Tau-recognizing antibodies

Abstract

To provide antibodies or antigen binding antibody fragments that specifically bind to human tau.SOLUTION: Provided herein is an antibody or antigen binding fragment thereof that specifically binds to human tau, the antibody comprising a mature heavy chain variable region and a mature light chain variable region, where the mature heavy chain variable region comprises CDR-H3 including sequences of CDR-H1, CDR-H2, and LDF having specific sequences respectively, except that Kabat position H28 can be occupied by N or T, Kabat position H54 can be occupied by N or D, Kabat position H56 can be occupied by D or E, and Kabat position H58 occupied by V or I, where the mature light chain variable region comprises CDRs L1, L2 and L3 comprising specific sequences respectively, except that Kabat position L24 can be occupied by K or R, where at least one of specified positions is occupied by the designated amino acid.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 62 / 758,421, filed November 9, 2018, and PCT Application No. PCT / US2018 / 059895, filed November 8, 2018. Each of these applications is incorporated by reference in its entirety into this application.

[0002] Array list reference This application contains an electronic sequence listing with file name 2019-11-08536322WO_ST25.TXT, which was created on November 8, 2019, and contains 96,408 bytes. This sequence listing is incorporated by reference in its entirety into this application. [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 be involved in the stabilization of microtubules, particularly in the central nervous system. Total tau (t-tau, i.e., phosphorylated and non-phosphorylated forms) and phospho-tau (p-tau, i.e., phosphorylated tau) are released by the brain in response to neuronal injury and neurodegeneration, and levels have been reported to be increased in the CSF of Alzheimer's disease patients compared to the general population (Jack et al., Lancet Neurol 9:119-28(2010)).

[0004] Tau is the major constituent of neurofibrillary tangles, which, together with plaques, are a hallmark of Alzheimer's disease. Tangles consist of abnormal fibrils 10 nm in diameter that occur in helically wound pairs with a regular period of 80 nm. Tau within neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated) with phosphate groups attached to specific sites on the molecule. Strong involvement of neurofibrillary tangles in Alzheimer's disease is found in entorhinal cortex layer II neurons, the CA1 and subiculum regions of the hippocampus, the amygdala, and deeper layers of the neocortex (layers III, V, and superficial VI). Hyperphosphorylated tau has also been reported to disrupt microtubule assembly, which may promote the destruction of neuronal networks.

[0005] Tau inclusions are a hallmark of the neuropathology of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy and Pick's disease. Summary of the Invention

[0006] In one aspect, the invention provides a mature heavy chain variable region comprising CDRs H1, H2 and H3 comprising SEQ ID NOs: 8, 9 and 10, respectively, except 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, and position H58 may be occupied by V or I; and CDRs H1, H2 and H3 comprising SEQ ID NOs: 12, 13 and 14, respectively, except that position L24 may be occupied by K or R. and a mature light chain variable region comprising L1, L2 and L3, wherein at least one of the following positions is occupied by the specified amino acids: 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, H58 is occupied by I, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L83 is occupied by L, L86 is occupied by H, L100 is occupied by A or Q and L106 is occupied by L.

[0007] In some such antibodies, CDR-H1 comprises SEQ ID NO:8 or SEQ ID NO:86, CDR-H2 comprises SEQ ID NO:9, SEQ ID NO:87, SEQ ID NO:88, or SEQ ID NO:92, CDR-H3 comprises SEQ ID NO:10, CDR-L1 comprises SEQ ID NO:12 or SEQ ID NO:89, CDR-L2 comprises SEQ ID NO:13, and CDR-L3 comprises SEQ ID NO:14. In some such antibodies, CDR-H1 comprises SEQ ID NO:8 or SEQ ID NO:86, CDR-H2 comprises SEQ ID NO:9, SEQ ID NO:87, or SEQ ID NO:88, CDR-H3 comprises SEQ ID NO:10, CDR-L1 comprises SEQ ID NO:12 or SEQ ID NO:89, CDR-L2 comprises SEQ ID NO:13, and CDR-L3 comprises SEQ ID NO:14. In some such antibodies, CDR-H1 comprises SEQ ID NO:86, CDR-H2 comprises SEQ ID NO:92, CDR-H3 comprises SEQ ID NO:10, CDR-L1 comprises SEQ ID NO:12 or SEQ ID NO:89, CDR-L2 comprises SEQ ID NO:13, and CDR-L3 comprises SEQ ID NO:14.

[0008] In some such antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86. In some such antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87. In some such antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 88. In some such antibodies, CDR-H2 has an amino acid sequence comprising SEQ ID NO: 92. In some such antibodies, CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89.

[0009] In some such antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87. In some such antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO: 88. In some such antibodies, CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86 and CDR-H2 has an amino acid sequence comprising SEQ ID NO: 92.

[0010] In some such antibodies, the antibody is a humanized antibody, a veneered antibody, or a chimeric antibody.

[0011] In some antibodies, the humanized mature heavy chain variable region has an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 76-80 and 90-91, and the humanized mature light chain variable region has an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 83-85. In some antibodies, the humanized mature heavy chain variable region has an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 76-80, and the humanized mature light chain variable region has an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 83-85. In some antibodies, the humanized mature heavy chain variable region has an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 90-91, and the humanized mature light chain variable region has an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 83-85.

[0012] In some such antibodies, at least one of the following positions in the VH region is occupied by the specified amino acids: H93 is occupied by S and H94 is occupied by T. In some such antibodies, positions H93 and H94 are occupied by S and T, respectively.

[0013] In some such antibodies, position H91 in the VH region is occupied by F.

[0014] In some such antibodies, at least one of the following positions in the VH region is occupied by a 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 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.

[0015] In some such 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 antibodies, positions H17, H80 and H83 in the VH region are occupied by T, M, and R, respectively.

[0016] In some such antibodies, position H58 in the VH region is occupied by I.

[0017] In some such 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 antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.

[0018] In some such 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 antibodies, positions H54 and H56 in the VH region are occupied by D and E, respectively.

[0019] In some such antibodies, at least one of the following positions in the VH region is occupied by a 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, and H54 is occupied by N or D. , H56 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.

[0020] In some antibodies, positions H91, H93, and H94 in the VH region are occupied, respectively, by F, S, and T. In some 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, respectively, by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T, L, and V. In some 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. In some 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. In some 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. In some 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.In some 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.

[0021] In some such antibodies, at least one of the following positions in the VL region is occupied by a 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 antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.

[0022] In some such antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L7 is occupied by T or S, L10 is occupied by T or S, L15 is occupied by I or L, L17 is occupied by Q or E, L24 is occupied by K or R, L37 is occupied by L or Q, L45 is occupied by K or R, L83 is occupied by L or V, L86 is occupied by H or Y, L100 is occupied by A or Q, and L106 is occupied by L or I.

[0023] In some antibodies, positions L7, L10, L15, L83, L86, and L106 in the VL region are occupied by S, S, L, V, Y, and I, respectively. In some 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.

[0024] In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 76 to 80 and SEQ ID NOs: 90 to 91, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 83 to 85. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 76 to 80, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 83 to 85. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 90 to 91, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 83 to 85.

[0025] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 76 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 76 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 76 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0026] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 77 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 77 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 77 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0027] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 78 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 78 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 78 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0028] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 79 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 79 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 79 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0029] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 80 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0030] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 90 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0031] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 91 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 83. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 91 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 84. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 91 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 85.

[0032] For example, the antibody can be a chimeric antibody. For example, the antibody can be a veneered antibody.

[0033] The antibody may be a fully murine, chimeric, veneered or humanized antibody or binding fragment, a single chain antibody, a single chain antibody, a Fab fragment, a Fab'2 fragment, or a single chain Fv. Some antibodies have a human IgG1 isotype, while other antibodies may have a human IgG2 or IgG4 isotype. Some antibodies have a mature light chain variable region fused to a light chain constant region and a mature heavy chain variable region fused to a heavy chain constant region. The heavy chain constant region of some antibodies is a variant of a native human constant region that has reduced binding to Fcγ receptors compared to the native human constant region. In some antibodies, the mature heavy chain variable region is fused to a heavy chain constant region having the sequence of SEQ ID NO: 103 with or without a C-terminal lysine, and / or the mature light chain variable region is fused to a light chain constant region having the sequence of SEQ ID NO: 104.

[0034] Some antibodies may have at least one mutation in the constant region, e.g., a mutation that reduces complement binding or activation by the constant region, e.g., a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331, EU numbering. Some antibodies have alanine at positions 318, 320, and 322. Some antibodies may be at least 95% w / w pure. The antibody may be conjugated to a therapeutic agent, a cytotoxic agent, a cytostatic agent, a neurotrophic agent, or a neuroprotective agent.

[0035] In another aspect, the invention provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharma- ceutically acceptable carrier.

[0036] In another aspect, the present invention provides a nucleic acid encoding any of the heavy chains and / or light chains disclosed herein, a recombinant expression vector comprising the nucleic acid, and a host cell transformed with the recombinant expression vector. For example, the nucleic acid may have a sequence comprising any one of SEQ ID NOs: 30-31, 93-99, 100-102, and 105-106. In another example, the nucleic acid may have a sequence comprising any one of SEQ ID NOs: 30-31, 93-97, 100-102, and 105-106. In another example, the nucleic acid may have a sequence comprising any one of SEQ ID NOs: 30-31, 98-99, 100-102, and 105-106.

[0037] In another aspect, the invention provides a vector comprising a nucleic acid encoding a mature heavy chain variable region and a mature light chain variable region operably linked to one or more regulatory sequences for effecting expression in a mammalian cell of any of the antibodies disclosed herein, a host cell comprising the nucleic acid, and a method of expressing an antibody in a mammalian cell comprising incorporating the nucleic acid into the genome of a transgenic animal in which the antibody is expressed.

[0038] Some vectors further comprise nucleic acid encoding a heavy chain constant region fused to the mature heavy chain variable region and a light chain constant region fused to the mature light chain variable region. In some vectors, the encoded heavy chain constant region has the sequence of SEQ ID NO: 103 with or without a C-terminal lysine, and the encoded light chain constant region has the sequence of SEQ ID NO: 104. In some vectors, the heavy chain constant region is encoded by the sequence of SEQ ID NO: 105, and the light chain constant region is encoded by the sequence of SEQ ID NO: 106.

[0039] In some vectors, the antibody expressed encoded by the nucleic acid is an scFv. In some vectors, the antibody expressed encoded by the nucleic acid is a Fab fragment.

[0040] In some vectors, the one or more regulatory sequences include one or more of a promoter, an enhancer, a ribosome binding site, and a transcription termination signal. In some vectors, the nucleic acid further encodes a signal peptide fused to the mature heavy and light chain variable regions. In some vectors, the nucleic acid is codon-optimized for expression in a host cell. In some vectors, the one or more regulatory sequences include a eukaryotic promoter. In some vectors, the nucleic acid further encodes a selectable gene.

[0041] In another aspect, the invention provides first and second vectors comprising nucleic acid encoding a mature heavy chain variable region and a mature light chain variable region, respectively, each vector operably linked to one or more regulatory sequences for effecting expression in a mammalian cell of any of the antibodies disclosed herein, a host cell comprising the nucleic acid, and a method of expressing an antibody in a mammalian cell comprising incorporating the nucleic acid into the genome of a transgenic animal in which the antibody is expressed.

[0042] In some vectors, the nucleic acid further encodes a heavy chain constant region fused to a mature heavy chain variable region and a light chain constant region fused to a mature light chain variable region. In some vectors, the encoded heavy chain constant region has the sequence of SEQ ID NO: 103, with or without a C-terminal lysine, and the encoded light chain constant region has the sequence of SEQ ID NO: 104. In some vectors, the heavy chain constant region is encoded by the sequence of SEQ ID NO: 105 and the light chain constant region is encoded by the sequence of SEQ ID NO: 106.

[0043] In yet another aspect, the invention provides a method of humanizing any of the non-human antibodies described herein, such as the murine antibody 3D6, which is characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11. Such a method may include selecting one or more acceptor antibodies, synthesizing a nucleic acid encoding a humanized heavy chain comprising the CDRs of the murine heavy chain and a nucleic acid encoding a humanized light chain comprising the CDRs of the murine antibody light chain, and expressing the nucleic acid in a host cell to produce the humanized antibody.

[0044] Methods for producing humanized, chimeric or veneered antibodies, such as humanized, chimeric or veneered versions of 3D6, are also provided. In such methods, cells transformed with nucleic acids encoding the heavy and light chains of the antibody are cultured to secrete the antibody. The antibody is then purified from the cell culture medium.

[0045] A cell line producing any of the antibodies disclosed herein can be produced by introducing into cells vectors encoding the antibody heavy and light chains and a selectable marker, growing the cells under conditions that select for cells with increased copy number of the vector, isolating single cells from the selected cells, and storing cloned cells from the selected single cells based on antibody yield.

[0046] Some cells were expanded under selective conditions and naturally expressed at least 100 mg / L / 10 6 Cell lines can be screened for those that secrete antibodies in amounts of cells / 24 hours. Single cells can be isolated from the selected cells. Cells cloned from the single cells can then be stored. Single cells can be selected based on desirable properties, such as antibody yield. An exemplary cell line is a cell line expressing 3D6.

[0047] The present invention also provides a method of inhibiting or reducing tau aggregation in a subject at risk of developing tau-mediated amyloidosis, the method comprising administering to the subject an effective dosing regimen of an antibody disclosed herein, thereby inhibiting or reducing tau aggregation in the subject. Exemplary antibodies include humanized versions of 3D6.

[0048] Also provided are methods of treating or preventing a tau-related disease in a subject, the methods comprising administering an effective dosing regimen of an antibody disclosed herein, thereby treating or preventing the disease. Examples of such diseases are 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some methods, the tau-associated disease is Alzheimer's disease. In some methods, the patient is an ApoE4 carrier.

[0049] Also provided is a method of reducing the aberrant propagation of tau, the method comprising administering an effective dosage regimen of an antibody disclosed herein, thereby reducing the propagation of tau.

[0050] Also provided is a method of inducing phagocytosis of tau, the method comprising administering an effective dosage regimen of an antibody disclosed herein, thereby inducing phagocytosis of tau.

[0051] Also provided is a method of inhibiting tau aggregation or deposition, the method comprising administering an effective dosage regimen of an antibody disclosed herein, thereby inhibiting tau aggregation or deposition.

[0052] Also provided is a method of inhibiting the formation of tau tangles, the method comprising administering an effective dosage regimen of an antibody disclosed herein.

