur-based linkers can be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Attachment of silver radioisotopes can include reducing immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used, which reacts with the isotope to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0362] Therapeutic moieties, other proteins, other antibodies, and / or detectable labels can be coupled or conjugated to the antibodies of the invention directly or indirectly through an intermediate (eg, a linker). For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);”Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers.Different linkers can be used that release the coupled therapeutic moiety, protein, antibody, and / or detectable label under acidic or reducing conditions upon exposure to a specific protease or other defined conditions.

[0363] VII. Pharmaceutical Compositions and Methods of Use In preventive applications, the antibody or the agent for inducing the antibody, or the pharmaceutical composition containing the same, is administered to a patient who is susceptible to or at risk of the disease (e.g., Alzheimer's disease) in a regime (dosage, frequency, and route of administration) effective to reduce the risk, reduce the severity, or delay the onset of at least one sign or symptom of the disease. In particular, the regime is preferably effective to inhibit or delay tau or phosphotau and paired filaments formed therefrom in the brain, and / or to inhibit or delay their toxic effects, and / or to inhibit or delay the onset of behavioral disorders. In therapeutic applications, the antibody or the agent for inducing the antibody is administered to a patient who is suspected of or already suffers from the disease (e.g., Alzheimer's disease) in a regime (dosage, frequency, and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease. In particular, the regime is preferably effective to reduce or at least inhibit further increase in the level of tau, phosphotau, or paired filaments formed therefrom, associated toxicity, and / or behavioral impairment. Behavioral impairment can be assessed from cognitive scales (such as ADAS Cog) or Mini-Mental State Examination. Treatment can be supported by improvement in such scales, which optionally results in such scales falling within the normal range, reducing the decline, or maintaining a constant value. Prevention can be supported by reduction or delay or absence of decline in such scales. Treatment and prevention can also be supported by changes in the level of one or more markers, including those disclosed in the examples.

[0364] A regime is considered to be therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the average outcome of a comparable control population of patients not treated with the methods of the invention, or if a favorable outcome is demonstrated in treated patients compared to control patients in controlled clinical trials at a level of P<0.05 or 0.01, or even 0.001 (e.g., Phase II, Phase II / III, or Phase III trials).

[0365] The effective dose will vary depending on many different factors, such as the means of administration, the target site, the physiological condition of the patient, whether the patient is an ApoE carrier, whether the patient is a human or an animal, other medicinal applications being taken, and whether the treatment is preventative or therapeutic.

[0366] Exemplary dosage ranges for the antibody are about 0.01-60 mg / kg, or about 0.1-3 mg / kg, or 0.15-2 mg / kg, or 0.15-1.5 mg / kg of patient body weight. The antibody may be administered at such dosages daily, every other day, weekly, every two weeks, monthly, quarterly, or by any other schedule determined by empirical analysis. Exemplary treatments entail administration in multiple doses over an extended period of time, for example, at least six months. Further exemplary treatment regimes entail administration once every two weeks or once a month, or once every 3-6 months.

[0367] The amount of agent for effective administration varies from 0.1 to 500 μg per patient, more commonly 1 to 100 or 1 to 10 μg per injection for human administration. The timing of injections can vary significantly, from once a day to once a year to once every few decades. A typical regime consists of booster injections at time intervals such as 6 week intervals or 2 months after immunization. Another regime consists of booster injections 1, 2, and 12 months after immunization. Another regime entails bimonthly injections until death. Alternatively, booster injections can be irregular as indicated by monitoring the immune response.

[0368] The antibody or the agent for inducing the antibody is preferably administered via a peripheral route (i.e., the antibody administered or induced crosses the blood-brain barrier to reach the intended site in the brain). The route of administration includes topical, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, or intramuscular. The preferred routes for administration of the antibody are intravenous and subcutaneous. The preferred routes for active immunization are subcutaneous and intramuscular. This type of injection is most typically performed in the arm or leg muscles. In some methods, the agent is injected directly into the specific tissue where the deposit has accumulated, for example, by intracranial injection.

[0369] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic, and are manufactured under GMP requirements. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dose for single administration). Pharmaceutical compositions may be manufactured using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulations vary according to the route of administration selected. For injection, the antibody may be formulated in an aqueous solution, preferably Hank's solution, Ringer's solution, or a physiologically compatible buffer such as saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain specific formulatory agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0370] The regime may be administered in combination with another agent effective in treating or preventing the disease being treated. For example, in the case of Alzheimer's disease, the regime may be combined with immunotherapy against Aβ (WO 2000 / 072880), cholinesterase inhibitors, or memantine, or in the case of Parkinson's disease, with immunotherapy against α-synuclein (WO 2008 / 103472), levodopa, dopamine receptor stimulants, COMT inhibitors, MAO-B inhibitors, amantadine, or anticholinergic agents.

[0371] The antibody is administered in an effective regime, meaning a dose, route of administration, and frequency of administration that delays the onset, reduces the severity, and even prevents the progression and / or ameliorates at least one sign or symptom of the disorder being treated. If the patient is already suffering from the disorder, the regime may represent a therapeutically effective regime. If the patient is at high risk for the disorder compared to the general population but has not yet experienced symptoms, the regime may represent a preventively effective regime. In some cases, therapeutic or preventive efficacy may be observed in an individual patient compared to historical controls or past experience of the same patient. In other examples, therapeutic or preventive efficacy may be demonstrated in preclinical or clinical trials in a population of treated patients compared to a control population of untreated patients.

[0372] Exemplary doses of the antibody can be 0.1-60 mg / kg (e.g., 0.5, 3, 10, 30, or 60 mg / kg), or 0.5-5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4, or 5 mg / kg), or 10-4000 mg or 10-1500 mg as a constant dose. Dosages will vary depending, among other things, on the patient's condition and response to prior treatment, if any, whether the treatment is preventative or therapeutic, and whether the disorder is acute or chronic.

[0373] Administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Some antibodies may be administered intravenously or subcutaneously into the systemic circulation. Intravenous administration may be by infusion over a period of time, such as, for example, 30-90 minutes.

[0374] The frequency of administration varies depending on the half-life of the antibody in circulation, the pathology of the patient, and the route of administration, among other factors.The frequency can be daily, weekly, quarterly, or at irregular intervals in response to changes in the pathology of the patient or the progression of the disorder being treated.The exemplary frequency of intravenous administration is weekly to quarterly over the course of continuous treatment, although more or less frequent administration is also possible.For subcutaneous administration, the exemplary frequency of administration is daily to monthly, although more or less frequent administration is also possible.

[0375] The number of doses administered varies depending on whether the disorder is acute or chronic and the response of the disorder to treatment. In acute disorders or acute exacerbations of chronic disorders, 1-10 doses are often sufficient. In some cases, a single bolus administration, optionally in divided form, is sufficient for acute disorders or acute exacerbations of chronic disorders. Treatment can be repeated for recurrence or acute exacerbations of acute disorders. In chronic disorders, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, quarterly, every 6 months, for at least 1, 5, or 10 years, or for the life of the patient.

[0376] A. Diagnostic and Monitoring Methods Optimizing in vivo imaging, diagnostic methods, and immunotherapy The present invention provides a method for in vivo imaging of tau protein deposits (e.g., neurofibrillary tangles and tau inclusions) in a patient. The method works by administering to the patient a reagent such as an antibody (e.g., murine, humanized, chimeric, or veneered 3D6 antibody) that binds to tau, and then detecting the agent after it has bound. Antibodies that bind to a tau epitope within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or within amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO:1, are preferred. In some methods, the antibody binds to an epitope within amino acid residues 199-213 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 of SEQ ID NO:1), or within amino acid residues 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 320-334 of SEQ ID NO:1). In some methods, the antibody binds to an epitope within amino acid residues 259-268 of SEQ ID NO:1, within amino acids 290-299 of SEQ ID NO:1, within amino acids 321-330 of SEQ ID NO:1, or within amino acids 353-362 of SEQ ID NO:1. Clearing responses of administered antibodies can be avoided or reduced by using antibody fragments lacking the full-length constant region, such as Fab. In some methods, the same antibody can act as both a treatment and a diagnostic reagent.

[0377] The diagnostic reagent can be administered by intravenous injection into the patient's body, or directly into the brain by intracranial injection or by drilling a hole through the skull. The dose of the reagent should be in the same range as for the treatment method. Usually the reagent is labeled, although in some methods the primary reagent with affinity for tau is unlabeled and a secondary labeling agent is used to bind to the primary reagent. The choice of label varies depending on the detection means. For example, fluorescent labels are suitable for optical detection. The use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Radioactive labels can also be detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0378] The method of imaging tau protein deposits in vivo is useful for diagnosing or confirming the diagnosis of tauopathies, such as Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and Pick's disease, or susceptibility to the disease. For example, the method can be used in patients who present with dementia symptoms. If the patient has abnormal neurofibrillary tangles, then the patient is likely to suffer from Alzheimer's disease. Alternatively, if the patient has abnormal tau inclusions, then depending on the location of the inclusions, the patient may suffer from frontotemporal lobar degeneration. The method can also be used in asymptomatic patients. The presence of abnormal tau protein deposits represents susceptibility to further symptomatic disease. The method is also useful for monitoring disease progression and / or response to treatment in patients who have previously been diagnosed with tau-related disease.

[0379] Diagnosis can be made by comparing the number, size, and / or intensity of the labeled loci with the corresponding baseline value. This baseline value can represent the average level of a population of individuals without the disease. The baseline value can also represent a previous level determined in the same patient. For example, a baseline value can be determined in a patient before starting tau immunotherapy treatment, and the value is then measured and compared to the baseline value. A decrease in the value compared to the baseline represents a positive response to treatment.

[0380] In some patients, the diagnosis of tauopathy can be aided by performing a PET scan. The PET scan can be performed, for example, using conventional PET imaging devices and auxiliary equipment. The scan usually includes one or more areas of the brain that are generally known to be associated with tau protein deposits, and one or more areas that have little, if any, deposits to act as a general control.

[0381] The signal detected by PET can be represented as a multi-dimensional image. The multi-dimensional image can be two-dimensional, representing a cross-section through the brain, three-dimensional, representing the brain in three dimensions, or four-dimensional, representing changes in the brain in three dimensions over time. A color scale can be used with different colors representing different amounts of label and, inferentially, tau protein deposits detected. The results of the scan can also be mathematically presented as a number related to the amount of label detected and, consequently, the amount of tau protein deposits. Label present in brain regions known to be associated with deposits in a particular tauopathy (e.g., Alzheimer's disease) can be compared to label present in regions known not to be associated with deposits to provide a ratio representing the degree of deposits in the previous region. With the same radiolabeled ligand, such a ratio provides a comparable measure of tau protein deposits and their changes between different patients.

[0382] In some methods, a PET scan is performed at the same time or in the same patient visit as an MRI or CAT scan. MRI or CAT scans provide more anatomical brain detail than PET scans. However, images from a PET scan can be overlaid with those from an MRI or CAT scan to more accurately depict the location of the PET ligand relative to brain anatomy, and by implication, tau deposits. Some machines can facilitate image overlay by performing PET scanning and MRI or CAT scanning without the patient changing position between scans.

[0383] Suitable PET ligands include radiolabeled antibodies of the invention (e.g., murine, humanized, chimeric, or veneered 3D6 antibodies). The radioisotopes used can be, for example, C 11 , N 13 , O 15 , F 18 , or I 123 The interval between administration of the PET ligand and performance of the scan will vary depending on the PET ligand, particularly its rate of uptake and clearance in the brain, and the half-life of its radiolabel.

[0384] PET scans may also be performed as a preventative measure in asymptomatic patients, or in patients who have symptoms of mild cognitive impairment but have not yet been diagnosed with a tauopathy but are at high risk of developing a tauopathy. In asymptomatic patients, scans are particularly useful for individuals who are considered at high risk for tauopathy due to medical history, genetic or biochemical risk factors, or advanced age. Preventative scans may begin in patients between the ages of, for example, 45 and 75 years. In some patients, the first scan is performed at age 50 years.

[0385] Preventive scans may be performed, for example, at intervals of 6 months to 10 years, preferably 1 to 5 years. In some patients, preventive scans are performed annually. If the PET scan performed as a preventive measure shows abnormally high levels of tau protein deposits, immunotherapy may be initiated and then a PET scan may be performed in patients diagnosed with tauopathy. If the PET scan performed as a preventive measure shows levels of tau protein deposits within normal levels, it may be performed at intervals of 6 months to 10 years, preferably 1 to 5 years, conventionally or in response to the appearance of signs and symptoms of tauopathy or mild cognitive impairment. If above normal levels of tau protein deposits are detected, by combining preventative scanning with tau-directed immunotherapy, the levels of tau protein deposits can be reduced to normal or near normal levels, or at least prevented from increasing further, and the patient can remain free of tauopathy for a longer period of time (e.g., at least 5, 10, 15, or 20 years, or for the remainder of the patient's life) than would be the case without preventative scanning and tau-directed immunotherapy.

[0386] Normal levels of tau protein deposits may be determined by the amount of neurofibrillary tangles or inclusion of tau in the brain of a representative sample of individuals from the general population (e.g., a representative sample of disease-free individuals under the age of 50) who have not been diagnosed with a particular tauopathy (e.g., Alzheimer's disease) and are not considered at high risk for developing the disease. Alternatively, normal levels may be recognized in an individual patient if the PET signal according to the present method in areas of the brain where tau protein deposits are known to develop is not different (within the accuracy of the measurement) from signals from areas of the brain where such deposits are known not to normally develop. Elevated levels in an individual may be recognized by comparison with normal levels (outside the mean and variance of the standard deviation) or simply from an increase in signal beyond experimental error in areas of the brain associated with tau protein deposits compared to areas not known to be associated with deposits. For the purpose of comparing the levels of tau protein deposits in individuals and populations, tau protein deposits should preferably be determined in the same regions of the brain, including at least one region where tau protein deposits associated with a particular tauopathy (e.g., Alzheimer's disease) are known to form. Patients with high levels of tau protein deposits are candidates for initiating immunotherapy.

[0387] After immunotherapy is initiated, a reduction in tau protein deposits can be seen first as an indication that the treatment is having the desired effect. The observed phenomenon can be within the range of, for example, 1-100%, 1-50%, or 1-25% of baseline values. Such effects can be measured in one or more areas of the brain where deposits are known to form, or can be measured from an average of said areas. The overall effect of the treatment can be approximated by adding the percentage reduction relative to baseline to the increase in tau protein deposits that would otherwise occur in the average of untreated patients.

[0388] Also, maintenance of tau protein deposits at a near constant level or even a small increase in tau protein deposits may be indicative of a response to treatment, albeit a suboptimal response. Such a response may be compared to the time course of tau protein deposit levels in patients with a particular tauopathy (e.g., Alzheimer's disease) who did not receive treatment to determine whether immunotherapy has the effect of inhibiting further increases in tau protein deposits.

[0389] Monitoring changes in tau protein deposits allows for adjustment of immunotherapy or other treatment regimes in response to treatment. PET monitoring provides an indication of the nature and degree of response to treatment. Thus, a decision can be made to adjust therapy, and if desired, treatment can be adjusted in response to PET monitoring. PET monitoring thus allows for adjustment of immunotherapy or other treatment regimes directed at tau before other biomarkers, MRI, or cognitive measures respond detectably. Significant changes provide some evidence that the treatment did or did not have a beneficial effect, as compared to the value of the parameter after treatment to the norm. In some cases, changes in the value of the patient's own parameter provide evidence that the treatment did or did not have a beneficial effect. In other examples, the change in value, if any, in the patient is compared to the change in value, if any, in a representative control population of patients who did not undergo immunotherapy. The difference between the normal response of the control patient and the response of a particular patient (e.g., the variance of the mean value + standard deviation) can also provide evidence that the immunotherapy regime has or has not achieved a beneficial effect in the patient.

[0390] In some patients, monitoring reveals a detectable decrease in tau protein deposits, but also reveals that the level of tau protein deposits remains above normal. In such patients, in the absence of unacceptable side effects, the treatment regime may be continued as is, or even increased in frequency and / or dose if not already at the maximum recommended dose.

[0391] If monitoring indicates that the level of tau protein deposits in the patient has already been reduced to normal or near-normal levels of tau protein deposits, the immunotherapy regime can be adjusted from an induction dose (i.e., a dose that reduces the level of tau protein deposits) to a maintenance dose (i.e., a dose that maintains tau protein deposits at a near constant level). Such a regime can be affected by reducing the dose or frequency of administration of the immunotherapy.

[0392] In other patients, monitoring may reveal that immunotherapy has some beneficial but suboptimal effects. Optimal effects may be defined as a percentage reduction in the level of tau protein deposits within the upper half or quartile of the change in tau protein deposits (measured or calculated over the whole brain or its representative regions known to form tau protein deposits) experienced by a representative sample of tauopathy patients undergoing immunotherapy at a given time point after starting treatment. Patients who undergo smaller reductions or whose tau protein deposits remain constant or even increase, but to a lesser extent than would be expected in the absence of immunotherapy (e.g. as inferred from a control group of patients not undergoing immunotherapy) may be classified as having undergone a positive but suboptimal response. Such patients may optionally be subjected to an adjustment regime in which the dose and / or frequency of administration of the agent is increased.

[0393] In some patients, tau protein deposits may increase in a manner similar to or greater than that in patients not receiving immunotherapy. If such an increase persists for a period of time, e.g., 18 months or 2 years, and even after an increase in either frequency or administration of the agent, immunotherapy may be discontinued in favor of other treatments, if desired.

[0394] The above description of the diagnosis, monitoring and treatment adjustment of tauopathy has mainly focused on PET scans. However, instead of PET scans, any other technique for visualizing and / or measuring tau protein precipitates that is suitable for use with the tau antibody of the present invention (e.g., mouse, humanized, chimeric, or veneered 3D6 antibody) can be used to carry out the method.

[0395] Also provided is a method for detecting an immune response against tau in a patient suffering from or susceptible to a tau-related disease. The method can be used to monitor the course of therapeutic and preventative treatment with the agents provided herein. The antibody profile from passive immunization usually shows an immediate peak in antibody concentration followed by an exponential decay. Without further doses, this decay reaches pretreatment levels within a period of days to months depending on the half-life of the antibody administered. For example, the half-life of some human antibodies is on the order of 20 days.