[0053] The invention also provides a method for detecting tau protein deposits in a subject suffering from or at risk for a disease associated with tau aggregation or deposition, comprising administering an antibody as disclosed herein to the subject and detecting antibody bound to tau in the subject. Examples of such diseases include 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some embodiments, the antibody is administered to the subject's body by intravenous injection. In some embodiments, the antibody is administered directly to the subject's brain by intracranial injection or by drilling a hole through the subject's skull. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

[0054] The present invention also provides a method for measuring efficacy of a treatment in a subject being treated for a disease associated with tau aggregation or deposition, the method comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering an antibody disclosed herein to the subject, and measuring a first amount of antibody bound to tau in the subject, administering the treatment to the subject, measuring a second level of tau protein deposits in the subject after treatment by administering the antibody to the subject, and detecting antibody bound to tau in the subject, wherein a reduction in the level of tau protein deposits indicates a positive response to the treatment.

[0055] The present invention also provides a method for measuring efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition, the method comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering an antibody disclosed herein to the subject, and measuring a first amount of antibody bound to tau in the subject, administering the treatment to the subject, measuring a second level of tau protein deposits in the subject after treatment by administering the antibody to the subject, and detecting the second amount of antibody bound to tau in the subject, wherein no change in the level of tau protein deposits or a small increase in tau protein deposits indicates a positive response to the treatment. [Brief description of the drawings]

[0056] [Figure 1] 1 shows a sequence comparison of the heavy chain variable region of the murine 3D6 antibody (sequence number 7) and the heavy chain variable region of the humanized version of the 3D6 antibody (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7), as well as the human germline heavy chain variable region sequence IGHV1-69-2*01 (sequence number 25) and the human receptor heavy chain variable region sequence 2RCS VH hFrwk (sequence number 75). hu3D6VHvb1 is SEQ ID NO: 76, hu3D6VHvb2 is SEQ ID NO: 77, hu3D6VHvb3 is SEQ ID NO: 78, hu3D6VHvb4 is SEQ ID NO: 79, hu3D6VHvb5 is SEQ ID NO: 80, hu3D6VHvb6 is SEQ ID NO: 90, and hu3D6VHvb7 is SEQ ID NO: 91. The CDRs defined by the Kabat / Chothia combination are shown in bold. [Diagram 2]Shown is a sequence comparison of the light chain variable region of the murine 3D6 antibody (SEQ ID NO: 11) and the light chain variable region of the humanized version of the 3D6 antibody (hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3), as well as the human germline light chain variable region sequence IGKV2-30*02 (SEQ ID NO: 27) and the human receptor ARX71335_VL_hFrwk (SEQ ID NO: 82). hu3D6VLvb1 is SEQ ID NO: 83, hu3D6VLvb2 is SEQ ID NO: 84, and hu3D6VLvb3 is SEQ ID NO: 85. The CDRs defined by Kabat are shown in bold. [Figure 3A] 1 shows the results of ELISA screening for selected mouse monoclonal anti-tau antibodies. [Figure 3B] 1 shows the results of ELISA screening for selected mouse monoclonal anti-tau antibodies. [Figure 3C] 1 shows the results of ELISA screening for selected mouse monoclonal anti-tau antibodies. [Figure 4] Figure 1 shows the binding kinetics of select mouse monoclonal anti-tau antibodies to recombinant human tau. [Diagram 5] 1 shows the results of a functional blocking assay for select mouse monoclonal anti-tau antibodies. [Figure 6] 1 shows the results of a disaggregation assay for select mouse monoclonal anti-tau antibodies. [Figure 7] 1 shows the results of experiments demonstrating that 3D6 and 5G8 immunocapture tau from human Alzheimer's disease tissue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] A brief description of the sequence SEQ ID NO: 1 shows the amino acid sequence of a human tau isoform (SwissProt P10636-8).

[0058] SEQ ID NO: 2 shows the amino acid sequence of a human tau isoform (SwissProt P10636-7).

[0059] SEQ ID NO: 3 shows the amino acid sequence of an isoform of human tau (Swissprot P10636-6), (4R0N human tau).

[0060] SEQ ID NO: 4 shows the amino acid sequence of a human tau isoform (SwissProt P10636-5).

[0061] SEQ ID NO: 5 shows the amino acid sequence of a human tau isoform (SwissProt P10636-4).

[0062] SEQ ID NO: 6 shows the amino acid sequence of a human tau isoform (SwissProt P10636-2).

[0063] SEQ ID NO: 7 shows the amino acid sequence of the heavy chain variable region of the murine 3D6 antibody.

[0064] SEQ ID NO: 8 shows the amino acid sequence of the combined Kabat / Chothia CDR-H1 of the murine 3D6 antibody.

[0065] SEQ ID NO: 9 shows the amino acid sequence of Kabat CDR-H2 of the murine 3D6 antibody.

[0066] SEQ ID NO: 10 shows the amino acid sequence of the Kabat CDR-H3 of the murine 3D6 antibody.

[0067] SEQ ID NO: 11 shows the amino acid sequence of the light chain variable region of the murine 3D6 antibody and the murine 6A10 antibody.

[0068] SEQ ID NO: 12 shows the amino acid sequence of Kabat CDR-L1 of the murine 3D6 and murine 6A10 antibodies.

[0069] SEQ ID NO: 13 shows the amino acid sequence of Kabat CDR-L2 of the murine 3D6 and murine 6A10 antibodies.

[0070] SEQ ID NO: 14 shows the amino acid sequence of the Kabat CDR-L3 of the murine 3D6 and murine 6A10 antibodies.

[0071] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1.

[0072] SEQ ID NO: 16 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv2.

[0073] SEQ ID NO: 17 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1b.

[0074] SEQ ID NO: 18 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1bA11.

[0075] SEQ ID NO: 19 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv5.

[0076] SEQ ID NO: 20 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv1.

[0077] SEQ ID NO: 21 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv2.

[0078] SEQ ID NO: 22 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv3.

[0079] SEQ ID NO: 23 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv4.

[0080] SEQ ID NO: 24 shows the amino acid sequence of heavy chain variable receptor accession number BAC01986.1.

[0081] SEQ ID NO: 25 is the heavy chain variable receptor IMGT accession number IGHV1-69-2 * The amino acid sequence of 01 is shown.

[0082] SEQ ID NO: 26 is the heavy chain variable receptor IMGT accession number IGKJ1 * The amino acid sequence of 01 is shown.

[0083] SEQ ID NO: 27 is the light chain variable receptor IMGT accession number IGKV2-30 * The amino acid sequence of 02 is shown.

[0084] SEQ ID NO: 28 is the light chain variable receptor IMGT accession number IGKJ2 * The amino acid sequence of 01 is shown.

[0085] SEQ ID NO: 29 shows the amino acid sequence of light chain variable receptor accession number AAZ09048.1.

[0086] SEQ ID NO:30 shows the nucleic acid sequence encoding the heavy chain variable region of the murine 3D6 antibody.

[0087] SEQ ID NO: 31 shows the nucleic acid sequence encoding the light chain variable region of the murine 3D6 antibody.

[0088] SEQ ID NO: 32 shows the amino acid sequence of Kabat CDR-H1 of the murine 3D6 antibody.

[0089] SEQ ID NO: 33 shows the amino acid sequence of Chothia CDR-H1 of the mouse 3D6 antibody.

[0090] SEQ ID NO: 34 shows the amino acid sequence of Chothia CDR-H2 of the mouse 3D6 antibody.

[0091] SEQ ID NO: 35 shows the amino acid sequence of AbM CDR-H2 of the murine 3D6 antibody.

[0092] SEQ ID NO: 36 shows the amino acid sequence of Contact CDR-L1 of mouse 3D6 antibody.

[0093] SEQ ID NO: 37 shows the amino acid sequence of Contact CDR-L2 of mouse 3D6 antibody.

[0094] SEQ ID NO: 38 shows the amino acid sequence of Contact CDR-L3 of mouse 3D6 antibody.

[0095] SEQ ID NO: 39 shows the amino acid sequence of Contact CDR-H1 of mouse 3D6 antibody.

[0096] SEQ ID NO: 40 shows the amino acid sequence of Contact CDR-H2 of mouse 3D6 antibody.

[0097] SEQ ID NO: 41 shows the amino acid sequence of Contact CDR-H3 of mouse 3D6 antibody.

[0098] SEQ ID NO: 42 shows the amino acid sequence of another Kabat-Chothia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3, hu3D6VHv1e, and hu3D6VHv1f).

[0099] SEQ ID NO: 43 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv5 and hu3D6VHv1bA11B6H3).

[0100] SEQ ID NO: 44 shows the consensus amino acid sequence in the heavy chain variable region of murine 3D6 and selected humanized 3D6 antibodies (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) (labeled "Majority" in Figure 2 of PCT / IB2017 / 052544).

[0101] SEQ ID NO: 45 shows the consensus amino acid sequence between the light chain variable regions of murine 3D6 and selected humanized 3D6 antibodies (labeled "Majority" in Figure 3 of PCT / IB2017 / 052544).

[0102] SEQ ID NO: 46 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1bA11B6G2.

[0103] SEQ ID NO: 47 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1bA11B6H3.

[0104] SEQ ID NO: 48 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1c.

[0105] SEQ ID NO: 49 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1d.

[0106] SEQ ID NO: 50 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1e.

[0107] SEQ ID NO: 51 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1f.

[0108] SEQ ID NO: 52 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv3.

[0109] SEQ ID NO: 53 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv3b.

[0110] SEQ ID NO: 54 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv3c.

[0111] SEQ ID NO: 55 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv4.

[0112] SEQ ID NO: 56 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv4b.

[0113] SEQ ID NO: 57 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv4c.

[0114] SEQ ID NO: 58 shows the amino acid sequence of another Kabat-Chothia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VH1c).

[0115] SEQ ID NO: 59 shows the amino acid sequence of another Kabat-Chothia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c).

[0116] SEQ ID NO: 60 shows the amino acid sequence of another Kabat-Chothia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv3b and hu3D6VHv4b).

[0117] SEQ ID NO: 61 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1bA11B6G2).

[0118] SEQ ID NO: 62 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b).

[0119] SEQ ID NO: 63 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c).

[0120] SEQ ID NO: 64 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1e).

[0121] SEQ ID NO: 65 shows the amino acid sequence of another Kabat CDR-H3 of the humanized 3D6 antibody (similar to hu3D6VHv1f).

[0122] SEQ ID NO: 66 shows the amino acid sequence of the heavy chain variable region of the murine 6A10 antibody.

[0123] SEQ ID NO: 67 shows the amino acid sequence of the combined Kabat / Chothia CDR-H1 of the murine 6A10 antibody.

[0124] SEQ ID NO: 68 shows the amino acid sequence of Kabat CDR-H2 of the murine 6A10 antibody.

[0125] SEQ ID NO: 69 shows the amino acid sequence of the Kabat CDR-H3 of the murine 6A10 antibody.

[0126] SEQ ID NO: 70 shows the amino acid sequence of the VH region of a murine antibody (pdb code 1CR9) used as a structural template for heavy chain humanization.

[0127] SEQ ID NO: 71 shows the consensus amino acid sequence in the heavy chain variable region of selected humanized 3D6 antibodies (VHv1, VHv1b, VHv1bA11, VHv1bA11B6G2, VHv1bA11B6H3, VHv1c, VHv1d, VHv1e, VHv1f, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) (labeled "Majority" in Figures 4A and 4B of PCT / IB2017 / 052544).

[0128] SEQ ID NO: 72 shows the amino acid sequence of the heavy chain of the chimeric 3D6 antibody.

[0129] SEQ ID NO: 73 shows the amino acid sequence of the light chain of the chimeric 3D6 antibody.

[0130] SEQ ID NO: 74 shows the amino acid sequence of the heavy chain variable structure model accession number 5MYX-VH_mSt.

[0131] SEQ ID NO: 75 shows the amino acid sequence of heavy chain variable receptor accession number 2RCS-VH_huFrwk.

[0132] SEQ ID NO: 76 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody.

[0133] SEQ ID NO: 77 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody.

[0134] SEQ ID NO: 78 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody.

[0135] SEQ ID NO: 79 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb4 of the humanized 3D6 antibody.

[0136] SEQ ID NO: 80 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb5 of the humanized 3D6 antibody.

[0137] SEQ ID NO: 81 shows the amino acid sequence of the light chain variable structure model accession number 5MYX-VL_mSt.

[0138] SEQ ID NO: 82 shows the amino acid sequence of light chain variable receptor accession number ARX71335-VL_huFrwk.

[0139] SEQ ID NO: 83 shows the amino acid sequence of the light chain variable region hu3D6VLvb1 of the humanized 3D6 antibody.

[0140] SEQ ID NO: 84 shows the amino acid sequence of the light chain variable region hu3D6VLvb2 of the humanized 3D6 antibody.

[0141] SEQ ID NO: 85 shows the amino acid sequence of the light chain variable region hu3D6VLvb3 of the humanized 3D6 antibody.

[0142] SEQ ID NO: 86 shows the amino acid sequence of another Kabat-Chothia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHvb4 and hu3D6VHvb5).

[0143] SEQ ID NO: 87 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb3 and hu3D6VHvb4).

[0144] SEQ ID NO: 88 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb5).

[0145] SEQ ID NO: 89 shows the amino acid sequence of another Kabat CDR-L1 of the humanized 3D6 antibody (similar to hu3D6VLvb3).

[0146] SEQ ID NO: 90 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb6 of the humanized 3D6 antibody.

[0147] SEQ ID NO: 91 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb7.

[0148] SEQ ID NO: 92 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb6 and hu3D6VHvb7).

[0149] SEQ ID NO: 93 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb1.

[0150] SEQ ID NO: 94 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb2.

[0151] SEQ ID NO: 95 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb3.

[0152] SEQ ID NO: 96 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb4.

[0153] SEQ ID NO: 97 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb5.

[0154] SEQ ID NO: 98 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb6.

[0155] SEQ ID NO: 99 shows the nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb7.

[0156] SEQ ID NO: 100 shows the nucleic acid sequence encoding the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb1.

[0157] SEQ ID NO: 101 shows the nucleic acid sequence encoding the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb2.

[0158] SEQ ID NO: 102 shows the nucleic acid sequence encoding the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb3.

[0159] SEQ ID NO: 103 shows the amino acid sequence of a representative IgG1 heavy chain constant region.

[0160] SEQ ID NO: 104 shows the amino acid sequence of a representative kappa light chain constant region.

[0161] SEQ ID NO: 105 shows a nucleic acid sequence encoding a representative IgG1 heavy chain constant region.

[0162] SEQ ID NO: 106 shows a nucleic acid sequence encoding a representative kappa light chain constant region.

[0163] definition Monoclonal antibodies or other biological entities are usually provided in isolated form. This means that the antibody or other biological entity is usually at least 50% w / w pure relative to interfering proteins and other contaminants resulting from its production or purification, but does not exclude the possibility that the monoclonal antibody may be combined with an excess of pharma- ceutically acceptable carrier(s) or other vehicles intended to facilitate use. Sometimes, the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95% or 99% w / w pure relative to interfering proteins and other contaminants resulting from its production or purification. An isolated monoclonal antibody or other biological entity is often the major macromolecular species remaining after its purification.