[0396] In some methods, a baseline measurement of antibody against tau in the subject is made before administration, followed immediately by a second measurement to determine peak antibody level, and one or more additional measurements are made periodically to monitor the decay of antibody level. When the antibody level has decreased to a predetermined percentage (e.g., 50%, 25%, or 10%) of baseline or baseline-subtracted peak, an additional dose of antibody is administered. In some methods, the peak or background-subtracted subsequent measured levels are compared to a reference level previously determined to constitute a useful preventive or therapeutic treatment regime in other subjects. If the measured antibody level is significantly lower than the reference level (e.g., lower than the mean value of the reference value in a population of subjects that will benefit from treatment-1 or preferably 2 standard deviations), administration of an additional dose of antibody is indicated.

[0397] Also provided is a method for detecting tau in a subject, for example by measuring tau in a sample from the subject or by imaging tau in vivo in the subject.Such a method is useful for diagnosing or confirming the diagnosis of tau-related disease or susceptibility thereto.The method can also be used in asymptomatic subjects.The presence of tau represents susceptibility to further symptomatic disease. The methods are also useful for monitoring disease progression and / or response to treatment in subjects previously diagnosed with Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism-Dementia Complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP).

[0398] A biological sample obtained from a subject having, suspected of having, or at risk of having Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP) can be contacted with an antibody disclosed herein to assess the presence of tau. For example, the level of tau in such a subject can be compared to the level present in a healthy subject. Alternatively, the level of tau in the subject treated for the disease can be compared to the level in a subject not treated for Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy, or progressive supranuclear palsy (PSP). Some such tests involve a biopsy of tissue obtained from the subject, and ELISA assays can be useful methods, for example, to assess tau in bodily fluid samples.

[0399] VIII. Kit The invention further provides kits (e.g., containers) containing the antibodies disclosed herein and related materials, such as instructions (e.g., package inserts). The instructions can include, for example, instructions for administration of the antibody and, optionally, one or more additional agents. The containers of antibody can be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses.

[0400] A package insert refers to instructions customarily included in a commercial package of a therapeutic product that contain information about the indications, use, dosage, administration, contraindications, and / or warnings regarding the use of that therapeutic product.

[0401] The kit may also comprise a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution, and may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0402] All patent applications, websites, other publications, deposit numbers, etc. mentioned above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually set forth as being incorporated by reference. Where different versions of a sequence are associated with deposit numbers at different times, it refers to the version associated with the deposit number at the effective filing date of this application. The effective filing date refers to the date prior to or the actual filing date of the priority application that refers to the deposit number, if applicable. Similarly, where different versions of a publication, website, etc. are published at different times, it refers to the most recent version published at the effective filing date of this application, unless otherwise stated. All features, steps, elements, embodiments, or aspects of the present invention may be used in conjunction with any other, unless otherwise stated. The present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, but it will be apparent that certain changes and modifications may be made within the scope of the appended claims. EXAMPLES

[0403] Example 1: Mouse 3D6 and the humanized variant hu3D6VHv1bA11 / L2-DIM4 block tau internalization

[0404] An internalization assay using fluorescence-activated cell sorting (FACS) was performed to evaluate various antibodies for their ability to block tau internalization into neurons. Antibodies that block internalization may block tau propagation.

[0405] Soluble tau aggregates were generated by incubating recombinant full-length tau with equimolar amounts of low molecular weight heparin at 37°C for 3 days. After incubation, insoluble and soluble tau were separated by centrifugation at 10,000 x g for 15 min. The supernatant was then separated by preparative size-exclusion chromatography, and the aggregate peak (>100 kDa) was collected and concentrated. To measure internalization, the soluble aggregate fraction was labeled with pHrodo Red succinimidyl ester, which fluoresces upon internalization into the endolysosomal pathway.

[0406] pHrodo-labeled 4R0N human tau P301L soluble oligomers (final concentration of 1.5 μg / ml) were pre-incubated with anti-tau antibodies (titration: starting concentration of 80 μg / ml, followed by 4-fold serial dilutions) in cell culture medium for 30 min at room temperature. The tau / antibody mixture was then added to B103 neuroblastoma cell line at a final concentration of 500,000 cells / ml and incubated in a tissue culture incubator (5% CO). 2) for 3-4 hours at 37°C. The cells were then washed three times with culture medium, followed by a 10 min incubation in culture medium and two washes with FACS buffer (1% FBS in PBS). The cells were resuspended in 100 μl of FACS buffer and the mean fluorescence intensity of Texas Red was measured by FACS LSR II. Texas Red fluorescence from pHrodo is activated by the low pH associated with the endolysosomal compartment after internalization. Since FACS detects cells and pHrodo only fluoresces after internalization, only tau internalized by cells is detected. A low mean fluorescence intensity indicates a low amount of internalized tau, suggesting a high blocking activity of the tested antibody.

[0407] (m) Both PRX005 (mouse 3D6) and PRX005 (hu3D6VHv1bA11 / L2-DIM4) showed high inhibitory activity in the model of tau internalization at comparable concentrations compared to the isotype control (isotype ctl) (Figure 1). All values ​​are means ± SD (n = 3-5). IC 50 = 9nM. [Tau] = 167nM.

[0408] Example 2 Mouse 3D6 reduces pathological tau development in an inducible tau seeding model using Alzheimer's disease extract

[0409] Murine 3D6 was examined for its ability to reduce the development of pathological tau in an inducible tau seeding model using Alzheimer's disease extract.

[0410] The study utilized mice expressing a clinical variant of human tau (P301S) under the control of the mouse PrP promoter (neuron-specific expression). These mice later develop promoter-induced tau pathology. hTauP301S mice show fibrous, neuritic tau foci by 6 months of age, which progressively accumulate with neuronal loss and atrophy of the hippocampus and entorhinal cortex by 9-12 months of age. Mice (average age 3 months) received a single stereotactic injection into the hippocampus. Mice (n=30 / group) received IP (intraperitoneal) injections (50 mg / kg) (once a week) of mouse 3D6 (mPRX005)-IgG2a or IgG2a negative control antibody, starting 7 days before the hippocampal injection and continuing for 2 months. At the end of the study, the extent of pathological progression was assessed using the AT8 antibody.

[0411] Preparation of AD brain extracts

[0412] Alzheimer's tissue was homogenized and tau protein enriched by first resuspending human gray matter in 9 volume equivalents (relative to the initial brain tissue mass) of Buffer A (10 mM Tris, 0.8 M NaCl, 10% sucrose, 2 mM DTT, 1 mM EGTA pH 7.4) using a motorized Dounce homogenizer with 20 strokes. This homogenate was then centrifuged at 10,000 x g for 10 minutes at 4 °C. The supernatant was filtered through Kimwipes and kept on ice until further use. The pellet obtained from the centrifugation step was resuspended again in 9 volume equivalents and centrifuged under the same conditions as before. The supernatant obtained from this centrifugation was again filtered through Kimwipes and combined with the other fraction. These pooled supernatants were then adjusted to 1% lauryl sarcosine content (using the 30% stock) and stirred at 180 RPM for 180 minutes at room temperature. The lysate was then centrifuged at 250,000×g for 90 min at 4°C. The supernatant was saved as the sarkosyl-soluble fraction, and the pellet was gently washed with 6 mL of PBS without dislodging the pellet from the tube. This wash was removed, and a further 2 mL pellet wash was performed. After removing this wash, the pellet was dislodged, resuspended and transferred to a clean, sterile microcentrifuge tube using 1 mL of PBS. At this point, the resuspended pellet was centrifuged again at 250,000×g for 30 min at 4°C. After centrifugation, the pellet was separated from the supernatant and the pellet was resuspended in 0.1 mL PBS / g starting weight. The pellet was broken up with a pipette tip and rotated end-over-end for 16 h at room temperature. After incubation, the resuspension was subjected to sonication using a tip probe sonicator (set at 15% power and 100% duty cycle) at 15 pulses and 0.5 s pulse width. The sonicated material was then passed through a 27G needle and rotated end-over-end at room temperature for 30 minutes. The solution was sonicated and the sample centrifuged at 100,000×g for 30 minutes at 4° C. The supernatant was saved as the high-g supernatant fraction and the pellet was resuspended in PBS at 50 uL / g starting material. The resuspended pellet was sonicated at 20% power, 100 pulses, and 0.5 second pulse width (with a 30 second break on ice after every 20 pulses). The homogenate was centrifuged at 10,000×g for 10 minutes at 4° C.The final supernatant was kept as the sarkosyl-insoluble enriched tau protein fraction and the pellet was discarded.

[0413] Dose Formulation and Administration

[0414] Preparation of materials

[0415] The required amount of Sarkosyl-enriched brain fractions from AD patients was thawed and subjected to sonication for 3 minutes in a sonicator water bath (QSonica) filled with ice water, with a pulse pattern of 10% power and 10 seconds on and 5 seconds off. After sonication, 1 μl of antibody (mouse 3D6 (mPRX005) or 6F10 (control antibody)) (all at 10 mg / ml without dilution) or PBS was mixed with 1 μl of Sarkosyl-enriched AD brain by pipetting immediately before stereotactic injection. Mouse IgG2a was used in this study because it promotes faster phagocyte clearance of tau in vitro compared to IgG1. IgG2a mPRX005 (mouse 3D6) showed superior efficacy in vivo compared to IgG1.