[0164] 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 binding that is detectably high in magnitude and is distinguishable from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of the formation of bonds between specific functional groups or specific spatial fits (e.g., lock-and-key type), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that the antibody binds to only one target.

[0165] 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 that are primarily responsible for antigen recognition. This variable region, linked to a cleavable signal peptide, is expressed first. The variable region without the signal peptide is often referred to as the mature variable region. Thus, for example, the mature variable region of a light chain means the light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function.

[0166] 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 the heavy chain also including a "D" region of about 10 or more amino acids. (For a general discussion, see Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7, which is incorporated by reference in its entirety in this application.)

[0167] 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 separated by three "complementarity determining regions" or "CDRs". The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs contain the amino acid residues of an antibody that are primarily involved in antigen binding. From the amino to carboxyl termini, both VL and VH domains contain the following framework (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. When the present application discloses a VL sequence having R as the C-terminal residue, this R can alternatively be considered to be the N-terminal residue of the light chain constant region, and therefore it should be understood that the present application also discloses VL sequences that do not have a C-terminal R.

[0168] The assignment of amino acids to the VL and VH domains, respectively, is consistent with any of the conventional definitions of CDRs, including those by Kabat (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), Chothia (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989), the combination of Chothia and Kabat CDRs, where CDR-H1 is the combination of the Chothia and Kabat CDRs; Oxford Molecular's antibody modelling and the definition of contact by 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 CDR definition (e.g., Kabat), that definition specifies the minimum number of CDR residues (i.e., Kabat CDRs) present in the antibody. It does not exclude that other residues that fall within another conventional CDR definition but are outside of that specified definition may exist 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 the combined Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues and no additional CDR residues according to other definitions. [Table 1] *The CDR-H1 from Chothia may end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places additional residues at 35A and 35B, while the Chothia numbering places them at 31A and 31B. If neither H35A nor H35B (Kabat numbering) are present, the Chothia CDR-H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0169] The term "antibody" includes complete antibodies and their binding fragments. Fragments, including isolated heavy and light chains, Fab, Fab', F(ab')2, F(ab)c, Dab, nanobody, and Fv, usually compete with the complete 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 complete immunoglobulins. The term "antibody" also includes bispecific and / or humanized antibodies. Bispecific or bivalent antibodies are artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites (see, for example, 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 than that bound by 3D6.

[0170] 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 on the blood-brain barrier, such as the insulin receptor, the insulin-like growth factor (IGF) receptor, the leptin receptor, or the lipoprotein receptor, or the 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 transported across the blood-brain barrier by receptor-mediated transcytosis. Brain uptake of bispecific antibodies can be further enhanced by engineering specific antibodies to reduce their affinity for blood-brain barrier receptors. The reduced affinity for the receptor results in a more widespread 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).

[0171] Representative bispecific antibodies may be: (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) TM)Molecule, In:Antibody Engineering, Springer Berlin Heidelberg (2010); (2) Tandab, which is the fusion of two single chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (3) flexibodies, which are a combination of scFvs and diabodies resulting in multivalent molecules; (4) so-called "dock and lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which can be applied to Fabs to obtain trivalent bispecific binding proteins consisting of two identical Fab fragments bound to different Fab fragments; or (5) so-called scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fc region. Examples of useful platforms for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc modified IgG1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

[0172] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids 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 on exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are usually lost on treatment with denaturing solvents. Epitopes typically include at least 3 or more, usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0173] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be determined by X-ray crystallography of the antibody bound to its antigen to identify contact 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. 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.

[0174] Competition between antibodies is determined by an assay in which a test antibody inhibits specific binding of a reference antibody to a common antigen (e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold or 100-fold) of the test antibody 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 for steric hindrance to occur.

[0175] The term "pharmaceutically acceptable" means the carrier, diluent or excipient or auxiliary material is compatible with other ingredients of the formulation and not substantially deleterious to the recipient.

[0176] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0177] An individual is at high risk for a disease if a subject has at least one known risk factor (e.g., genetic, biochemical, family history, situational exposure) that places an individual with that risk factor at a statistically significantly higher risk of developing the disease than individuals without that risk factor.

[0178] The term "biological sample" refers to a sample of biological material that can be obtained from or in a biological source, e.g., a human or mammalian subject. Such samples can be organs, organelles, tissues, tissue sections, body fluids, peripheral blood, plasma, serum, cells, molecules such as proteins and peptides, and any portion or combination thereof derived therefrom. The term "biological sample" can also encompass any material obtained by processing a sample. Derived materials can include cells or their progeny. Processing of a biological sample can include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.

[0179] The term "control sample" refers to a biological sample that is not known or suspected to contain a tau-related disease region, or at least not known or suspected to contain a disease region of a given type. The control sample can be obtained from an individual that does not suffer from a tau-related disease. Alternatively, the control sample can be obtained from a patient that does not suffer from a tau-related disease. Such a sample can be obtained at the same time or at another time that the biological sample is suspected to contain a tau-related disease. Both the biological sample and the control sample can be obtained from the same tissue. Preferably, the control sample is essentially or entirely composed of healthy regions and can be used to compare with the biological sample suspected to contain a tau-related disease region. Preferably, the tissue of the control sample is of the same type as the tissue in the biological sample. Preferably, the tau-related disease-affected cells suspected to be in the biological sample preferably originate from the same cell type (e.g., neuron or glia) as the cell type in the control sample.

[0180] The term "disease" refers to any abnormal condition that impairs physiological function. This term is used in a broad sense to encompass any disorder, disease, abnormality, pathology, pathological condition, state, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

[0181] The term "symptom" means subjective evidence of disease, such as a change in gait, that is noticed by a subject. "Sign" means objective evidence of disease, such as that recognized by a physician.

[0182] The term "positive response to treatment" refers to a more favorable response or average response in an individual patient in a patient population compared to the average response in a control population not receiving the treatment.

[0183] For the purpose of classifying amino acid substitutions as conservative or non-conservative amino acid substitutions, 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 are the exchange of a member of one of these classes for a member of another class.

[0184] Percent sequence identity is determined using maximally aligned antibody sequences according to the Kabat numbering convention. After alignment, when a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody regions divided by the total number of positions in the two aligned regions (not counting gaps) and converted to a percentage by multiplying by 100.

[0185] A composition or method that "comprising" or "including" one or more of the described elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" an antibody may include the antibody alone or the antibody in combination with other components.

[0186] The representation of a range of values ​​includes every integer within or defining that range, and every subrange defined by an integer within that range.

[0187] Unless otherwise clear from the context, the term "about" encompasses minor variations, such as the standard tolerance of error (eg, SEM) of measurement of the indicated value.

[0188] Statistical significance means p<0.05.

[0189] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0190] Detailed Description I. Overview The present invention provides antibodies that bind to tau. Some antibodies specifically bind epitopes within the microtubule binding region (MTBR) region of human tau. Some antibodies bind to tau regardless of phosphorylation state. Some antibodies of the present invention are useful for preventing or slowing tau-associated disease progression and tau-associated pathological progression. Although understanding of the mechanism is not necessary to practice the present invention, reduced toxicity may result from the antibodies inducing phagocytosis of tau, inhibiting inter- or intra-molecular aggregation of tau or inhibiting its binding to other molecules, by stabilizing non-toxic conformations, by inhibiting inter- or intracellular spread of pathogenic tau forms, by inhibiting phosphorylation of tau, by preventing tau binding to cells, or by inducing proteolytic cleavage of tau, among other mechanisms. Antibodies or agents of the present invention that induce such antibodies can be used in the treatment or effective prevention of Alzheimer's disease and other diseases associated with tau.

[0191] II.Target molecule Unless otherwise clear from the context, reference to tau refers to native human tau, including all isoforms, whether or not post-translational modifications (e.g., phosphorylation, glycosylation, or acetylation) are present. Six major isoforms (splice variants) of tau occur in the human brain. The longest of these variants has 441 amino acids with the first methionine residue truncated. The 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 Swissprot numbers of the isoforms are shown below. P10636-8 (SEQ ID NO: 1) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA 100 110 120 AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 130 140 150 HVTQARMVSK SKDGTGSDDK KAKGADGKTK 160 170 180 IATPRGAAPP GQKGQANATR IPAKTPPAPK 190 200 210 TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS 220 230 240 RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 250 260 270 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP 280 290 300 GGGKVQIINK KLDLSNVQSK CGSKDNIKHV 310 320 330 PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH 340 350 360 KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 370 380 390 THVPGGGNKK IETHKLTFRE NAKAKTDHGA 400 410 420 EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 430 440 DSPQLATLAD EVSASLAKQG L P10636-7 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA 100 110 120 GHVTQARMVS KSKDGTGSDD KKAKGADGKT 130 140 150 KIATPRGAAP PGQKGQANAT RIPAKTPPAP 160 170 180 KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 190 200 210 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA 220 230 240 KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 250 260 270 PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH 280 290 300 VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 310 320 330 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN 340 350 360 ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 370 380 390 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM 400 410 VDSPQLATLA DEVSASLAKQ GL P10636-6 (4R0N human tau) (SEQ ID NO: 3) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 70 80 90 AGHVTQARMV SKSKDGTGSD DKKAKGADGK 100 110 120 TKIATPRGAA PPGQKGQANA TRIPAKTPPA 130 140 150 PKTPPSSGEP PKSDGDRSGYS SPGSPGTPGS 160 170 180 RSRTPSLPTP PTREPKKVAV VRTPKPSPSS 190 200 210 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH 220 230 240 QPGGGKVQII NKKLDLSNVQ SKCGSKDNIK 250 260 270 HVPGGGSVQI VYKPVDLSKV TSKCGSLGNI 280 290 300 HHKPGGGQVE VKSEKLDFKD RVQSKIGSLD 310 320 330 NITHVPGGGN KKIETHKLTF RENAKAKTDH 340 350 360 GAEIVYKSPV VSGDTSPRHL SNVSSTGSID 370 380 MVDSPQLATL ADEVSASLAK QGL P10636-5 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA 100 110 120 AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 130 140 150 HVTQARMVSK SKDGTGSDDK KAKGADGKTK 160 170 180 IATPRGAAPP GQKGQANATR IPAKTPPAPK 190 200 210 TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS 220 230 240 RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 250 260 270 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP 280 290 300 GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK 310 320 330 PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT 340 350 360 HVPGGGNKKI ETHKLTFREN AKAKTDHGAE 370 380 390 IVYKSPVVSG DTSPRHLSNV SSTGSIDMVD 400 410 SPQLATLADE VSASLAKQGL P10636-4 (SEQ ID NO:5) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA 100 110 120 GHVTQARMVS KSKDGTGSDD KKAKGADGKT 130 140 150 KIATPRGAAP PGQKGQANAT RIPAKTPPAP 160 170 180 KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 190 200 210 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA 220 230 240 KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 250 260 270 PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH 280 290 300 KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 310 320 330 THVPGGGNKK IETHKLTFRE NAKAKTDHGA 340 350 360 EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 370 380 DSPQLATLAD EVSASLAKQG L P10636-2 (Sequence number 6) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 70 80 90 AGHVTQARMV SKSKDGTGSD DKKAKGADGK 100 110 120 TKIATPRGAA PPGQKGQANA TRIPAKTPPA 130 140 150 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS 160 170 180 RSRTPSLPTP PTREPKKVAV VRTPKPSPSS 190 200 210 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH 220 230 240 QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 250 260 270 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN 280 290 300 ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 310 320 330 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM 340 350 VDSPQLATLA DEVSASLAKQ GL

[0192] Reference to tau includes the approximately 30 known naturally occurring variants listed in the SwissProt database and their permutations, as well as mutations associated with tau pathologies such as dementia, Pick's disease, supranuclear palsy, etc. (see, e.g., the SwissProt database and Poorkaj, et al. Ann Neurol. 43:815-825 (1998)). Some examples of tau mutations numbered by the 441 isoform include 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 (P301S), and a glycine to valine mutation at amino acid position 272 (G272V). L), 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 (K369l), a glycine to arginine mutation at amino acid position 389 (G389R), and an arginine to tryptophan mutation at position 406 (R406W).

[0193] 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 fragments thereof includes the naturally occurring human amino acid sequence, including isoforms, mutants, and allelic variants thereof.

[0194] III. Antibodies A. Binding Specificity and Functional Properties The present invention provides antibodies that bind to tau. Some antibodies specifically bind to epitopes within the microtubule binding region (MTBR) region of human tau. Some antibodies bind to tau regardless of phosphorylation state. Some antibodies bind to epitopes that do not contain residues that are subject to phosphorylation. These antibodies can be obtained by immunization with tau polypeptides purified from natural sources or recombinantly expressed. Antibodies can be screened for binding to unphosphorylated forms of tau and to forms of tau that are phosphorylated at one or more residues that are subject to phosphorylation. Such antibodies preferably bind with indistinguishable affinity or bind to phosphorylated tau within a factor of at least 1.5-fold, 2-fold, or 3-fold (i.e., are "pan-specific") compared to unphosphorylated tau. 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. Also included are antibodies that compete for binding to tau with any of the above antibodies, e.g., those that compete with 3D6.

[0195] Reference to 3D6 should be understood to refer to any of the murine, chimeric, veneered and humanized forms of this antibody, unless otherwise clear from the context. This antibody has been deposited under [Accession Number]. This antibody specifically binds within the MTBR region of SEQ ID NO:1. This antibody is further characterized by its ability to bind both phosphorylated and non-phosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded / aggregated forms of tau. The antibody designated 6A10 is another such representative murine antibody. Reference to 6A10 should be understood to refer to any of the murine, chimeric, veneered and humanized forms of this antibody, unless otherwise clear from the context. The combined Kabat / Chothia 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 and 100% VL sequence identity with the murine 3D6 VH and VL chains, respectively.

[0196] Some of the antibodies of the invention bind to the same or overlapping epitopes as an antibody designated 3D6, the sequences of the heavy and light chain mature variable regions of which are shown in SEQ ID NOs: 7 and 11, respectively.

[0197] The combined Kabat / Chothia CDRs of the heavy chain of 3D6 are shown in SEQ ID NOs:8, 9, and 10, respectively, and the Kabat CDRs of the light chain of 3D6 are shown in SEQ ID NOs:12, 13, and 14, respectively.

[0198] Table 2 shows the 3D6 CDRs defined by Kabat, Chothia, the combination of Chothia and Kabat (also referred to herein as the "Kabat / Chotia combination"), AbM, and Contact. [Table 2]

[0199] Other antibodies can be obtained by mutagenesis of cDNAs encoding the heavy and light chains of representative antibodies such as 3D6. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence of the mature heavy and / or light chain variable regions to 3D6 or any other representative antibody or antibody chain and retain their functional properties, and / or that differ from the respective antibodies 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 according to any conventional definition, preferably Kabat's definition, that are 90%, 95%, 99% or 100% identical to the corresponding CDRs of 3D6 as defined by Kabat.