[0416] Prior to testing, test and control articles were formulated in a sterile vehicle (1x phosphate buffered saline (1x PBS)) to a concentration of 5 mg / mL, allowing administration in a dose volume of 10 mL / kg.

[0417] Stereotactic injection

[0418] Mice were anesthetized using isoflurane and placed in a stereotaxic apparatus (Kopf instruments) so that the skull was horizontal. The surgical area was shaved, disinfected using 70% alcohol and iodine, and the skin was incised. A microdrill and a drill bit with a head diameter of 0.9 mm were used to drill a hole in the skull at the correct rostral and lateral position relative to the bregma (coordinates listed in Table 3). A 30-gauge cannula, stored in a holder, was lowered into the appropriate position (coordinates listed in Table 3). Preincubated AD brain extract (1 μl of AD brain extract) was injected at a rate of 1 μl / min (WPI, AL-1000, infusion pump). The injection volume was injected via PE10 tubing attached to an airtight 10 μl Hamilton syringe (#1701) mounted on an infusion / aspiration pump. After injection, the needle was left stationary for 5 minutes and then slowly withdrawn. The skin incision was closed with sutures. Carprofen was injected subcutaneously as an analgesic. A heating pad was used to maintain the body temperature of the mice throughout the procedure until they recovered from anesthesia. [Table 3]

[0419] Sample collection and processing

[0420] At 5 months of age (2 months after stereotaxic injection), 2 Mice were sacrificed using a cephalosporin A (ESA) and subjected to transcardial perfusion with ice-cold 1×PBS via the left ventricle for 5 min (performed by a peristaltic pump at 3 ml / min). The right atrium was cut as an outflow pathway. Brains were removed from the skull. Whole brains were fixed in 10% neutral buffered formalin (NBF) for 24 h and stored in 1×PBS at 4° C. until further processing.

[0421] histological staining

[0422] Reagents for immunofluorescence staining were supplied as described in Table 4. [Table 4]

[0423] Brains were shipped to Neuroscience Associates (Knoxville, TN) and treated overnight with 20% glycerol and 2% dimethyl sulfoxide to prevent freezing artifacts. Specimens were then embedded in gelatin matrix using MultiBrain® / MultiCord® Technology (NeuroScience Associates, Knoxville, TN). Blocks were flash frozen and hardened by immersion in 2-methylbutane cooled with crushed dry ice before mounting on the freezing stage of an AO860 sliding microtome. MultiBrain® / MultiCord® blocks were sectioned coronally at 35μ to obtain sections containing the hippocampus (bregma -0.5 to -4.0). All sections were cut to pass through the entire length of the specimen segment and collected consecutively in a series of 24 containers. All containers contained antigen preservation solution (50% PBS pH 7.0, 50% ethylene glycol, 1% polyvinylpyrrolidone). For immunohistochemistry, free-floating sections were stained with AT8 (1:5000, Thermo Scientific). All subsequent incubation solutions from blocking serum used Triton® X-100 in Tris-buffered saline (TBS) as the medium, and all rinses were performed in TBS. After hydrogen peroxide treatment and blocking serum, sections were immunostained with biotinylated AT8 (1:5000) overnight at room temperature. The medium solution contained Triton® X-100 for permeabilization. After rinsing, Vector Lab's ABC solution (avidin-biotin-HRP complex; VECTASTAIN® Elite ABC, Vector, Burlingame, CA) was applied. Sections were rinsed again and then treated with diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide to create a visible reaction product. After further rinsing, sections were mounted on gelatin-coated slides and air-dried. Slides were dehydrated in alcohol, cleared in xylene, and coverslipped. Each slide was laser etched with block number and staining information.Slides were sequentially sequenced from rostral to caudal for each stain, then numbered in indelible ink in the upper right corner and digitally scanned at 10× on a Huron Digital Pathology Tissuescope LE120.

[0424] Immunohistochemical analysis

[0425] A8-positive neurons were quantified in the ipsilateral and contralateral hippocampus (Ammon's cornu, dentate gyrus, and subiculum) using the particle counting function of Image J. A total of 15 sections spaced 210 μm apart were quantified. Only neurons >5 μm in size with identifiable nuclei and neuronal projections were included.

[0426] Statistical significance was calculated using two-way ANOVA with hemisphere (within-subjects) and treatment (between-subjects) factors. All statistical analyses and figure preparations were performed using GraphPad Prism9.

[0427] result

[0428] Figure 2A shows a view of a brain section from a mouse treated with control (upper panel) and mouse 3D6 [(m)PRX005; lower panel]. The contralateral side (contra) is on the left side of each figure, and the ipsilateral side (ipsi) is on the right side of each figure.

[0429] The results are shown in Figure 2B. Overall tau pathology burden was lower in the contralateral hippocampus (relative to injection) compared to the ipsilateral hippocampus (relative to injection). This is expected to be due to contralateral hippocampal pathology resulting from the propagation of tau through efferent neurons from the injection site hippocampus. Systemic mouse 3D6 treatment resulted in a significant decrease in AT8 staining in both the ipsilateral and contralateral hippocampi as measured by immunohistochemistry compared to treatment with IgG2a isotype control (Figure 2B). These results demonstrate the efficacy of systemically administered mouse 3D6 in suppressing the uptake and propagation of tau pathology induced by pathogenic species from AD. All values ​​are mean ± SE (n = 30).

[0430] Example 3: Treatment with mouse 3D6 reduces pathological tau and ameliorates behavioral deficits in a transgenic tau model

[0431] The efficacy of murine 3D6 (mPRX005) was evaluated in a transgenic tau aging model. Utilizing this model provides another independent efficacy testing approach in that the development of tau pathology occurs due to aging and overexpression, and any tau species that are sensitive to antibody treatment are secreted by neurons. This removes the bias inherent in the selection of the particular tau seed used in an induced disease model.

[0432] In this study, we evaluated age-dependent pathological changes, tau post-translational changes, and behavioral changes in a human tau transgenic mouse line (PS19 line) carrying the clinical frontotemporal dementia-associated P301S mutation under the control of the mouse prion promoter. PS19 mice exhibit age-dependent tau hyperphosphorylation (detected by AT8 and AT100) in the spinal cord, brainstem, midbrain, cortex, amygdala, and hippocampus, as well as associated motor deficits. Onset of tau pathology appears by 6 months and progresses with neurodegeneration until death at 10-14 months of age.

[0433] Mice were injected once a week (50 mg / kg IP (intraperitoneal)) with PBS, IgG1 isotype control, and mouse 3D6 (mPRX005) for 3 months (age range 6-9.7 months) and various endpoints of tau pathology and associated behavioral deficits were measured.

[0434] Behavioral assessment

[0435] The inverted grid hanging test was performed at 3, 6, and 9 months of age, and before sacrifice at 9.7 months of age. Inverted grid hanging tests coordination and muscle status. A grid (40 x 20 cm / 0.5 x 0.5 cm mesh) was placed 50 cm above a flat soft surface, and the time it took the animal to fall was measured.

[0436] Free-floating vibratome sections

[0437] A total of approximately 32 sagittal sections (40 μm) containing the appropriate regions of interest were cut from each right hemisphere. Brain sections of interest were selected at 200 μm intervals between 2.64 and 0.84 lateral from the bregma based on a stereotaxic mouse brain atlas (Paxinos and Franklin). A set of five sections per mouse was prepared, and the ROIs were subjected to staining with AT8 and AT100, respectively. Sections from all animals selected for a particular stain were randomized for each stain and quantified blindly. Section numbers were derived from the mouse number with subnumbers 1 to 5 for different sections per mouse.

[0438] Immunohistochemistry procedure

[0439] Sagittal brain sections (40 μm) were cut with a vibrating blade HM650V microtome (Thermo Scientific, Waltham, MA, USA) and stored in 1× PBS / 0.1% sodium azide until use.

[0440] After two 5-minute washes with PBS, the brain sections were incubated in a 1×PBS:methanol (1:1) solution for 10 minutes, followed by three washes (5 minutes each) with PBS-0.1% Triton®-100 (PBST). After 30 minutes of blocking (5% milk in PBST), the brain sections were incubated with a specific primary mouse anti-tau antibody (AT8 or AT100, see Table 5 below for specifications) at room temperature for 2 hours (or overnight at 4° C.), then three 5-minute washes with PBST, followed by incubation with the appropriate Alexa-conjugated secondary antibody in 5% milk-PBST (1:500; Invitrogen; ThermoFisher) at room temperature for 1 hour. After three washes with PBST and two washes with PBS (5 minutes each), the brain sections were mounted on microscope slides (Menzel, Superfrost+), dried, embedded with Fluoromount (Sigma-Aldrich), and coverslipped. Immunoreactive areas in ROIs were determined with Image J. Statistical analysis was performed in GraphPad Prism v9.0 using Kruskal-Wallis followed by Dunn post-hoc analysis for multiple comparisons (when applicable). Outliers were identified using the ROUT method in GraphPad Prism based on FDR with a very stringent Q=0.1% (maximum desired FDR) (when applicable).