[0200] The present invention also provides antibodies having some or all (e.g., 3, 4, 5, and 6) CDRs that are completely or substantially derived from 3D6. Such antibodies can include a heavy chain variable region with at least two, and usually all three CDRs, completely or substantially derived from the heavy chain variable region of 3D6 and / or a light chain variable region with at least two, and usually all three CDRs, completely or substantially derived from the light chain variable region of 3D6. The antibody can include both a heavy chain and a light chain. The CDRs are substantially derived from the corresponding 3D6 CDRs, except that CDR-H2 (as defined by Kabat) can have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions, when it contains no more than 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 in amino acid sequence to 3D6 in the mature heavy and / or light chain variable regions and may retain its functional properties and / or may differ from the 3D6 antibodies by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0201] Some antibodies identified by such assays can bind to monomeric, misfolded, aggregated, phosphorylated, or non-phosphorylated forms of tau, etc. Similarly, some antibodies are immunoreactive to non-pathological and pathological forms and conformations of tau.

[0202] B. Humanized Antibodies A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted onto a human "acceptor" antibody sequence (see, for example, U.S. Patent Nos. 5,530,101 and 5,585,089 to Queen; U.S. Patent No. 5,225,539 to Winter, U.S. Patent No. 6,407,213 to Carter, U.S. Patent No. 5,859,205 to Adair, and U.S. Patent No. 6,881,557 to Foote). The sequence of the acceptor antibody can be, for example, a mature human antibody sequence, 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 a donor antibody, as well as variable region framework sequences and, if present, constant regions 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 a donor antibody heavy chain, and if present, a heavy chain variable region framework sequence and a heavy chain constant region substantially from 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 a donor antibody light chain, and if present, a light chain variable region framework sequence and a light chain constant region substantially from human light chain variable region framework and constant region sequences. Besides nanobodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. The CDRs in a humanized antibody are substantially derived from the corresponding CDRs in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (defined by any conventional method, but preferably by Kabat) are identical between the respective CDRs. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical.To be classified as humanized according to the 2014 World Health Organization (WHO) International Nonproprietary Name (INN) definition of humanized antibodies, there must be at least 85% identity with the human germline antibody sequence (i.e., prior to somatic hypermutation). A mixed antibody is an antibody in which one antibody chain (e.g., heavy chain) meets the threshold and the remaining chain (e.g., light chain) does not. If neither chain meets the threshold, the antibody is classified as chimeric, even if the variable framework regions of both chains are substantially human with some back mutations. See Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1,1-9, DOI:10.1080 / 19420862.2015.1114320. Also available at "WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review)" (Internet) 2014. http: / / www.who.int / medicines / services / inn / BioRev2014.pdf), which is incorporated herein 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 humanized antibodies provided herein have at least 85% sequence identity to human germline sequences, and some humanized antibodies provided herein have less than 85% sequence identity to human germline sequences. Some heavy chains of the humanized antibodies provided herein have between about 60% and 100% sequence identity to human germline sequences, such as between about 60% and 69%, 70% and 79%, 80% and 84%, or 85% and 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, while other heavy chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or more sequence identity to human germline sequences. Some light chains of the humanized antibodies provided herein have sequence identities of about 60%-100% to human germline sequences, e.g., in the range of 80%-84%, or 85%-89%. Some light chains fall below the 2014 WHO INN definition, for example, having 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" according to 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 at least 85% identity to human germline sequences paired with a light chain that has less than 85% identity to human germline sequences are "mixed" according to the 2014 WHO INN definition, or vice versa. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized" and have a heavy chain with at least 85% identity to the human germline sequence paired with a light chain with at least 85% identity to the human germline sequence. Additional humanized antibodies of the invention meet the 2014 WHO INN definition of "mixed."

[0203] Humanized antibodies often include all six CDRs (defined by any conventional method, but preferably as defined by Kabat) from a murine antibody, but 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).

[0204] In some antibodies, only a portion of the CDRs, i.e., a subset of the CDR residues required for binding, referred to as SDRs, are required to retain binding in the humanized antibody. CDR residues that do not contact the antigen and are not present in the SDRs can be identified by molecular modeling and / or empirical analysis from regions of the Kabat CDRs outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987) based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), or as described in Gonzales et al., Mol. Immunol. 41:863, 2004. In such humanized antibodies, in positions where one or more donor CDR residues are absent or where the entire donor CDR has been removed, the amino acid occupying that position may be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids to include in the CDR reflects a balance of competing considerations. Such substitutions are advantageous in that they potentially reduce the number of mouse amino acids in the humanized antibody, thereby reducing potential immunogenicity, and / or to comply with the WHO INN definition of "humanization". However, substitutions may also result in changes in affinity, and it is preferable to avoid significant decreases in affinity. The position of substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0205] The human acceptor antibody sequence can be selected from among many known human antibody sequences, if necessary, to provide a high degree of sequence identity (e.g., 65-85% identity) between the variable region framework of the human acceptor sequence and the corresponding variable region framework of the donor antibody chain.

[0206] An example of a heavy chain receptor sequence is the human mature heavy chain variable region of humanized 48G7Fab with PDB accession code 2RCS-VH_huFrwk (SEQ ID NO: 75). The variable domains of 3D6 and 48G7Fab also share identical lengths for the CDR-H1, H2 loops. Another example of a heavy chain receptor sequence is IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25). IMGT No. IGHV1-69-2 * 01 (SEQ ID NO: 25) shares the canonical mouse 3D6 heavy chain CDR-H1 and H2. IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25) belongs to human heavy chain subgroup 1. An example of a light chain receptor sequence is the human mature light chain variable region of human antibody ARX71335VL (SEQ ID NO: 82) with PDB accession code. The variable light chain domains of the 3D6 and ARX71335VL antibodies also share identical lengths for the CDR-L1, L2 and L3 loops. Another example of a light chain receptor sequence is IMGT number IGKV2-30 * 02 (SEQ ID NO: 27). IMGT number IGKV2-30 * 02 (SEQ ID NO: 27) has the same canonical class of CDR-L1, CDR-L2 and L3 as mouse 3D6. IMGT No. IGKV2-30 * 02 (SEQ ID NO:27) belongs to human kappa subgroup 2.

[0207] If more than one human receptor antibody sequence is selected, a complex or hybrid of these receptors can be used, and the amino acids used at the different positions of the humanized light and heavy chain variable regions can be selected from any of the human receptor antibody sequences used. For example, IMGT No. IGHV1-69-2* The human mature heavy chain variable region of IMGT number IGKV2-3001 (SEQ ID NO: 25) and PDB accession code number 2RCS-VH_huFrwk (SEQ ID NO: 75) were used as acceptor sequences for humanization of the 3D6 mature heavy chain variable region. An example of a position where these two acceptors differ is position H17 (T or S). Humanized versions of the 3D6 heavy chain variable region can contain either amino acid at this position. For example, the human mature light chain variable region IMGT number IGKV2-3001 (SEQ ID NO: 25) and PDB accession code number 2RCS-VH_huFrwk (SEQ ID NO: 75) were used as acceptor sequences for humanization of the 3D6 mature heavy chain variable region. * 02 (SEQ ID NO: 27) and PDB code number ARX71335VL_huFrwk (SEQ ID NO: 82) were used as acceptor sequences for humanization of the 3D6 mature light chain variable region. An example of a position where these two acceptors differ is position L100 (Q or A). The humanized version of the 3D6 light chain variable region may contain either amino acid at this position.

[0208] Particular amino acids from the human variable region framework residues can be selected for substitution based on their possible effect on CDR conformation and / or binding to antigen. Such possible effects can be studied by modeling, by examination of the characteristics of the amino acid at a particular position, or by empirical observation of the effects of substitution or mutagenesis of a particular amino acid.

[0209] For example, if an amino acid differs between the murine variable region framework residue and the selected human variable region framework residue, the human framework amino acid can be replaced with the equivalent framework amino acid from the murine antibody, where the amino acid is: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR regions or within the CDRs defined by Chochia but not Kabat; (3) otherwise interacts with the CDR region (e.g., is within about 6 Å of the CDR region) (e.g., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain), or (4) Residues involved in the VL-VH interface: It is reasonable to assume that.

[0210] The present invention provides humanized versions of the murine 3D6 antibody, including seven 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), and hu3D6VHvb7 (SEQ ID NO: 91)) and three exemplary humanized light chain mature variable regions (hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), and hu3D6VLvb3 (SEQ ID NO: 85)).

[0211] In one embodiment, humanized sequences are generated using a two-step PCR protocol that allows for the introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682].

[0212] As defined by U.S. Patent No. 5,530,101 to Queen, framework residues of classes (1)-(3) are sometimes alternately referred to as canonical and vernier residues. Framework residues that help determine 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 support the conformation of the antigen-binding loops and serve to fine-tune the fit of the antibody to the antigen are sometimes referred to as vernier residues (Foote & Winter, J Mol. Bio 224:487-499 (1992)).

[0213] Other framework residues that are candidates for substitution create potential glycosylation sites. Still other substitution candidates are recipient human framework amino acids that are unusual for that position for human immunoglobulins. These amino acids can be substituted with amino acids from the equivalent positions of the mouse donor antibody or with amino acids from the equivalent positions of more typical human immunoglobulins.

[0214] Other framework residues that are candidates for substitution are the N-terminal glutamine residues (Q), which may be replaced with glutamic acid (E) to minimize the possibility of pyroglutamic acid conversion [Y. Diana Liu, et al., 2011, J. Biol. Chem., 286:11211-11217]. Conversion of glutamic acid (E) to pyroglutamic acid (pE) occurs more slowly than from glutamine (Q). Due to the loss of primary amines upon conversion of glutamine to pE, the antibody becomes more acidic. Incomplete conversion leads to antibody heterogeneity, which can be observed as multiple peaks by charge-based analytical methods. Differences in heterogeneity may indicate poor process control.

[0215] An exemplary humanized antibody is a humanized version of murine 3D6, designated Hu3D6.

[0216] Murine antibody 3D6 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NOs: 7 and 11, respectively. The invention provides seven exemplary humanized mature heavy chain variable regions: hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7. The invention further provides three exemplary human mature light chain variable regions: hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3. Figures 1 and 2 show sequence comparisons of the heavy and light chain variable regions, respectively, of murine 3D6 and various humanized antibodies.

[0217] For reasons such as their potential impact on CDR conformation and / or binding to antigen, mediating interactions between heavy and light chains, interacting with the constant region, being sites for desirable or undesirable post-translational modifications, potentially immunogenic due to being an unusual residue for its position in a human variable region, acquiring aggregation potential, and other reasons as further demonstrated by the examples, the following 31 variable region framework positions have been identified as having similar functionality to the three exemplified human variable region framework positions: The following substitutions were considered in the mouse mature light chain variable region and seven exemplified human mature heavy chain variable regions: L7 (T7S, from germline), L10 (T10S, from germline), L15 (I15L, from germline), L17 (Q17E, to enhance stability), L37 (L37Q, from germline), L45 (K45R, from germline), L83 (L83V, from germline), L86 (H86Y, from mouse 3D6), L100 (A100Q, from germline), and L20 (A200Q, from germline). row), L106 (L106I, from germline), H1 (Q1E, from mouse 3D6), H5 (Q5V, from germline), H11 (L11V, from germline), H17 (S17T, from germline), H20 (L20I, from germline), H23 (T23K, from germline), H38 (K38R, from mouse 3D6), H42 (E42G, from germline), H43 (Q43K, from germline), H66 (K66R, from germline), H6 7 (A67V, from germline), H75 (S75T, from germline), H76 (N76D, from germline), H80 (L80M, from germline), H81 (Q81E, from germline), H83 (T83R, from germline), H91 (Y91F, from mouse 3D6), H93 (A93S, from mouse 3D6), H94 (S94T, from mouse 3D6), H108 (T108L, from germline), and H109 (L109V, from germline). In describing substitutions here and elsewhere, comments in parentheses indicate one rationale for the substitution. Some substitutions have multiple rationales.The following five variable region CDR positions were considered as substitution candidates in the three exemplary human mature light chain variable regions and the seven exemplary human mature heavy chain variable regions, as further demonstrated by the examples: L24 (K24R, from germline), H28 (N28T, from germline), H54 (N54D, from germline), H56 (D56E, from germline), and H58 (V58I, from germline). 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-Chothia combination 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 combination 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 combination 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.

[0218] Here, as elsewhere, the first mentioned residue is the residue of the humanized antibody formed by grafting the Kabat CDR, or in the case of CDR-H1, the combined Chothia-Kabat CDR, onto a human acceptor framework, and the second mentioned residue is the residue being considered to replace such residue. Thus, the first mentioned residue in the variable region framework is human and the first mentioned residue in the CDR is murine.

[0219] Exemplary antibodies include any permutation or combination of the following exemplary mature heavy and light chain variable regions: VHvb1 / VLvb1, VHvb1 / VLvb2, VHvb1 / VLvb3, VHvb2 / VLvb1, VHvb2 / VLvb2, VHvb2 / VLvb3, VHvb3 / VLvb1, VHvb3 / VLvb2, VHvb3 / VLvb2, VHvb3 / VLvb1, VHvb3 / VLvb2, VHvb3 / VLvb3 ...3, VHvb3 / VLvb3, VHvb3 / VLvb3, VHvb3 / VLvb3, VHv VLvb3, VHvb4 / VLvb1, VHvb4 / VLvb2, VHvb4 / VLvb3, VHvb5 / VLvb1, VHvb5 / VLvb2, VHvb5 / VLv b3, VHvb6 / VLvb1, VHvb6 / VLvb2, VHvb6 / VLvb3, VHvb7 / VLvb1, VHvb7 / VLvb2, VHvb7 / VLvb3.

[0220] The exemplified antibodies include permutations or combinations of the exemplified 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), and hu3D6VHvb7 (SEQ ID NO: 91) with 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 exemplary mature light chain variable regions hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), or hu3D6VLvb3 (SEQ ID NO: 85), and humanized 3D6V6 heavy chain variable regions 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); hu3D6VHv1 This includes any permutation or combination of bA11b6H3 (sequence number 47); hu3D6VHv1c (sequence number 48); 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).

[0221] The present invention provides variants of the 3D6 humanized antibody, in which the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity to 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), or hu3D6VHvb7 (SEQ ID NO: 91), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), or hu3D6VLvb3 (SEQ ID NO: 85). Some such antibodies retain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or all 36 backmutations or other mutations in SEQ ID NOs: 76-80, 90-91, and 83-85. Some such humanized antibodies contain the same set of backmutations or other mutations as the exemplified sequences that define sequence identity.

[0222] Thus, for example, the invention includes a humanized antibody having a mature heavy chain variable region having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 77, and the same set of mutations as listed in Table 6 for SEQ ID NO: 77, and the three CDRs of SEQ ID NO: 77, and a mature light chain sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 84 or 85, and the same set of mutations as listed in Table 7 for SEQ ID NO: 84 or 85, respectively, and the three CDRs of SEQ ID NO: 84 or 85, respectively. Some antibodies comprise a mature heavy chain variable region of SEQ ID NO: 77 and a mature light chain variable region of SEQ ID NO: 84 or 85.

[0223] The invention also includes humanized antibodies having a mature heavy chain variable region having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:90, and the same set of mutations as listed in Table 6 for SEQ ID NO:90, and the three CDRs of SEQ ID NO:90, and a mature light chain sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:84 or 85, and the same set of mutations as listed in Table 7 for SEQ ID NO:84 or 85, respectively, and the three CDRs of SEQ ID NO:84 or 85, respectively. Some antibodies comprise a mature heavy chain variable region of SEQ ID NO:90, and a mature light chain variable region of SEQ ID NO:84 or 85.

[0224] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acids: H93 is occupied by S and H94 is occupied by T. In some humanized 3D6 antibodies, positions H93 and H94 (Vernier residues) are occupied by S and T, respectively, as in, for example, huVHvb1, huVHvb2, huVHvb3, huVHvb4, huVHvb5, huVHvb6, and huVHvb7.

[0225] In some humanized 3D6 antibodies, position H91 in the VH region (an interface residue) is occupied by F, as is the case for example in huVHvb1, huVHvb2, and huVHvb6.

[0226] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by a 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, as in, for example, huVHvb2, huVHvb3, huVHvb4, huVHvb5, huVHvb6, and huVHvb7.

[0227] 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, as in, for example, huVHvb3, huVHvb4, huVHvb5, huVHvb6, and huVHvb7.

[0228] In some humanized 3D6 antibodies, position H58 in the VH region (a CDR-H2 residue) is occupied by I, as is the case for example in huVHvb3, huVHvb4, and huVHvb5.

[0229] 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, as in, for example, huVHvb4, huVHvb5, huVHvb6, and huVHvb7.

[0230] 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 (CDR-H2 residues) are occupied by D and E, respectively, as for example in huVHvb6 and huVHvb7.

[0231] 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, and H54 is occupied by N or D. H56 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.

[0232] 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, similar to 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, similar to 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 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, similar to 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 in huVHvb7.

[0233] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acids: 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, as in, for example, huVHvb2 and huVHvb3.

[0234] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L7 is occupied by T or S, L10 is occupied by T or S, L15 is occupied by I or L, L17 is occupied by Q or E, L24 is occupied by K or R, L37 is occupied by L or Q, L45 is occupied by K or R, L83 is occupied by L or V, L86 is occupied by H or Y, L100 is occupied by A or Q, and L106 is occupied by L or I.

[0235] 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, similar to 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, similar to huVLvb3.

[0236] In some humanized 3D6 antibodies, the heavy chain variable region has ≧85% identity to human sequences. In some humanized 3D6 antibodies, the light chain variable region has ≧85% identity to human sequences. In some humanized 3D6 antibodies, each of the heavy and light chain variable regions has ≧85% identity to human germline sequences. 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) and the three light chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 12, 13, and 14), with the proviso 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, and position L24 is occupied by K or R. In some humanized 3D6 antibodies, the Kabat / Chothia combination 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, or SEQ ID NO: 92. In some humanized 3D6 antibodies, the Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89.

[0237] 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.

[0238] Unless otherwise specified, variable region framework positions correspond to Kabat numbering. Other such variants typically differ from the exemplary Hu3D6 heavy and light chain sequences by only a small number (e.g., typically 1, 2, 3, 5, 10, or 15) of substitutions, deletions, or insertions. Such differences are usually in the framework, but can also occur in the CDRs.

[0239] One possibility for additional diversity in humanized 3D6 variants is additional backmutations in the variable region framework. Many of the framework residues that do not contact the CDRs in the humanized mAb can be amenable to substitution with amino acids at the corresponding positions of the donor mouse mAb or other mouse or human antibodies, and many more potential CDR contact residues are also amenable to substitution. Even amino acids within the CDRs can be changed, for example, with residues found at the corresponding positions of the human acceptor sequence used to provide the variable region framework. In addition, a different human acceptor sequence can be used, for example, for the heavy and / or light chain. If a different acceptor sequence is used, one or more of the above recommended backmutations may not be performed, since the corresponding donor and acceptor residues are already free of backmutations.

[0240] Preferably, the substitutions or backmutations in the humanized 3D6 variants (whether conservative or not) do not substantially affect the binding affinity or potency of the humanized mAb, ie, its ability to bind to tau.

[0241] Humanized 3D6 antibodies are further characterized by their ability to bind both phosphorylated and non-phosphorylated tau, as well as misfolded / aggregated forms of tau.Some humanized antibodies are characterized by their ability to bind to human tau similarly to mouse 3D6 or stronger (e.g., up to 2-fold, 5-fold, 10-fold or 20-fold) or possess other functional properties, such as inhibiting tau binding to neuronal cells or disaggregating tau.Such properties can be compared by any of the assays described in the Examples.

[0242] C. Selection of Constant Regions The variable regions of the heavy and light chains of chimeric, veneer, or humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are 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 can 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).

[0243] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chains, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in part or all of the molecule. 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 Winter 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 extend half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Representative substitutions to increase antibody half-life include Gln at position 250 and / or Leu at position 428 (EU numbering of the constant region is used in this paragraph). 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, 234, 236 and / or 237 in 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 of human IgG1, according to EU numbering. In some antibodies, mutations at one or more of human IgG1 positions 318, 320, and 322, EU numbering, are used. 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.

[0244] A representative human light chain kappa constant region has the amino acid sequence of SEQ ID NO: 104 (with or without an N-terminal arginine). A representative human IgG1 heavy chain constant region has the amino acid sequence of SEQ ID NO: 103 (with or without a C-terminal lysine). 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 mature variable domains of the heavy and light chains are linked via a spacer.

[0245] Human constant regions show allotypic and isoallotypic variation between different individuals. That is, constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that serum that recognizes the isoallotype binds to the non-polymorphic regions of one or more other isotypes. Thus, for example, another heavy chain constant region is IgG1 G1m3 with or without a C-terminal lysine. Reference to human constant region includes constant regions with any natural allotype or any permutation of residues that occupy positions in natural allotypes.

[0246] D. Expression of Recombinant Antibodies Several methods are known for the production of 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 for the VH region leader peptide encompassing the translation initiation as the 5' primer and the g2b constant region as the specific 3' primer. Representative primers are described in U.S. Patent Application Publication No. 2005 / 0009150 by Schenk et al. (hereinafter "Schenk"). Sequences from multiple independently derived clones can be compared to ensure that no changes were 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.

[0247] The light chain variable VL region can be cloned in a similar manner. In one approach, a consensus primer set is designed for amplification of the VL region with a 5' primer designed to hybridize to a 3' primer specific for the VL region encompassing the translation initiation codon and the Ck region downstream of the VJ joining region. In a second approach, a 5' RACE RT-PCR method is used to clone the VL-encoding cDNA. Representative primers are described in Schenk (see above). The cloned sequence is then combined with sequences encoding human (or other non-human species) constant regions. Representative sequences encoding human constant regions include SEQ ID NO: 105, which encodes a human IgG1 constant region (SEQ ID NO: 103), and SEQ ID NO: 106, which encodes a human kappa light chain constant region (SEQ ID NO: 104).

[0248] In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of their respective VDJ or VJ junctions and inserted 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. Culture supernatants are collected 48 hours after gene transfection and analyzed for antibody production by Western blot analysis or antigen binding by ELISA. The chimeric antibodies are humanized as described above.

[0249] Chimeric, veneered, humanized and human antibodies are usually produced by recombinant expression. Recombinant polynucleotide constructs usually contain expression control sequences operably linked to antibody chain coding sequences, including naturally associated or heterologous expression control sequences, e.g., promoters. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. After the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and collection and purification of cross-reacting antibodies.

[0250] These expression vectors are usually replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker, e.g., ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the DNA sequence of interest.

[0251] E. coli is one type of prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is a yeast host with suitable vectors, including expression control sequences, origins of replication, and termination sequences, as needed. Representative promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization.

[0252] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting complete 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)), as well as necessary information processing 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).

[0253] Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of the transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Patent 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 from a mammary gland-specific gene, e.g., casein or beta-lactoglobulin.

[0254] The vector containing the DNA segment of interest can be introduced into the host cell using a method that depends on the type of host cell. For example, calcium chloride gene transfer is usually used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene gun, or viral gene transfer 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 production of transgenic animals, the transgene can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, and the nucleus of such cells can be introduced into enucleated oocytes.

[0255] Vectors (single or multiple) encoding antibody heavy and light chains can be introduced into cell cultures and cell pools can be screened for growth productivity and product quality in serum-free medium. The highest producing cell pools can then be subjected to FACS-based single cell cloning to generate monoclonal lines. Specific productivities of over 50 pg or 100 pg per cell per day can be used, corresponding to product titers of over 7.5 g / L of culture. Antibodies produced by single cell clones can also be tested for turbidity, filtration characteristics, PAGE, IEF, UV scan, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. Selected clones can then be placed into multiple vials and stored frozen for later use.

[0256] Once expressed, antibodies can be purified according to standard procedures in the art, including Protein A capture, HPLC purification, column chromatography, gel electrophoresis, etc. (for review, see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0257] Methodologies for commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single cell cloning, cell storage, use of selection markers for copy number amplification, CHO terminators, or improved protein titer (see, for example, U.S. Patent Nos. 5,786,464, 6,114,148, 6,063,598, 7,569,339, WO 2004 / 050884, WO 2008 / 012142, WO 2008 / 012142, WO 2005 / 019442, WO 2008 / 107388, and WO 2009 / 027471, and U.S. Patent No. 5,888,809). Antibodies can also be administered in the form of nucleic acids encoding the heavy and / or light chains of the antibody. If both heavy and light chains are present, the chains are preferably linked as a single chain antibody. Antibodies for passive administration can also be prepared, for example, by affinity chromatography from sera of patients treated with the peptide immunogen.

[0258] DNA can be delivered in naked form (i.e., without colloidal or encapsulating material), or by retroviral systems, including vectors derived from retroviruses such as MMLV, HIV-1, and ALV (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated viral vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)); lentiviral vectors, such as those based on HIV or FIV gag sequences, viral vectors of the pox family, including vaccinia virus and avian poxviruses, viral vectors of the alphavirus genus, such as those derived from Sindbis and Semliki Forest viruses (see, e.g., Dubensky et al., J. Exp. Med. 179, 1867 (1994)). Several viral vector systems can be used, including rhabdoviruses such as Venezuelan equine encephalitis virus (see U.S. Pat. No. 5,643,576) and vesicular stomatitis virus (see WO 96 / 34625), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al, WO 94 / 12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).

[0259] The DNA coding for immunogen, or the DNA coding for antibody heavy and / or light chain, or the vector containing it can be packaged into liposome.Suitable lipids and related analogues are described in U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185.The vector and DNA coding for immunogen, or the DNA coding for antibody heavy and / or light chain can also be adsorbed or bound to microparticle carrier, examples of which include polymethylmethacrylate polymers and polylactides and poly(lactide-co-glycolides) (see, for example, McGee et al., J.Micro Encap.1996).

[0260] Vectors or segments therefrom encoding antibody heavy and / or light chains can be incorporated into cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, which can then be reimplanted into the patient, usually after selection of cells that have incorporated the transgene (see, e.g., WO 2017 / 091512). Exemplary patient-derived cells include patient-derived pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal).

[0261] Vectors or segments thereof encoding antibody heavy and / or light chains can be introduced ex vivo into any region of interest in cells, such as albumin genes or other safe harbor genes. Cells incorporating the vectors can be transplanted with or without prior differentiation. Cells can be transplanted into specific tissues, such as secretory tissues or locations of lesions, or systemically, such as by infusion into the blood. For example, cells can be transplanted into a patient's secretory tissue, such as the liver, optionally with prior differentiation into cells present in that tissue, such as hepatocytes in the case of the liver. Expression of the antibody in the liver results in the secretion of the antibody into the blood.

[0262] E. Antibody Screening Assays As noted above, antibodies can first be screened for the desired binding specificity. Active immunogens can similarly be screened for the ability to induce antibodies with such binding specificity. In this case, the active immunogen is used to immunize laboratory animals and the resulting sera are tested for the appropriate binding specificity.

[0263] Antibodies with the desired binding specificity can then be tested in cell and animal models. Neuronal cells should be preferred as the cells used for such screening. Cellular models of tau pathology have been reported in which neuroblastoma cells are transfected with the 4 repeat domain 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 the neuroblastoma N2a cell line by adding doxycycline. This cellular model allows studying the toxicity of tau to cells in the 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 efficiency of antibodies in inhibiting the formation of tau aggregates or disaggregating them.

[0264] Antibodies can also be screened in transgenic animal models of tau-associated diseases. Such transgenic animals can include tau transgenes (e.g., any of the human isoforms) and, optionally, human APP transgenes, such as tau, ApoE, presenilin, or alpha-synuclein phosphorylating kinases. Such transgenic animals are treated to develop at least one sign or symptom of tau-associated diseases.

[0265] A representative 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 the Thy1.2 promoter. This model shows a rapid course of neurodegeneration, motor impairment, and degeneration of afferent fibers and cerebellar granule cells. Another representative 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 the 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, which is incorporated herein by reference in its entirety. This is an excellent model for studying the consequences of tangle development and for screening therapeutics that can inhibit the production of these aggregates. Another advantage of these animals is the relatively early onset of pathology. In homozygous strains, tau pathology and associated behavioral abnormalities can be observed, at least, as early as 3 months of age, but these animals remain relatively healthy until at least 8 months of age. That is, at 8 months of age, these animals are able to walk around, feed themselves, and perform behavioral tasks well enough to allow monitoring of treatment effects. Active immunization of these mice with AI wI KLH-PHF-1 for 6-13 months resulted in titers of approximately 1,000, with fewer neurofibrillary tangles, less pSer422, and reduced weight loss compared to untreated control mice.

[0266] Antibody activity can be assessed by a variety of criteria, including reduction in the amount of total or phosphorylated tau, reduction in other pathological features such as amyloid deposits of Aβ, and inhibition or delay or behavioral deficits. Antibodies can be tested for passage of the antibody through the blood-brain barrier into the brain of transgenic animals. Antibodies or antibody-inducing fragments can also be tested in non-human primates that naturally or through induction develop symptoms of a disease characterized by tau. Antibody testing is usually performed simultaneously with a control experiment that is performed in parallel except that the antibody or active agent is not present (e.g., replaced by vehicle). Reduction, delay or inhibition of signs or symptoms of the disease due to the antibody or active agent under test can then be assessed compared to the control.