[0441] [Table 5]

[0442] automatic quantification

[0443] Images were acquired with a Leica DM400 B LED fluorescence microscope and analyzed with ImageJ. All acquired images were subjected to the same computer subroutine to minimize investigator bias. For quantification of AT8- and AT100-positive areas, an automatic thresholding method was applied throughout the analysis.

[0444] A region of interest (mid-rostral pons) was selected for brainstem quantification. Five brain sections per mouse were included in the analysis for each staining with AT100 or AT8, and the average values ​​were calculated. If the region of interest contained mechanical, structural, and / or staining artifacts, images were excluded or manually corrected when possible.

[0445] The upper panel of Figure 3 is a schematic diagram of the transgenic tau model experimental protocol. [Tx:ipq1w 50mpk refers to intraperitoneal injection of 50 mg per kg mouse body weight once a week]

[0446] result

[0447] Systemic passive immunization with mouse 3D6 promoted the reduction of tau pathology in the brainstem, as measured by immunostaining with an antibody directed against tau hyperphosphorylation sites ( Fig. 3 , bottom right panel; * p<0.05). Furthermore, treatment with mouse 3D6 reduced tau pathology-associated motor impairment as measured by the grid hanging assay (Figure 3, bottom left panel; * p<0.05). Treatment with mPRX005 initiated at the onset of pathology (therapeutic modality) delayed brainstem tau pathology and the resulting behavioral impairment. Treatment with mouse 3D6 also reduced tau pathological accumulation in the cortex and hippocampus as measured by immunohistochemistry and biochemistry. Evaluation of trough antibody levels at two time points (before the sixth dose and at the end of the study) showed that the mean level of mouse 3D6 was 280 μg / mL. Collectively, these results demonstrate the efficacy of treatment with mouse 3D6 in an aging transgenic model of tauopathy and provide confidence that treatment with mouse 3D6 is effective against tau progression induced by non-fibrillar forms of tau.

[0448] Example 4: Mouse 3D6 protects mouse primary cortical neurons from tau-induced toxicity

[0449] Cortical neurons are prepared from C57Bl6 / J mouse fetuses from embryonic days 16-17 as previously reported in Pillot, T., Drouet, B., Queille, S., et al., The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain. J Neurochem. 73(4):1626-34 (1999). Briefly, dissociated cortical cells are plated (50,000 cells / well) in 48-well plates precoated with 1.5 μg / mL polyornithine (Sigma). Cells are cultured in chemically defined DMEM / F-12 medium without serum and supplemented with hormones, proteins, and salts. Cultures are maintained in a humidified 6% CO 2 Store at 35°C in an ambient atmosphere.

[0450] Neuron Processing

[0451] All treatments were performed in triplicate in 48-well plates on days in vitro (DIV) 6–7. Neurons were incubated for 24 h with either vehicle or human tau oligomers (hTO) (final concentration of 1 μM based on monomer) in a final volume of 140 μL per well in the presence of five increasing concentrations of test article.

[0452] Final antibody:hTO ratios were 1:5, 1:3, 1:1, 3:1, and 5:1. hTO concentrations are based on monomer molar equivalents since the exact composition of the oligomers and epitope presentation are unknown. Antibodies were incubated with hTO for 30 min at room temperature before being added to neurons.

[0453] Measurement of neuronal viability

[0454] After being placed under the test conditions, mouse cortical neurons were incubated for 24 hours, after which neuronal viability was monitored using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay.

[0455] To measure the MTT signal, cells were incubated with MTT (Sigma, Cat. No. M2128-10G, Lot No. MKBH7489V) for 1 h at 35° C. For this purpose, MTT was solubilized in PBS at 5 mg / mL. 14 μL of MTT solution was added to each well. After incubation, the medium was removed and cells were dissolved in 150 μL of DMSO for 10 min and protected from light. After complete solubilization of the formazan product, the absorbance was determined at 570 nm in a FLUOSTAR-Omega plate reader (BMG-LABTECH).

[0456] For measurement of LDH release, culture medium (110 μL) from each well was transferred to a 1.5 mL Eppendorf tube and replaced with untreated medium for the MTT assay. The collected medium was centrifuged at 800 g for 5 min and the supernatant (100 μL cell-free culture medium) was transferred to a 48-well plate stored at 4°C and protected from light for further analysis. Quantification of LDH in the culture medium was performed according to the manufacturer's recommendations (Cytotoxicity Detection Kit [LDH], Roche Ref 11 644 793 001).

[0457] result

[0458] Effect of mouse 3D6 (mPRX005) on neuronal viability: LDH assay

[0459] To test mouse 3D6(mPRX005) for its ability to protect neurons from tau-induced neurotoxicity, primary mouse cortical neurons were treated with various concentrations of mouse 3D6(mPRX005) along with tau oligomers and viability was measured by MTT assay. A molar equivalent of antibody:hTO was used because (a) the specific composition and molecular weight of tau species is heterogeneous and unknown, and (b) the experimental duration is limited and there are differences between in vitro and in vivo environments, and the concentration of tau used to induce measurable toxicity in this particular model is higher than that expected to be present in the extracellular environment in AD brains. Treatment with mouse 3D6(mPRX005) reduced tau-induced toxicity in a dose-dependent manner, and increasing concentrations returned neuronal viability to near baseline levels (Figure 4, left panel; all values ​​are mean ± SD (n=3-5)).

[0460] Effect of mouse 3D6 (mPRX005) on neuronal viability: LDH assay

[0461] As another independent method to assess neuronal viability, we also assessed mouse 3D6(mPRX005)'s ability to block tau-induced neurotoxicity using LDH release. Lactate dehydrogenase (LDH) release is an indicator of cell death. A decrease in LDH indicates decreased cell death, which occurs due to decreased internalization of tau. Similar to the treatments and results seen in the MTT assay, mouse 3D6(mPRX005) demonstrated the ability to block tau neurotoxicity in a dose-dependent manner, indicating that neuronal membrane integrity was preserved after treatment with tau (Figure 4, right panel; all values ​​are mean ± SD (n=3-5)).

[0462] In summary, in vitro antibody screening across the full length of tau protein demonstrated superior activity of MTBR R1 / R2 against tau uptake and neurotoxicity. The mouse precursor of PRX005 (mouse 3D6) has high affinity for the MTBR tau epitope and has a superior profile compared to other antibodies. Direct inhibition of tau-heparan sulfate proteoglycan interaction may contribute to blocking tau internalization, toxicity, and the development of intracellular tau pathology. In vivo treatment with mPRX005 (mouse 3D6) in transgenic tau mice and seeding models reduces intraneuronal tau pathology and downstream behavioral impairments. The consistently superior profile of PRX005(hu3D6VHv1bA11 / L2-DIM4) across a broad range of in vitro and in vivo systems supports the advancement of PRX005(hu3D6VHv1bA11 / L2-DIM4) as a clinical candidate for the potential treatment of tauopathies (e.g., Alzheimer's disease).

[0463] Example 5. Exemplary CDRs

[0464] Exemplary CDRs of the antibodies of the invention are shown in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0465] List of Arrays P10636-8 (SEQ ID NO: 1) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPT REPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHH KPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0466] P10636-7 (SEQ ID NO: 2) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKS PSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0467] P10636-6 (4RON human tau) (SEQ ID NO: 3) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVP MPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0468] P10636-5 (SEQ ID NO: 4) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGT PGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0469] P10636-4 (SEQ ID NO:5) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPP TREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0470] P10636-2 (SEQ ID NO: 6) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0471] SEQ ID NO:7; Mouse 3D6 VH amino acid sequence: EVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0472] SEQ ID NO:8; Kabat / Chothia HCDR1: GFNIKDYYLH

[0473] SEQ ID NO:9; Kabat HCDR2: WIDPENGDTVYDPKFQG

[0474] SEQ ID NO: 10; Kabat HCDR3: LDF

[0475] SEQ ID NO:11; Mouse 3D6 VL amino acid sequence: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK

[0476] SEQ ID NO:12; Mouse Kabat LCDR1: KSSQSLLDSDGKTYLN

[0477] SEQ ID NO:13; Mouse Kabat LCDR2: LVSKLDS

[0478] SEQ ID NO:14; Mouse Kabat LCDR3: WQGTHFPYT

[0479] SEQ ID NO: 15; hu3D6VHv1: EVQLVQSGAEVVRPGALVKVSCKASGFNIKDYYLHWVRQAPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0480] SEQ ID NO: 16; hu3D6VHv2: EVQLVQSGAEVKKPGASVKVSCKVSGFNIKDYYLHWVRQAPEQGLEWMGWIDPENGDTVYDPKFQGRVTITADTSNTAYMELSSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0481] SEQ ID NO: 17; hu3D6VHv1b: EVQLVQSGAEVVRPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0482] SEQ ID NO: 18; hu3D6VHv1bA11: EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0483] SEQ ID NO: 19; hu3D6VHv5: EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0484] SEQ ID NO:20; hu3D6VLv1: DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0485] SEQ ID NO:21; hu3D6VLv2: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0486] SEQ ID NO:22; hu3D6VLv3: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0487] SEQ ID NO:23; hu3D6VLv4: DIVMTQTPLSLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0488] SEQ ID NO:24; Heavy Chain Variable Acceptor Acc.#BAC01986.1 QVQLQQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAPGQGLEWMGRIIPILGIATYAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARGKGEFEGMDVWGQGTTVTVSS