[0267] IV. Patients Eligible for Treatment The presence of neurofibrillary tangles has been noted in several diseases, including Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathies, 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), and progressive supranuclear palsy (PSP). The present dosing regimen can also be used to treat or prevent any of these diseases. Due to the widespread relationship between neurological diseases and conditions and tau, the present dosing regimen can be used to treat or prevent any subject that shows elevated levels of tau or phosphorylated tau (e.g., in CSF) compared to the average value of individuals without neurological disease.The present dosing regimen 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, and in particular in patients.

[0268] Patients eligible for treatment include individuals at risk of disease but who do not show symptoms and those who currently show symptoms. Patients at risk of disease include those with a known genetic risk of disease. Such individuals include individuals who have relatives who have experienced the disease and individuals whose risk has been determined by analysis of genetic or biochemical markers. Genetic markers of risk include tau mutations, such as those mentioned above, as well as mutations in other genes associated with neurological diseases. For example, ApoE4 alleles in heterozygous form and also in homozygous form are associated with risk of Alzheimer's disease. Other markers of risk of Alzheimer's disease include mutations in the APP gene, particularly at position 717 and at positions 670 and 671, called the Hardy and Swedish mutations, mutations in PS1 and PS2 of the presenilin genes, family history of AD, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by PET imaging due to the characteristic dementia and the presence of the risk factors mentioned above. In addition, several diagnostic tests are available to identify individuals with AD. These include measuring CSF tau or phosphorylated tau and Aβ42 levels. Elevated levels of tau or phosphorylated tau and decreased levels of Aβ42 signify the presence of AD. Several mutations are associated with Parkinson's disease. Mutations in other genes, such as Ala30Pro or Ala53, or PARK8, a leucine-rich repeat kinase, are associated with Parkinson's disease. Individuals can also be diagnosed with any of the above-mentioned neurological disorders by DSM IV TR criteria.

[0269] 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 requires multiple doses over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response drops, booster doses are required. In cases of potential Down's syndrome patients, treatment can begin prenatally by administering therapeutic drugs to the mother or shortly after birth.

[0270] V. Nucleic acids The present invention further provides nucleic acids encoding any of the above heavy and light chains (e.g., SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 76-80, SEQ ID NO: 90-91, and SEQ ID NO: 83-85). Representative nucleic acids include SEQ ID NO: 30-31, 93-99, 100-102, and 105-106. Optionally, such nucleic acids further encode a signal peptide and are capable of expressing a signal peptide linked to the heavy or light chain variable region. The coding sequence of the nucleic acid is operably linked to regulatory sequences that can ensure expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The regulatory sequences may include a promoter, e.g., a prokaryotic promoter or a eukaryotic promoter. The nucleic acids encoding the heavy or light chains can be codon-optimized for expression in a host cell. The nucleic acids encoding the heavy and light chains can encode selectable genes. The nucleic acids encoding the heavy and light chains can occur in isolated form or can be inserted into one or more vectors. The nucleic acids can be synthesized, for example, by solid phase synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains can be linked, for example, as one contiguous nucleic acid in an expression vector, or can be inserted separately, for example, each into its own expression vector.

[0271] VI. Conjugated Antibodies Conjugated antibodies that specifically bind to antigens such as tau can be used to detect the presence of tau; 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-Parkinson's disease / dementia complex; monitor and evaluate the efficacy of therapeutic agents used to treat patients diagnosed with corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP); and detect the aggregation of tau. inhibiting or reducing tau fibril formation; reducing or clearing tau deposits; stabilizing non-toxic conformations of tau; or treating or preventing 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). For example, such antibodies can be conjugated to other therapeutic moieties, other proteins, other antibodies, and / or detectable labels. See WO 03 / 057838; U.S. Patent No. 8,455,622.Such therapeutic moieties may be any agent that can be used to treat, combat, alleviate, 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 disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0272] The conjugated therapeutic moiety may include a cytotoxic drug, a cytostatic drug, a neurodifferentiator, a neuroprotectant, a radiotherapeutic drug, an immunomodulatory agent, or any bioactive agent that promotes or enhances the activity of the antibody. A cytotoxic drug may be any agent that is toxic to cells. A cytotoxic drug may be any agent that inhibits cell proliferation. A neurodifferentiator may be any agent, including chemical or protein substances, that promotes neuronal maintenance, proliferation, or differentiation. A neuroprotectant may be an agent, including chemical or protein substances, that protects neurons from acute insults or degenerative processes. An immunomodulatory agent may be any agent that stimulates or suppresses 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 conjugated to a tau-specific antibody, such as the antibodies described herein, the conjugated therapeutic moiety will have a specific affinity for tau-related disease affected cells over normal cells. Thus, administration of the conjugated antibody directly targets cancer cells with minimal damage to the surrounding healthy tissue. This may be particularly useful when the therapeutic moieties are too toxic to be administered alone. Additionally, smaller amounts of the therapeutic agent moiety can be used.

[0273] Some such antibodies can be modified to act as immunotoxins. See, e.g., U.S. Patent No. 5,194,594. For example, ricin, a plant-derived cytotoxin, can be conjugated to antibodies using the bifunctional reagents S-acetylmercaptosuccinic anhydride for antibodies and succinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz et al., Cancer Res. 48(16):4469-4476 (1998). Conjugation reduces the B-chain binding activity of ricin, but does not impair either the toxic potential of the ricin A-chain or the antibody activity. Similarly, saporin, an inhibitor of ribosome assembly, can be conjugated to antibodies via disulfide bonds between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

[0274] Some such antibodies can be linked to a radioisotope. Examples of radioisotopes include, for example, yttrium. 90 (90Y) Indium 111 (111In), 131 I, 99 mTc, radioactive silver-111, radioactive silver-199, and bismuth 213 The linking of radioisotopes to antibodies can be carried out with conventional bifunctional chelates. For the linking of radioactive silver-111 and radioactive silver-199, sulfur-based linkers can be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linking of silver radioisotopes may require reduction of immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used, which reacts with such isotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0275] Some such antibodies can be linked to other therapeutic moieties. Such therapeutic moieties can be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective. For example, the antibodies can be linked to toxic chemotherapeutic agents such as tubulin inhibitors, such as maytansine, geldanamycin, tubulin binding agents (e.g., auristatins), or minor groove binding agents, such as calicheamicin. Other representative therapeutic moieties include agents known to be useful in treating, managing, or ameliorating a patient's 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0276] Antibodies may also be conjugated to other proteins. For example, antibodies may be conjugated to finomers. Fynomers are small binding proteins (e.g., 7 kDa) derived from the human oncogene Fyn SH3 domain. They may be stable and soluble, and may lack cysteine ​​residues and disulfide bonds. Fynomers can be genetically engineered to bind target molecules with the same affinity and specificity as antibodies. They are suitable for generating antibody-based multispecific fusion proteins. For example, finomers can be fused to the N-terminus and / or C-terminus of antibodies to generate bi- or trispecific FynomAbs with different structures. Fynomers can be selected through screening techniques using FACS, Biacore, and cell-based assays, which allow efficient selection of finomers with optimal properties against finomer libraries. 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).

[0277] The antibodies disclosed herein can also be linked or conjugated to one or more other antibodies (e.g., to form an antibody heteroconjugate). Such other antibodies can bind to a different epitope within tau or can bind to a different target antigen.

[0278] The antibody may also be conjugated to a detectable label. Such an antibody can be used to assess the efficacy of, for example, the diagnosis and / or treatment of 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Such antibodies are particularly useful for making such determinations in subjects with or susceptible to 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), or in suitable biological samples obtained from such subjects. Representative detectable labels which can be bound or linked 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 silver-111, radioactive silver-199, bismuth, etc. 213 , iodine ( 131 I, 125 I, 123 I, 121I), 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 These include radioactive materials such as tin; positron-emitting metals using various positron emission tomography techniques; non-radioactive paramagnetic metal ions; and molecules that are radiolabeled or conjugated to specific radioisotopes.

[0279] The linking of radioisotopes to antibodies can be carried out with conventional bifunctional chelates. For the linking of radioactive silver-111 and radioactive silver-199, sulfur-based linkers can be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linking of silver radioisotopes may require reduction of immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used, which reacts with such isotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0280] Therapeutic drug moieties, other proteins, other antibodies, and / or detectable labels may be attached or conjugated directly or indirectly through an intermediary (e.g., a linker) to an antibody of the invention. 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); Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Hellstrom et al., “Antibodies For Drug Delivery,”; Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985), Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,”; Therapy,Baldwin et See, Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in J. Chem. Soc., 1985, pp. 303-16; 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 attached therapeutic agent moiety, protein, antibody, and / or detectable label upon exposure to specific proteases under acidic or reducing conditions, or under other defined conditions.

[0281] VII. Pharmaceutical Compositions and Methods of Use In prophylactic applications, the antibody or pharmaceutical composition thereof is administered to a patient susceptible to or at risk of a disease (e.g., Alzheimer's disease) in a dosage regimen (dosage, frequency and route of administration) effective to reduce the risk of the disease, reduce the severity of the disease, or delay the onset of at least one sign or symptom of the disease. In particular, the dosage regimen is preferably effective to inhibit or delay tau or phosphorylated tau and filament pairs formed therefrom in the brain, and / or inhibit or delay their toxic effects, and / or inhibit or delay the onset of behavioral deficits. In therapeutic applications, the antibody is administered to a patient suspected of or already suffering from a disease (e.g., Alzheimer's disease) in a dosage regimen (dosage, frequency and route of administration) effective to improve or at least inhibit further deterioration of at least one sign or symptom of the disease. In particular, the dosage regimen is preferably effective to reduce or at least inhibit further increases in the levels of tau, phosphorylated tau, or filament pairs formed therefrom, associated toxicity and / or behavioral deficits.

[0282] A dosing regimen is considered to be therapeutically or prophylactically effective if each treated patient achieves a better therapeutic outcome than the average therapeutic outcome in a control population of comparable patients not treated by the methods of the invention, or if a controlled clinical trial (e.g., a Phase II, Phase II / III or Phase III trial) demonstrates a better therapeutic outcome for treated patients versus control patients at a level of p<0.05 or 0.01, or even 0.001.

[0283] The effective dose will vary depending on many different factors, including 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, whether other agents are administered, and whether the treatment is prophylactic or therapeutic.

[0284] 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 can be administered at such dosages daily, every other day, weekly, biweekly, monthly, quarterly, or according to any other schedule determined by empirical analysis. Exemplary treatments entail administration in multiple doses over an extended period of time, e.g., for at least six months. Further exemplary dosing regimes entail administration every two weeks, or once a month, or once every three to six months.

[0285] The antibody is preferably administered via a peripheral route (i.e., a route in which the administered or induced antibody crosses the blood-brain barrier and reaches the intended site in the brain). Routes of administration include topical, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular or intramuscular administration. Preferred routes of administration of antibodies are intravenous and subcutaneous. Preferred routes for active immunization are subcutaneous and intramuscular. This type of injection is most typically given in the arm or leg muscles. In some methods, the agent is injected directly into a particular tissue where deposits have accumulated, e.g., intracranially.

[0286] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., dosage for single administration). Pharmaceutical compositions can be formulated with one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution can contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibody can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0287] This dosage regimen can be administered in combination with another drug that is effective in treating or preventing the disease being treated.For example, in the case of Alzheimer's disease, this dosage regimen can be combined with immunotherapy against Aβ (WO2000 / 072880), cholinesterase inhibitors or memantine, or in the case of immunotherapy for Parkinson's disease, it can be combined with immunotherapy against α-synuclein (WO2008 / 103472), levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, amantadine or anticholinergic drugs.

[0288] The antibody is administered in an effective dosing regimen, which refers to a dosing regimen including a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and / or ameliorates at least one sign or symptom of the disorder being treated. If the patient is already suffering from the disorder, the dosing regimen may also be referred to as a therapeutically effective dosing regimen. If the patient is at high risk for the disorder compared to the general population, but is not yet symptomatic, the dosing regimen may also be referred to as a prophylactically effective dosing regimen. In some cases, therapeutic or prophylactic efficacy may be observed in an individual patient against historical controls or past experience of the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated during preclinical or clinical trials of a treated patient population compared to a control population of untreated patients.

[0289] Exemplary dosages of antibodies are 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 as a fixed dose, 10-4000 mg or 10-1500 mg. Dosage will depend, among other things, on the patient's condition and response to previous treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0290] Administration can be parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal or intramuscular. Some antibodies can be administered intravenously or subcutaneously into the systemic circulation. Intravenous administration can be, for example, by infusion over 30-90 minutes.

[0291] The frequency of administration depends, inter alia, on the half-life of the antibody in circulation, the condition of the patient, and the route of administration. The frequency can be daily, weekly, monthly, every three months, or at irregular intervals, depending on the change in the patient's condition or the progression of the disorder being treated. A typical frequency for intravenous administration is every week to every three months, depending on the cause of the treatment, although more or less frequent administration is possible. For subcutaneous administration, a typical frequency is daily to monthly, although more or less frequent administration is possible.

[0292] The number of doses depends on whether the disorder is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, often 1 to 10 doses. Sometimes, for acute disorders or acute exacerbations of chronic disorders, a single rapid dose, optionally in divided form, is sufficient. For recurrence of acute disorders or acute exacerbations, treatment may be repeated. For chronic disorders, the antibody may be administered at regular intervals, for example, weekly, biweekly, monthly, every 3 months, every 6 months, for at least 1, 5 or 10 years, or for the life of the patient.

[0293] A. Diagnostic and Monitoring Methods In vivo imaging, diagnostic methods, and optimized 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 a humanized antibody of the present invention to a patient and then detecting the antibody after binding. Clearance reactions of the administered antibody can be avoided or reduced by using antibody fragments that lack the full-length constant region, such as Fab. In some methods, the same antibody can function as both a treatment and a diagnostic reagent.

[0294] 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 in the skull. The dosage of the reagent should be in the same range as for the treatment method. Typically, the reagent is labeled, but in some methods, the primary reagent with affinity for tau is not labeled and a secondary labeling agent is used to bind to the primary reagent. The choice of label depends on the means of detection. 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).

[0295] The method of in vivo imaging of tau protein deposits 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 such diseases. For example, the method can be used in patients who show symptoms of dementia. If the patient has abnormal neurofibrillary tangles, the patient may have Alzheimer's disease. Alternatively, if the patient has abnormal tau inclusions, the patient may have frontotemporal lobar degeneration, depending on the location of the inclusions. The method can also be used in asymptomatic patients. The presence of abnormal tau protein deposits indicates a susceptibility to future symptomatic disease. The method is also useful for monitoring disease progression and / or response to treatment in patients previously diagnosed with tau-related diseases.

[0296] Diagnosis can be made by comparing the number, size, and / or intensity of the labeled loci with the corresponding baseline value. The baseline value can represent the average level in a population of non-disease individuals. The baseline value can also represent a previous level determined in the same patient. For example, the patient's baseline value can be measured before starting tau immunotherapy treatment, and the measured value can then be compared to the baseline value. A decrease in the value relative to the baseline signal indicates a positive response to treatment.

[0297] In some patients, the diagnosis of tauopathy may be aided by performing a PET scan. The PET scan may be performed, for example, using conventional PET imaging equipment and auxiliary equipment. The scan typically includes one or more areas of the brain commonly known to be associated with tau protein deposits, and one or more areas where few, if any, deposits are typically present to serve as controls.

[0298] The signals detected in a PET scan can be represented as a multi-dimensional image. A multi-dimensional image can represent a cross-section of the brain in two dimensions, a three-dimensional brain in three dimensions, or a change in the three-dimensional brain over time in four dimensions. A color scale can be performed using different colors to indicate different amounts of label and putative tau protein deposits detected. The results of the scan can also be presented numerically in numbers that relate to the amount of label and the resulting amount of tau protein deposits detected. Label present in brain regions known to be associated with deposits of a particular tauopathy (e.g., Alzheimer's disease) can be compared to label present in regions not known to be associated with deposits to provide a ratio representing the extent of deposits in the former region. Such ratios for the same radiolabeled ligand provide a comparable measure of tau protein deposits and their changes between different patients.

[0299] In some methods, a PET scan is performed at the same time as an MRI or CAT scan, or in the same patient visit as an MRI or CAT scan. An MRI or CAT scan provides more anatomical detail of the brain than a PET scan. However, images from a PET scan can be overlaid onto the MRI or CAT scan images, which more accurately shows the location of the PET ligand and putative tau deposits relative to anatomical structures in the brain. Some devices can perform both a PET scan and an MRI or CAT scan of a patient without the patient changing position between scans, facilitating the overlay of the images.

[0300] 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 administering the PET ligand and performing the scan may depend on the PET ligand, particularly its rate of uptake and clearance from the brain, and the half-life of its radiolabel.

[0301] PET scans can also be performed as a preventative measure in asymptomatic patients or those with symptoms of mild cognitive impairment but who have not been diagnosed with a tauopathy and who are nevertheless at high risk for developing a tauopathy. In asymptomatic patients, scans are particularly useful for individuals who are considered at high risk for tauopathy due to family history, genetic or biochemical risk factors, or advanced age. Prophylactic scans can begin in patients between the ages of 45 and 75, for example. In some patients, the first scan is performed at age 50.

[0302] 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 PET scans may be initiated as in patients diagnosed with tauopathy. If the PET scan performed as a preventive measure shows levels of tau protein deposits within normal levels, further PET scans may be performed, as described above, at intervals of 6 months to 10 years, preferably 1 to 5 years, 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 prophylactic scanning with administration of tau-targeted immunotherapy, the levels of tau protein deposits can be reduced to or closer to 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 if the patient had not received prophylactic scanning and tau-targeted immunotherapy.

[0303] Normal levels of tau protein deposits can be determined by the amount of neurofibrillary tangles or tau inclusions in the brain of a representative sample of individuals from a population that have not been diagnosed with a particular tauopathy (e.g., Alzheimer's disease) and are not considered to be at high risk of developing such a disease (e.g., a representative sample of disease-free individuals under the age of 50). Alternatively, normal levels can be recognized in an individual patient if the PET signal by the method of the present invention in brain regions where tau protein deposits are known to occur does not differ (within the precision of measurement) from signals from brain regions where such deposits are known to normally occur. Elevated levels in an individual can be recognized by comparison with normal levels (e.g., levels outside the variance of the mean + standard deviation) or simply from elevated signals beyond experimental error in brain regions associated with tau protein deposits compared to regions 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 region(s) of the brain, including at least one region known to form tau protein deposits associated with a particular tauopathy (e.g., Alzheimer's disease). Patients with elevated levels of tau protein deposits are candidates for initiating immunotherapy.

[0304] A reduction in the level of tau protein deposits after starting immunotherapy can initially be seen as an indication that the treatment is having the desired effect. The observed reduction can be, for example, within the range of 1-100%, 1-50%, or 1-25% of baseline values. Such effects can be measured in one or more areas of the brain known to form deposits, or can be measured from the average values ​​of such areas. The overall effect of the treatment can be estimated by adding the percentage reduction relative to baseline to the increase in tau protein deposits that would occur in the average untreated patient in the absence of treatment.

[0305] Maintenance of a near constant level of tau protein deposits or even a small increase in tau protein deposits may be indicative of a response to treatment, albeit a suboptimal response, which can be compared to the time course of levels of tau protein deposits in untreated patients with a particular tauopathy (e.g., Alzheimer's disease) to determine whether immunotherapy is effective in inhibiting further increases in tau protein deposits.

[0306] Monitoring changes in tau protein deposits allows for adjustments to immunotherapy or other treatment regimens in response to the treatment. PET monitoring provides an indication of the nature and extent of the response to the treatment. It can then be determined whether or not treatment adjustments should be made, and if necessary, the treatment can be adjusted in response to the PET monitoring. Thus, PET monitoring allows for adjustments to the administration regimen of tau-targeted immunotherapy or other treatments before other biomarkers, MRI or cognitive measures respond detectably. A significant change means that comparing the value of the parameter after treatment to the baseline provides evidence that the treatment has or has not produced a beneficial effect. In some cases, the change in the value of the parameter in the patient itself provides evidence that the treatment has or has not produced a beneficial effect. In other examples, the change in value in the patient is compared to the change in value, if any, in a representative control population of patients not receiving immunotherapy. The difference between the response of a particular patient and the normal response of control patients (e.g., the variance of the mean plus standard deviation) can also provide evidence that the administration regimen of the immunotherapy has or has not achieved a beneficial effect in the patient.

[0307] In some patients, monitoring shows a detectable reduction in tau protein deposits, but levels of tau protein deposits remain above normal, and such patients may continue the treatment regimen at the same dosing frequency and / or dose in the absence of unacceptable side effects, or at an increased dosing frequency and / or dose if not at the maximum recommended dose.

[0308] If monitoring indicates that the levels of tau protein deposits in the patient have been reduced to normal or near normal, the immunotherapy regimen may be adjusted from an induction regimen (i.e., a regimen that reduces the levels of tau protein deposits) to a maintenance regimen (i.e., a regimen that maintains tau protein deposits at a near constant level). Such a regimen may be accomplished by reducing the dose and / or frequency of administration of the immunotherapy.

[0309] In other patients, monitoring may show that immunotherapy has some beneficial effect, but suboptimally. Optimal effect may be defined as the percentage reduction in the level of tau protein deposits within the upper half or quartile of change in tau protein deposits (measured or calculated over the whole brain or representative region(s) where tau protein deposits are known to form) of a representative sample of tauopathy patients receiving immunotherapy at a given time point after the start of treatment. Patients whose tau protein deposits have a small decrease, or whose tau protein deposits remain constant, or whose tau protein deposits increase, but to a lesser extent than would be expected in the absence of immunotherapy (e.g., when extrapolated from a control group of patients not receiving immunotherapy) may be classified as patients experiencing a positive, but suboptimal, response. Such patients may be given an adjustment in the dosing regimen to increase the dose and / or frequency of administration of the drug, if necessary.

[0310] In some patients, tau protein deposits may increase similarly to or exceed those in patients not receiving immunotherapy. If such an increase continues over a period of time, such as 18 months or 2 years, even after the frequency or dose of the drug has been increased, immunotherapy may be discontinued in favor of other treatments, if necessary.

[0311] The above discussion of diagnosing, monitoring, and adjusting treatment for tauopathies has focused primarily on the use of PET scans, however, any other technique for visualizing and / or measuring tau protein deposits that is amenable to the use of the tau antibodies of the invention (e.g., murine, humanized, chimeric, or veneered 3D6 antibodies) can be used in place of PET scans to perform such methods.

[0312] Also provided is a method for detecting immune responses against tau in patients suffering from or susceptible to tau-related diseases. This method can be used to monitor the course of therapeutic and prophylactic treatment with the agents provided herein. The antibody profile after passive immunization usually shows an immediate peak in antibody concentration followed by an exponential decay. Without further administration, the decay approaches pre-treatment levels within a period of several days to several months, depending on the half-life of the administered antibody. For example, the half-life of some human antibodies is on the order of 20 days.

[0313] In some methods, a baseline measurement of the subject's antibody against tau is taken before administration, a second measurement is taken shortly thereafter to measure the peak level of the antibody, and one or more additional measurements are taken at intervals to monitor the decay of the antibody level. When the antibody level falls to a predetermined percentage (50%, 25% or 10%) of the baseline or baseline-subtracted peak, an additional dose of the antibody is administered. In some methods, the peak or background-subtracted subsequent measured levels are compared to a predetermined reference level to generate a beneficial prophylactic or therapeutic treatment regimen for other subjects. If the measured antibody level is significantly lower than the reference level (e.g., less than the mean minus one standard deviation, preferably two standard deviations, of the reference value of the subject population that will benefit from the treatment), an additional antibody dose is required.

[0314] Also provided is a method for detecting tau in a subject, for example, by measuring tau in a sample from the subject or by in vivo imaging of tau in the subject.Such a method is useful for diagnosing or confirming the diagnosis of tau-related disease or susceptibility thereto.This method can also be used in asymptomatic subjects.The presence of tau indicates susceptibility to future symptomatic disease. The methods are also useful for monitoring disease progression and / or response to treatment in subjects who have been 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0315] Biological samples from subjects having, suspected of having, or at risk of having 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), 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 subjects can be compared to the level of tau present in healthy subjects. Alternatively, the levels of tau in such subjects undergoing treatment for a disease may be compared to the levels of tau in subjects who have not been 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Some such tests require a biopsy of tissue from such subjects. ELISA assays can also be a useful method for assessing tau in, for example, fluid samples.

[0316] VII. Kit The present invention provides a kit (e.g., a container) that contains the antibodies and related items disclosed herein, such as instructions for use (e.g., a package insert). The instructions can include, for example, instructions for administration of the antibody and, optionally, one or more additional agents. The container of antibody can be a unit dose, bulk package (e.g., a multi-dose package), or a sub-unit dose.

[0317] Package insert means instructions customarily included in commercial packages of therapeutic agents that contain information about the indications, uses, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic agent.

[0318] The kit may also contain a second container containing a pharma- ceutically acceptable buffer, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0319] VIII. Other Uses For clinical diagnosis or treatment or research, the antibodies can be used to detect tau or fragments thereof. For example, the antibodies can be used to detect the presence of tau in a biological sample as an indication that the biological sample contains tau deposits. The binding of the antibody to the biological sample can be compared to the binding of the antibody to a control sample. The control sample and the biological sample can include cells of the same tissue origin. The control sample and the biological sample can be obtained from the same individual or different individuals, at the same time or at different times. If necessary, multiple biological samples and multiple control samples are evaluated at multiple times to protect against random variations unrelated to differences between the samples. A direct comparison is then made between the biological sample(s) and the control sample(s) to determine whether the antibody binding (i.e., the presence of tau) to the biological sample(s) is increased, decreased, or the same as the antibody binding to the control sample(s). An increase in the binding of the antibody to the biological sample(s) compared to the binding to the control sample(s) indicates the presence of tau in the biological sample(s). In some cases, the increase in antibody binding is statistically significant. Optionally, antibody binding to the biological sample is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-fold greater than antibody binding to a control sample.

[0320] Additionally, the antibodies can be used to detect the presence of tau in biological samples to monitor and evaluate the efficacy of therapeutic agents being used to treat patients 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 disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). A biological sample from a patient 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) is evaluated to establish baseline binding of the antibody to the sample (i.e., baseline for the presence of tau in the sample) prior to initiating treatment with the therapeutic agent. In some cases, multiple biological samples from a patient are evaluated at multiple time points to establish both a baseline and a criterion of random variation unrelated to treatment. A therapeutic agent is then administered according to a dosing regimen. The dosing regimen may include multiple administrations of the agent over a period of time. In some cases, antibody binding (i.e., the presence of tau) is evaluated in multiple biological samples at multiple time points to establish a criterion of random variation and indicate a tendency to respond to immunotherapy. Various comparative evaluations of antibody binding to the biological samples are then performed. If only two evaluations are performed, a direct evaluation is performed between the two evaluations to determine whether antibody binding (i.e., the presence of tau) increases, decreases, or remains the same between the two evaluations.If three or more measurements are taken, the measurements can be analyzed as a time course beginning before treatment with the therapeutic agent and as a time course progressing through the course of treatment. If antibody binding (i.e., the presence of tau) to the biological sample is reduced in the patient, it can be concluded that the therapeutic agent is effective in treating the patient's 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). The reduction in antibody binding is statistically significant. In some cases, binding is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Evaluation of opponent binding may be performed in conjunction with evaluation of signs and symptoms 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT) or progressive supranuclear palsy (PSP).

[0321] Antibodies can also be used as research reagents for studies detecting tau or its fragments. For such applications, the antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, radioisotopes, and provided in kit form with all the reagents required to perform a detection assay. Antibodies can also be used to purify tau or binding partners of tau, for example, by affinity chromatography.

[0322] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety in this application to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date means a date earlier than the actual filing date or, if applicable, the filing date of the priority application for the accession number. Similarly, where different versions of a publication, website, etc. were published at different times, the most recently published version as of the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other unless otherwise indicated. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0323] Working Example Example 1. Identification of Tau Monoclonal Antibodies Monoclonal antibodies against tau were generated as follows: Immunizations were performed with recombinant N-terminal His-tagged 383a.a. human tau (4R0N) containing the P301S mutation [Immunogen A] or recombinant 383a.a. human tau (4R0N) containing the P301S mutation and lacking the N-terminal His-tag [Immunogen B]. The immunogens were emulsified in RIBI adjuvant.

[0324] Five-week-old female Balb / c mice were immunized intraperitoneally with 25 μg of Immunogen A on day 0 and 10 μg of Immunogen A on days 7, 14, 21, 27, 34, 48, 55, and 62, respectively. Mice were immunized with 10 μg of Immunogen B on days 76 and 90. Mice were bled and titers against Immunogen A were measured on days 43 and 98; animals with the highest titers were boosted with a terminal immunization of 50 μg of Immunogen B on day 101. This boost was delivered 1 / 2 intraperitoneally and 1 / 2 intravenously. Fusion hybridomas were screened against both immunogens by ELISA, and those with the highest signals were epitope mapped (see Example 2).