[0489] SEQ ID NO:25; Heavy chain variable acceptor Acc.#IMGT#IGHV1-69-2 * 01 EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCAT

[0490] SEQ ID NO:26; Heavy chain variable acceptor Acc.#IMGT#IGKJ1 * 01 QHWGQGTLVTVSS

[0491] SEQ ID NO:27; Light chain variable acceptor Acc.#IMGT#IGKV2-30 * 02 Acc.#IMGT#IGKV2-30 * 02 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWP

[0492] SEQ ID NO:28; Light chain variable acceptor Acc.#IMGT#IGKJ2 * 01 YTFGQGTKLEIK

[0493] SEQ ID NO:29; Light Chain Variable Acceptor Acc.#AAZ09048.1 DVVMTQSPLSLTVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYRVSHWDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTYWPLTFGQGTKLEIK

[0494] SEQ ID NO:30; Mouse 3D6 VH nucleic acid sequence: GAGGTTCAGCTGCAGCAGTCTGGGGCTGACCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATTTGCACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGT GATACTGTATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAATACAGCCTACCTGCAGCTCGGCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTTCTACCCTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0495] SEQ ID NO:31; Mouse 3D6 VL nucleic acid sequence: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGT CTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGT

[0496] SEQ ID NO:32; Mouse CDR-H1 Kabat DYYLH

[0497] SEQ ID NO:33; Mouse CDR-H1 Chothia GFNIKDY

[0498] SEQ ID NO:34: Mouse CDR-H2 Chothia DPENGD

[0499] SEQ ID NO:35; Mouse CDR-H2 AbM WIDPENGDTV

[0500] SEQ ID NO:36: Mouse CDR-L1 Contact KTYLNWL

[0501] SEQ ID NO: 37: Mouse CDR-L2 Contact RLIYLVSKLD

[0502] SEQ ID NO: 38: Mouse CDR-L3 Contact WQGTHFPY

[0503] SEQ ID NO:39: Mouse CDR-H1 Contact KDYYLH

[0504] SEQ ID NO: 40: Mouse CDR-H2 Contact WIGWIDPENGDTV

[0505] SEQ ID NO: 41: Mouse CDR-H3 Contact STLD

[0506] SEQ ID NO: 42; Alternative Kabat-Chothia CDR-H1 GFTIKDYYLH

[0507] SEQ ID NO: 43; Alternative Kabat CDR-H2 WIDPEDGDTVYAPKFQG

[0508] SEQ ID NO: 44; consensus VH amino acid sequence of FIG. 2 of PCT / IB2017 / 052544 EVQLVQSGAEVVXPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGXATITADTSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0509] SEQ ID NO: 45; consensus VL amino acid sequence of FIG. 3 of PCT / IB2017 / 052544 DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR

[0510] SEQ ID NO: 46; hu3D6VHv1bA11B6G2: EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWVDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0511] SEQ ID NO: 47; hu3D6VHv1bA11B6H3: EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0512] SEQ ID NO: 48; hu3D6VHv1c: EVQLVQSGAEVKRPGALVKISCKASGFNFKDYYLHWVRQRPEQGLEWMGWIDPENGDTVYDEKFQGRVTITADTSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0513] SEQ ID NO: 49; hu3D6VHv1d: EVQLVQSGAEVKRPGALVKISCKASGYTFTDYYLHWVRQRPEQGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0514] SEQ ID NO: 50; hu3D6VHv1e: EVQLVQSGADVvkPGALVKISCKASGFTIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYAEKFQGRVTITADTSNTAYLeLGSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0515] SEQ ID NO:51; hu3D6VHv1f: EVQLVQSGADVVKPGALVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYMELGSLTSEDTAVYFCSTLDYWGQGTTLTVSS

[0516] SEQ ID NO:52; hu3D6VHv3: EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0517] SEQ ID NO:53; hu3D6VHv3b: EVQLVQSGAEVKKPGALVKISCKVSGYNFKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0518] SEQ ID NO:54; hu3D6VHv3c: EVQLVQSGAEVKKPGALVKISCKVSGYTFTDYYLHWVRQAPGKGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0519] SEQ ID NO:55; hu3D6VHv4: EVQLVQSGAEVVKPGATVKISCKVSGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0520] SEQ ID NO:56; hu3D6VHv4b: EVQLVQSGAEVVKPGALVKISCKVSGYNFKDYYLHWVRQRPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0521] SEQ ID NO:57; hu3D6VHv4c: EVQLVQSGAEVVKPGALVKISCKVSGYTFTDYYLHWVRQRPGKGLEWWMGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0522] SEQ ID NO:58; Alternative Kabat-Chothia CDR-H1 (as found in hu3D6VH1c, etc.) GFNFKDYYLH

[0523] SEQ ID NO:59; Alternative Kabat-Chothia CDR-H1 (such as found in hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c) GYTFTDYYLH

[0524] SEQ ID NO:60; Alternative Kabat-Chothia CDR-H1 (as found in hu3D6VHv3b and hu3D6VHv4b, etc.) GYNFKDYYLH

[0525] SEQ ID NO:61; alternative Kabat CDR-H2 (as found in hu3D6VHv1bA11B6G2, etc.) WVDPEDGDTVYAPKFQG

[0526] SEQ ID NO:62, alternative Kabat CDR-H2 (such as found in hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b) WIDPENGDTVYDEKFQG

[0527] SEQ ID NO:63; Alternative Kabat CDR-H2 (such as found in hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c) WVDPEDGDTVYAEKFQG

[0528] SEQ ID NO:64; Alternative Kabat CDR-H2 (as found in hu3D6VHv1e, etc.) WIDPENGDTVYAEKFQG

[0529] SEQ ID NO:65; alternative Kabat CDR-H3 (as found in hu3D6VHv1f, etc.) L.D.Y. SEQ ID NO:66; heavy chain variable region of mouse 6A10 antibody EVQLQQSGAELVRSGASVKLSCTASGLNIKDYYIHWVKQRPEQGLEWIGWIDPENDDTEYAPKFQGRATLTTDTSSNTAYLQLSSLTSEDTAVYYCTPLDYWGQGTSVTVSS

[0530] SEQ ID NO: 67; Kabat / Chothia composite CDR-H1 of mouse 6A10 antibody GLNIKDYYIH

[0531] SEQ ID NO:68; Kabat CDR-H2 of mouse 6A10 antibody WIDPENDDTEYAPKFQG

[0532] SEQ ID NO:69; Kabat CDR-H3 of mouse 6A10 antibody L.D.Y.

[0533] SEQ ID NO: 70; Mus VH structural template (PDB#1CR9_H) KVKLQQSGAELVRSGASVKLSCTASGFNIKDYYIQWVKQRPEQGLEWIGWIDPENGNSEYAPRFQGKATMTADTLSNTAYLQLSSLTSEDTAVYYCNADLHDYWGQGTTLTVSS

[0534] SEQ ID NO: 71; consensus VH amino acid sequence of Figures 4A and 4B of PCT / IB2017 / 052544 EVQLVQSGAEVVKPGALVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRVTITADTSNTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0535] SEQ ID NO:72; heavy chain of chimeric 3D6 antibody EVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0536] SEQ ID NO:73; light chain of chimeric 3D6 antibody DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0537] SEQ ID NO: 74: Amino acid sequence of heavy chain variable structure model Acc.#5MYX-VH_mSt EVQLQQSGAELVRPGSSVKISCKASGYIFNNYWINWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREGYIVYWGQGTLVTVSA

[0538] SEQ ID NO: 75; Amino acid sequence of heavy chain variable acceptor Acc.#2RCS-VH_huFrwk QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCASYYGIYWGQGTTLTVSS

[0539] SEQ ID NO: 76: Amino acid sequence of the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0540] SEQ ID NO: 77: Amino acid sequence of the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLELSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0541] SEQ ID NO: 78: Amino acid sequence of the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRATITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0542] SEQ ID NO: 79; Amino acid sequence of the heavy chain variable region hu3D6VHvb4 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0543] SEQ ID NO: 80: Amino acid sequence of the heavy chain variable region hu3D6VHvb5 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0544] SEQ ID NO: 81: Amino acid sequence of light chain variable structure model Acc.#5MYX-VL_mSt DVVLTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYVVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIK

[0545] SEQ ID NO: 82; Amino acid sequence of light chain variable acceptor Acc.#ARX71335-VL_huFrwk DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCEQGTHFPLTFGAGTKLELK

[0546] SEQ ID NO: 83: Amino acid sequence of the light chain variable region hu3D6VLvb1 of the humanized 3D6 antibody DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCWQGTHFPYTFGAGTKLELK

[0547] SEQ ID NO: 84: Amino acid sequence of the light chain variable region hu3D6VLvb2 of the humanized 3D6 antibody DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGAGTKLEIK

[0548] SEQ ID NO: 85: Amino acid sequence of the light chain variable region hu3D6VLvb3 of the humanized 3D6 antibody DVVMTQSPLSLSVTLGEPASISCRSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0549] SEQ ID NO: 86; Amino acid sequence of the alternative Kabat-Chothia composite CDR-H1 of the humanized 3D6 antibody (as found in hu3D6VHvb4 and hu3D6VHvb5, etc.) GFTIKDYYLH

[0550] SEQ ID NO:87; Amino acid sequence of alternative Kabat CDR-H2 of humanized 3D6 antibody (as found in hu3D6VHvb3 and hu3D6VHvb4, etc.) WIDPENGDTIYDPKFQG

[0551] SEQ ID NO: 88; Amino acid sequence of alternative Kabat CDR-H2 of humanized 3D6 antibody (as found in hu3D6VHvb5, etc.) WIDPEDGETIYDPKFQG

[0552] SEQ ID NO:89; Amino acid sequence of the alternative Kabat CDR-L1 of the humanized 3D6 antibody (as found in hu3D6VLvb3, etc.) RSSQSLLDSDGKTYLN

[0553] SEQ ID NO: 90: Amino acid sequence of the heavy chain variable region hu3D6VHvb6 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYFCSTLDFWGQGTLVTVSS

[0554] SEQ ID NO: 91: Amino acid sequence of the heavy chain variable region hu3D6VHvb7 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0555] SEQ ID NO: 92; Amino acid sequence of alternative Kabat CDR-H2 of humanized 3D6 antibody (as found in hu3D6VHvb6 and hu3D6VHvb7, etc.) WIDPEDGETVYDPKFQG

[0556] SEQ ID NO: 93; Light chain variable region of hu3D6VLv2 variant L54D (also known as L2-DIM21) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKDDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0557] SEQ ID NO: 94; Light chain variable region of hu3D6VLv2 variant L54G (also known as L2-DIM7) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0558] SEQ ID NO: 95; light chain variable region of hu3D6VLv2 variant L45N DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKNDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0559] SEQ ID NO: 96; light chain variable region of hu3D6VLv2 variant L54E DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKEDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0560] SEQ ID NO: 97; Light chain variable region of hu3D6VLv2 variant L50E DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYEVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0561] SEQ ID NO: 98; light chain variable region of hu3D6VLv2 variant L54Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKQDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0562] SEQ ID NO: 99; light chain variable region of hu3D6VLv2 variant L50D DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYDVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0563] SEQ ID NO: 100; light chain variable region of hu3D6VLv2 variant L54K DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKKDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0564] SEQ ID NO: 101; light chain variable region of hu3D6VLv2 variant L54R DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0565] SEQ ID NO: 102; light chain variable region of hu3D6VLv2 variant L54T DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKTDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0566] SEQ ID NO: 103; Light chain variable region of hu3D6VLv2 variant L50G (also known as L2-DIM22) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYGVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0567] SEQ ID NO: 104; light chain variable region of hu3D6VLv2 variant I48G DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLGYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0568] SEQ ID NO: 105; light chain variable region of hu3D6VLv2 variant I48D DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLDYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0569] SEQ ID NO: 106; light chain variable region of hu3D6VLv2 variant L47G DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRGIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0570] SEQ ID NO: 107; Light chain variable region of hu3D6VLv2 variant Y49E DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIELVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0571] SEQ ID NO: 108; light chain variable region of hu3D6VLv2 variant L54V DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKVDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0572] SEQ ID NO: 109; light chain variable region of hu3D6VLv2 variant L54S DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKSDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0573] SEQ ID NO:110; Light chain variable region of hu3D6VLv2 variant S52G (also known as L2-DIM9) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVGKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0574] SEQ ID NO:111; light chain variable region of hu3D6VLv2 variant L47N DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRNIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0575] SEQ ID NO: 112; Light chain variable region of hu3D6VLv2 variant L47D DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRDIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0576] SEQ ID NO: 113; Light chain variable region of hu3D6VLv2 variant L47E DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRREIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0577] SEQ ID NO: 114; Light chain variable region of hu3D6VLv2 variant L47P DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRPIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0578] SEQ ID NO: 115; light chain variable region of hu3D6VLv2 variant L47T DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRTIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0579] SEQ ID NO: 116; light chain variable region of hu3D6VLv2 variant L47S DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRSIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0580] SEQ ID NO: 117; Light chain variable region of hu3D6VLv2 variant L47A DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRAIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0581] SEQ ID NO: 118, Light chain variable region of hu3D6VLv2 variant L50V DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYVVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0582] SEQ ID NO: 119; Light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R (also known as L2-DIM1) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYGVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0583] SEQ ID NO: 120; Light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G (also known as L2-DIM2) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYEVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0584] SEQ ID NO: 121; Light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54G (also known as L2-DIM3) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0585] SEQ ID NO: 122; Light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R (also known as L2-DIM4) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0586] SEQ ID NO: 123; Light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54T (also known as L2-DIM5) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKTDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0587] SEQ ID NO: 124; Light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D (also known as L2-DIM6) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKDDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0588] SEQ ID NO: 125; light chain variable region of hu3D6VLv2 variant L37Q_L54R DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0589] SEQ ID NO: 126; Light chain variable region of hu3D6VLv2 variant L37Q_L54G DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0590] SEQ ID NO: 127; Light chain variable region of hu3D6VLv2 variant L37Q_L54D (also known as L2-DIM12) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKDDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0591] SEQ ID NO: 128; Light chain variable region of hu3D6VLv2 variant L37Q_L50G (also known as L2-DIM13) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYGVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0592] SEQ ID NO: 129; Light chain variable region of hu3D6VLv2 variant L37Q_L50D (also known as L2-DIM14) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0593] SEQ ID NO: 130; light chain variable region of hu3D6VLv2 variant L37Q_L54T DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0594] SEQ ID NO: 131; Light chain variable region of hu3D6VLv2 variant L37Q_S52G DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0595] SEQ ID NO: 132; Light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54G (also known as L2-DIM17) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0596] SEQ ID NO: 133; Light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54R (also known as L2-DIM18) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0597] SEQ ID NO: 134; Light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54G (also known as L2-DIM19) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYEVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0598] SEQ ID NO: 135; Light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54R (also known as L2-DIM20) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYEVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0599] SEQ ID NO: 136; light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYGVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0600] SEQ ID NO: 137; light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYGVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0601] SEQ ID NO: 138; light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0602] SEQ ID NO: 139; light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKDDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0603] SEQ ID NO: 140; light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54G_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKGDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0604] SEQ ID NO: 141; Light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54R_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYDVSKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0605] SEQ ID NO: 142; Light chain variable region of hu3D6VLv2 variant L37Q_L50V_L54D_G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYVVSKDDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0606] SEQ ID NO: 143; Light chain variable region of hu3D6VLv2 variant L37Q (also known as L2-DIM8) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0607] SEQ ID NO: 144 Light chain variable region of hu3D6VLv2 variant G100Q DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0608] SEQ ID NO: 145 Light chain variable region of hu3D6VLv2 variant L37Q_L54E DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKEDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIK

[0609] SEQ ID NO: 146; Heavy chain variable region of hu3D6VHv1bA11 variant D60E (also known as h3D6VHvb8) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYEPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0610] SEQ ID NO: 147 Heavy chain variable region of hu3D6VHv1bA11 variant L82cV EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSVTSEDTAVYFCSTLDFWGQGTLVTVSS

[0611] SEQ ID NO: 148; Heavy chain variable region of hu3D6VHv1bA11 variant D60E_L80M_Q81E_L82cV_T83R (also known as h3D6VHvb9) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYEPKFQGRATITADTSTDTAYMELGSVRSEDTAVYFCSTLDFWGQGTLVTVSS

[0612] SEQ ID NO: 149; Amino acid sequence of alternative Kabat CDR-H2 of humanized 3D6 antibody (as found in h3D6VHvb8 and h3D6VHvb9, etc.) WIDPENGDTVYEPKFQG

[0613] SEQ ID NO: 150; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L54D and hu3D6VLv2 L37Q_L54D, etc.): LVSKDDS

[0614] SEQ ID NO: 151; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L54G and hu3D6VLv2 L37Q_L54G, etc.): LVSKGDS

[0615] SEQ ID NO: 152; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L54N): LVSKNDS

[0616] SEQ ID NO: 153; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L54E and hu3D6VLv2 L37Q_L54E, etc.): LVSKEDS

[0617] SEQ ID NO:154; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L50E): EVSKLDS

[0618] SEQ ID NO: 155; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L54Q): LVSKQDS

[0619] SEQ ID NO: 156; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L50D and hu3D6VLv2 L37Q_L50D, etc.): DVSKLDS

[0620] SEQ ID NO: 157; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L54K): LVSKKDS

[0621] SEQ ID NO: 158; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L54R and hu3D6VLv2 L37Q_L54R, etc.): LVSKRDS

[0622] SEQ ID NO: 159; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L54T and hu3D6VLv2 L37Q_L54T, etc.): LVSKTDS

[0623] SEQ ID NO: 160; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L50G and hu3D6VLv2 L37Q_L50G, etc.): GVSKLDS

[0624] SEQ ID NO: 161; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L54V, etc.): LVSKVDS

[0625] SEQ ID NO: 162; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L54S): LVSKSDS

[0626] SEQ ID NO: 163; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 S52G and hu3D6VLv2 L37Q_S52G, etc.): LVGKLDS

[0627] SEQ ID NO: 164; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L50V, etc.): VVSKLDS

[0628] SEQ ID NO: 165; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L37Q_L50G_L54R and hu3D6VLv2 L37Q_L50G_L54R_G100Q, etc.): GVSKRDS

[0629] SEQ ID NO: 166; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L37Q_L50G_L54G and hu3D6VLv2 L37Q_L50G_L54G_G100Q, etc.): GVSKGDS

[0630] SEQ ID NO: 167; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_S52G_L54G): LVGKGDS

[0631] SEQ ID NO: 168; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L37Q_S52G_L54R and hu3D6VLv2 L37Q_S52G_L54R_G100Q, etc.): LVGKRDS

[0632] SEQ ID NO: 169; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_S52G_L54T): LVGKTDS

[0633] SEQ ID NO: 170; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L37Q_S52G_L54D and hu3D6VLv2 L37Q_S52G_L54D_G100Q, etc.): LVGKDDS

[0634] SEQ ID NO: 171; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L37Q_L50D_L54G and hu3D6VLv2 L37Q_L50D_L54G_G100Q, etc.): DVSKGDS

[0635] SEQ ID NO: 172; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (as found in hu3D6VLv2 L37Q_L50D_L54R and hu3D6VLv2 L37Q_L50D_L54R_G100Q, etc.): DVSKRDS

[0636] SEQ ID NO: 173; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50E_L54G): EVSKGDS

[0637] SEQ ID NO: 174; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50E_L54R): EVSKRDS

[0638] SEQ ID NO: 175; Amino acid sequence of alternative Kabat CDR-L2 of humanized 3D6 antibody (such as found in hu3D6VLv2 L37Q_L50V_L54D_G100Q): VVSKDDS

[0639] SEQ ID NO: 176; Amino acid sequence of heavy chain constant region (IgG1: allotype G1m17,1): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0640] SEQ ID NO:177; Amino acid sequence of light chain constant region (kappa): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0641] SEQ ID NO: 178; Amino acid sequence of the mature heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0642] SEQ ID NO: 179; Amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0643] SEQ ID NO: 180; Amino acid sequence of the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with the bovine alpha-lactalbumin signal peptide at the N-terminus MMSFVSLLLVGILFHATQAEVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFC STLDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0644] SEQ ID NO: 181; Amino acid sequence of the light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with bovine alpha-lactalbumin signal peptide at the N-terminus MMSFVSLLLVGILFHATQADVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVGKRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFP YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0645] SEQ ID NO: 182; Nucleotide sequence encoding the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with the bovine alpha-lactalbumin signal peptide at the N-terminus

[0646] SEQ ID NO: 183; Nucleotide sequence encoding the light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with the bovine alpha-lactalbumin signal peptide at the N-terminus ATGATGTCCTTTGTCTCTCTGCTCCTGGTTGGCATCCTATTCCATGCCACCCAGGCCGATGTTGTGATGACCCAGTCTCCACTCTCTTTGCCCGTTACCCTTGGACAACCTGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGCA ACAGAGGCCAGGCCAGTCTCCACGGCGCCTAATCTATCTGGTGGGCAAACGGGACTCTGGAGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTGGGAGTTTATTATTGCTGGCAAGGCACACATTTTCCGTACACGT TCGGAGGGGGGACCAAGCTGGAAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGCTTAAGTCCGGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCACAGGGGAGAGTGTTAGTGAGATCTCGAG

[0647] SEQ ID NO:184: Amino acid sequence of tau microtubule-binding repeat domain 1 (amino acid residues 255 to 271 of SEQ ID NO:1) NVKSKIGSTENLKHQPG

[0648] SEQ ID NO:185: Amino acid sequence of tau microtubule-binding repeat domain 2 (amino acid residues 286 to 302 of SEQ ID NO:1) NVQSKCGSKDNIKHVPG

[0649] SEQ ID NO:186: Amino acid sequence of tau microtubule-binding repeat domain 3 (amino acid residues 317 to 333 of SEQ ID NO:1) KVTSKCGSLGNIHHKPG

[0650] SEQ ID NO:187: Amino acid sequence of tau microtubule-binding repeat domain 4 (amino acid residues 349 to 365 of SEQ ID NO:1) RVQSKIGSLDNITHVPG

[0651] SEQ ID NO: 188: Amino acid sequence of the tau core motif to which 3D6 binds KIGSTENLKH

[0652] SEQ ID NO: 189: Amino acid sequence of the N-terminal tau sequence of the tau core motif to which 3D6 binds NVKS

[0653] SEQ ID NO: 190: Amino acid sequence of the C-terminal tau sequence of the tau core motif to which 3D6 binds QPG

[0654] SEQ ID NO: 191; Amino acid sequence of the epitope of 3D6 KXXSXXNX(K / H)H

[0655] SEQ ID NO: 192: Amino acid sequence of the tau core motif to which 3D6 binds KCGSKDNIKH

[0656] SEQ ID NO: 193: Amino acid sequence of the tau core motif to which 3D6 binds KCGSLGNIHH

[0657] SEQ ID NO: 194: Amino acid sequence of the tau core motif to which 3D6 binds KIGSLDNITH

Claims

1. 1. A composition for use in a method of reducing internalization of tau by cells in a subject, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

2. 1. A composition for use in a method for reducing tau-induced toxicity in a subject, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

3. 1. A composition for use in a method for reducing or delaying the onset of a behavioral disorder in a subject, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

4. 1. A composition for use in a method for reducing the level of a marker of tau pathology in a subject, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

5. 1. A composition for use in a method of reducing the incidence of tau pathology in a subject, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

6. A composition for use in a method for reducing intraneuronal tau pathology by cells in a subject in need thereof, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

7. A composition for use in a method for reducing or preventing toxicity in cortical neurons in a subject in need thereof, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

8. A composition for use in a method for reducing tau pathology in the hippocampus in a subject in need thereof, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

9. A composition for use in a method for reducing cortical tau pathology in a subject in need thereof, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

10. A composition for use in a method for reducing brainstem tau pathology in a subject in need thereof, the composition comprising a therapeutically effective amount of an antibody or antigen-binding antibody fragment comprising a heavy chain variable domain comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the sequence of LDF, and a light chain variable domain comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:13 or SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:

14.

11. The composition of any one of claims 1 to 10, wherein the subject has pathological hallmarks of Alzheimer's disease.

12. The composition of any one of claims 1 to 11, wherein the subject has Alzheimer's disease.

13. The composition of any one of claims 1 to 12, wherein the CDR-L2 of the antibody or antigen-binding antibody fragment comprises SEQ ID NO:

13.

14. The composition of any one of claims 1 to 12, wherein the CDR-L2 of the antibody or antigen-binding antibody fragment comprises SEQ ID NO:

168.

15. The composition of any one of claims 1 to 12, wherein the heavy chain variable domain of the antibody or antigen-binding antibody fragment comprises a mature heavy chain variable domain of SEQ ID NO: 18 and the light chain variable domain of the antibody or antigen-binding antibody fragment comprises a mature light chain variable domain of SEQ ID NO:

122.

16. The composition of any one of claims 1 to 12, wherein the antibody or antigen-binding antibody fragment is a humanized version of a murine antibody characterized by a mature heavy chain variable domain of SEQ ID NO: 7 and a mature light chain variable domain of SEQ ID NO:

11.

17. 13. The composition of any one of claims 1 to 12, wherein the antibody comprises a light chain comprising the mature light chain variable domain fused to a light chain constant region and a heavy chain comprising the mature heavy chain variable domain fused to a heavy chain constant region.

18. The composition of claim 17, wherein the heavy chain constant region of the antibody comprises the amino acid sequence of SEQ ID NO: 176, with or without the C-terminal lysine.

19. 18. The composition of claim 17, wherein the mature heavy chain variable domain fused to the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 178, with or without the C-terminal lysine.

20. The composition of claim 17 , wherein the antibody further comprises a signal peptide fused to the mature heavy chain variable domain and / or the mature light chain variable domain.

21. 21. The composition of claim 20, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 180, with or without the C-terminal lysine.

22. The composition of claim 17, wherein the light chain constant region of the antibody comprises the amino acid sequence of SEQ ID NO:

177.

23. 18. The composition of claim 17, wherein the mature light chain variable domain fused to a light chain constant region comprises the amino acid sequence of SEQ ID NO:

179.

24. 24. The composition of claim 23, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

181.

25. 20. The composition of claim 19, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 178, with or without the C-terminal lysine, and the light chain comprises the amino acid sequence of SEQ ID NO:

179.

26. 22. The composition of claim 21 , wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 180, with or without the C-terminal lysine, and the light chain comprises the amino acid sequence of SEQ ID NO:

181.

27. The composition of claim 17 , wherein the antibody comprises at least one mutation in the heavy chain constant region.

28. The composition of claim 27, wherein the at least one mutation in the heavy chain constant region reduces complement binding or activation by the heavy chain constant region or reduces binding to an Fcγ receptor compared to a native human heavy chain constant region.

29. 29. The composition of claim 28, wherein the antibody comprises a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 according to the EU numbering, or comprises an alanine at positions 318, 320, and 322 according to the EU numbering.

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