[0325] Example 2. Epitope mapping of antibody 3D6 A series of overlapping biotinylated peptides spanning the 383aa 4R0N human tau protein were used to map the mouse 3D6 antibody, and additional peptides were used to model potential post-translational modifications at the C- and N-termini of the protein.

[0326] Biotinylated peptides were bound to separate wells of streptavidin-coated ELISA plates. Plates were blocked and treated with mouse 3D6 followed by incubation with horseradish peroxidase-labeled anti-mouse antibody. After extensive washing, OPD was applied to the plates and developed. Plate absorbance was read at 450 nm. Background subtraction of absorbance values ​​from wells without primary antibody was performed and a threshold of 0.2 absorbance units for positive binding was set. Binding was mapped to sites within the MTBR region.

[0327] Example 3. Design of humanized 3D6 The starting point or donor antibody for humanization was the murine antibody 3D6. The heavy chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:7. The light chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:11. The heavy chain Kabat / Chothia combination CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs:8-10, respectively. The light chain Kabat CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs:12-14, respectively. Kabat numbering is used throughout.

[0328] The variable kappa (Vκ) of the 3D6 antibody belongs to mouse Kabat subgroup 2, which corresponds to human Kabat subgroup 2, and the variable heavy (Vh) belongs to mouse Kabat subgroup 2c, which corresponds to human Kabat subgroup 1 [Kabat EA, et al., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. The 16-residue Chothia CDR-L1 belongs to canonical class 4 of Vκ, the 7-residue Chothia CDR-L2 belongs to class 1, and the 9-residue Chothia CDR-L3 belongs to class 1 [Martin AC, and Thornton JM (1996) J. Mol. Biol. 263: 800-15. [Martin & Thornton, 1996]. The 10-residue Chothia CDR-H1 belongs to class 1, and the 17-residue Chothia CDR-H2 belongs to class 2 [Martin & Thornton, 1996]. CDR-H3 has no canonical class. To find a structure that would provide a rough structural model of the 3D6 antibody, we searched against protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33: D233-7]. To build the Fv model of 3D6, we used the structure of mouse anti-pyroglutamic acid Abeta antibody Fab c number 24 (pdb code 5MYX) with a resolution of 1.4 Å [Piechotta, A. et al., 2017, J Biol Chem 292: 12713-12724], which retained the same canonical structure as the loops of 3D6.

[0329] The framework of 3D6 VH shares a high degree of sequence similarity with the corresponding region of humanized 48G7 Fab PDB:2RCS designed by Wedemayer, GJ, et al. (1997; Science 276: 1665-1669). The variable domains of 3D6 and 48G7 Fab also share the same length for CDR-H1, H2 loops. Similarly, the framework of 3D6 VL shares a high degree of sequence similarity with the corresponding region of human antibody ARX71335 VL cloned by Dafferner, AJ, et al. (2017;. Direct Submission). The variable light chain domains of 3D6 and ARX71335 antibodies also share the same length for CDR-L1, L2 and L3 loops. Therefore, the framework regions of 48G7 VH (2RCS-VH) and ARX71335 VL were selected as the acceptor sequences for the CDRs of 3D6. A model of the 3D6 CDRs grafted onto each human framework of VH and VL was constructed and used as guidance for further backmutation.

[0330] The heavy and light chain variant sequences resulting from the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains as outlined in the WHO INN Committee guidelines. (WHO-INN: International Nonproprietary Nomenclature for Biological and Biotechnological Substances (INN) (Overview) (Internet) 2014. Available at http: / / www.who.int / medicines / services / inn / BioRev2014.pdf) When possible, residues were changed to correspond to the corresponding human germline sequence to increase the degree of humanization and reduce potential immunogenicity. For the humanized VLvb2 and VLvb3 variants, the sequences were aligned to the human germline gene IGKV2-30. * Mutations were introduced to make VHvb2, VHvb3, VHvb4, VHvb5, VHvb6, and VHvb6 variants more similar to the human germline gene IGHV1-69-2 (SEQ ID NO: 27).* Mutations were introduced to make it more similar to 01 (SEQ ID NO:25).

[0331] The hu3D6-VH and hu3D6-VL versions were designed to allow evaluation of various framework residues for their contribution to antigen binding, thermal stability, and immunogenicity, as well as for optimization of glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface exposed charged patches, deamination, and proteinase susceptibility. Positions considered for mutation included: - the positions that define the canonical CDR conformation (summarised in Martin, ACR (2010) Protein sequence and structure analysis of antibody variable domains. in Kontermann R and Duebel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.), - Position within the Vernier zone (Foote J and Winter G.(1992) Antibody framework residues affecting the conformation of the hypervariable loops.J Mol Biol.224(2):487-99.) - localization at the VH / VL domain interface (summarized in Le'ger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanisation by CDR grafting. in Shepherd P and Dean C (eds). Monoclonal Antibodies: a Practical Approach. Oxford ,UK: Oxford University Press.), - positions susceptible to post-translational modifications, such as glycosylation or pyroglutamylation; - positions occupied by residues predicted to clash with the CDRs according to a model of the 3D6 CDRs grafted onto the VH and VL frameworks, or - positions occupied by rare residues in sequenced human antibodies, where either the parent murine 3D6 residue or some other residue is much more prevalent in the human antibody repertoire; Examples include:

[0332] Sequence comparisons of murine 3D6 and various humanized antibodies are shown for the light chain variable region (Table 4 and Figure 2) and the heavy chain variable region (Table 3 and Figure 1).

[0333] Seven humanized heavy chain variable region variants and three humanized light chain variable region variants were constructed containing different substitution permutations hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, or hu3D6VHvb7 (SEQ ID NOs: 76-80 and 90-91, respectively), and hu3D6VLvb1, hu3D6VLvb2, or hu3D6VLvb3 (SEQ ID NOs: 83-85, respectively) (Tables 3 and 4). Representative humanized Vk and Vh designs with backmutations and other mutations based on selected human frameworks are shown in Tables 3 and 4, respectively. The bolded regions in Tables 3 and 4 indicate the CDRs defined by the Kabat / Chothia combination. SEQ ID NOs: 76-80 and SEQ ID NOs: 90-91 contain backmutations and other mutations as shown in Table 5. The amino acids at the positions in hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 are shown in Table 6. The amino acids at the positions in hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3 are shown in Table 7. The percentage humanization degree of the humanized VH chains hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 (SEQ ID NOs: 76 to 80 and 90 to 91, respectively), and the percentage humanization degree of the humanized VL chains hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3 (SEQ ID NOs: 83 to 85, respectively) are shown in Table 8. [Table 3] JPEG2025063136000005.jpg255159JPEG2025063136000006.jpg212159JPEG20250631360 00007.jpg255159JPEG2025063136000008.jpg254159JPEG2025063136000009.jpg133159 [Table 4] JPEG2025063136000011.jpg239159JPEG2025063136000012.jpg244159JPEG20250631360 00013.jpg243159JPEG2025063136000014.jpg244159JPEG2025063136000015.jpg123159 [Table 5] [Table 6] JPEG2025063136000018.jpg209159 [Table 7] [Table 8]

[0334] Positions where the canonical, Vernier, or interface residues differ between the mouse and human receptor sequences are candidates for substitution. Examples of canonical / CDR interacting residues include Kabat residues H54 and H94 in Table 3. Examples of Vernier residues include Kabat residues H28, H67, H93, and H94 in Table 3. Examples of interface / packing (VH+VL) residues include Kabat residues H91 and H93 in Table 3.

[0335] The principles for selecting the positions shown in Table 3 in the heavy chain variable region as replacement candidates are as follows.

[0336] Heavy chain variable region hu3D6VHvb1 - consists of the CDR-H1, H2 and H3 loops of 3D6-VH grafted onto the framework of 48G7-VH (RCS-VH) with back mutations at positions H91 (Y91F), H93 (A93S) and H94 (S94T).

[0337] hu3D6VHvb2 -Restores all framework substitutions at positions that are important for defining the Chothia canonical class, that are part of the Vernier zone, or that are located at the VH / VL domain interface, or that contribute to structural stability. 3D6-VH_vb2 incorporates back mutations or substitutions Q1E, Q5V, L11V, L20I, T23K, K38R, E42G, Q43K, K66R, S75T, N76D, Q81E, Y91F, A93S, S94T, T108L, and L109V, allowing evaluation of the contribution of these positions to antigen binding affinity and immunogenicity.

[0338] hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 consists of further substitutions and increases antibody stability and / or optimization of glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface exposed charge patch, surface exposed charge patch, deamination and proteinase susceptibility.

[0339] Q1E is a stability enhancing mutation that reduces the ability to form pyroglutamic acid (Liu, supra). Q1E is a back mutation.

[0340] Q5V is a frequency-based and germline-aligning mutation. Val is the most abundant mutation in human sequences at this position. Val is the most abundant mutation in human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO:25).

[0341] L11V is a germline alignment mutation. Val is a mutation in the human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO:25).

[0342] S17T is a germline alignment mutation. Thr is located at this position in the human germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO:25).

[0343] L20I is a germline alignment mutation. Ile is a mutation in the human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO:25).

[0344] T23K is a frequency-based and germline aligned mutation. Lys is the most common mutation at this position. Lys is the most common mutation at this position in the human germline gene IMGT number IGHV1-69-2. * 01 (SEQ ID NO:25).

[0345] N28T: This is a CDR-H1 residue substitution for Thr. N28T is a germline alignment mutation. Thr is a CDR-H1 residue substitution for Thr at this position in human germline gene IMGT number IGHV1-69-2. * 01 (SEQ ID NO:25).

[0346] K38R ​​is a frequency-based back mutation. Arg is the most abundant at this position. Arg at this position is predicted to generate one H-bond plus two H-bonds with Glu46, Asp86, and Tyr90, respectively, in the heavy chain, and therefore Arg may enhance stability over Lys at this position.

[0347] E42G is a frequency-based and germline aligned mutation. Gly is a mutation in the human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO: 25). Gly is the most abundant site at this position. Gly substitution is not predicted to affect stability.

[0348] Q43K: The Lys side chain at this position is predicted to form H-bonds with the main chain as well as the G42 side chain and to form H-bonds with Gln39 and Arg40, which may enhance the stability of the Lys substitution at this position compared to Q. Q43K is a germline aligned mutation. Lys is a nucleotide variant of the human germline gene IMGT number IGHV1-69-2 at this position. *01 (SEQ ID NO:25).

[0349] N54D and D56E are substitutions of CDR residues and are predicted to be in non-antigen contact positions according to the homology model. The N54D and D56E substitutions are predicted to stabilize the antibody structure. N54D and D56E are germline aligned mutations. Human germline gene IMGT number IGHV1-69-2 * In 01 (SEQ ID NO:25), Asp is at position H54 and Glu is at position H56.

[0350] V58I is a substitution in the CDR-H2 residue. Germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO:25) has an Ile at this position, a residue that is not predicted to contact the antigen.

[0351] K66R: Arg at this position is predicted to form H-bonds with Ser82a and Thr83 in addition to forming H-bonds and salt bridges with Asp86. K66R is a germline aligned mutation. Arg at this position is predicted to form H-bonds with Ser82a and Thr83 in addition to forming H-bonds and salt bridges with Asp86. * 01 (SEQ ID NO:25).

[0352] A67V is a substitution of a Vernier zone residue. Germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO:25) has a Val at this position.

[0353] S75T: Ser at this position is predicted to make H-bonds with Asp72 and Tyr76. Thr at this position is also predicted to make these contacts, but Thr, being a surface-exposed residue, may enhance antibody stability. S75T is a germline aligned mutation. Thr at this position is predicted to make H-bonds with Asp72 and Tyr76. * 01 (SEQ ID NO:25).

[0354] N76D:Asp is a germline alignment mutation. Asp is located at this position in the human germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO:25).

[0355] L80M:Met is a germline alignment mutation. Met is a mutation in the human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO:25).

[0356] Q81E: Glu is predicted to form an H-bond + salt bridge with K19, thus Glu at this position enhances antibody stability. Q81E is a germline aligned mutation. Glu is predicted to form an H-bond + salt bridge with K19 at this position, thus enhancing antibody stability. * 01 (SEQ ID NO:25).

[0357] T83R enhances thermostability and increases the degree of humanization. Arg is a germline alignment mutation. Arg is a human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO: 25). Arg is the most abundant at this position.

[0358] Y91F is a mutation of an interface residue and is a back mutation. Tyr at this position may enhance antibody stability.

[0359] A93S is a backmutation of a Vernier zone and interface zone residue.

[0360] S94T is a backmutation of the canonical structural residue defined by Chothia and the Vernier residue.

[0361] T108L:Leu is a germline aligned mutation. Leu is located at this position in the human germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25). Leu at this position is predicted to reduce the immunogenicity of the antibody and have no effect on antibody stability.

[0362] L109V is a frequency-based mutation. Val is the most common mutation at this position. L109V is a germline alignment mutation. Val is a mutation that occurs most frequently at this position in the human germline gene IMGT number IGHV1-69-2. * 01 (SEQ ID NO:25).

[0363] The principles for selecting the positions shown in Table 4 in the light chain variable region as substitution candidates are as follows.

[0364] Kappa light chain variable region

[0365] hu3D6VLvb1 - consists of the CDR-L1, L2, and L3 loops of 3D6-VL grafted onto the framework of ARX71335VL.

[0366] hu3D6VLvb2 and hu3D6VLvb3 -Restore all framework substitutions at positions that are important for defining the Chothia canonical class, that are part of the Vernier zone, or that are located at the VH / VL domain interface. Hu3D6-VLvb2 and hu3D6-VLvb3 also contain substitutions that contribute to structural stability, and hu3D6-VL-vb2 incorporates back mutations T7S, I15L, L83V, H86Y, and L106I, allowing evaluation of the contribution of these positions to antigen binding affinity and immunogenicity. All substitutions in Hu3D6-VL-vb3 are described for vb2 with additional changes at Q17E, K24R, L37Q, K45R, and L106I.

[0367] T7S is a germline alignment mutation. Ser is a mutation that confers a nucleotide sequence to the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO:27).

[0368] T10S is a frequency-based and germline aligned mutation. Ser is abundant at this position. Ser is abundant at this position in the human germline gene IGKV2-30. *02 (SEQ ID NO:27).

[0369] I15L is a germline alignment mutation. Leu is a nucleotide variant of the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO:27).

[0370] Q17E: Glu at this position is predicted to form an H-bond with T14 and a salt bridge with Lys107, both light chain residues, predicted to enhance antibody stability.

[0371] K24R is a mutation in a CDR residue. Both Lys and Arg are predicted to form H-bonds and salt bridges with Asp70 in the light chain. Arg is predicted to fit better in the conformation. Arg is also a germline alignment mutation. Arg is the only mutation in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO:27).

[0372] L37Q: This is predicted to be a deeply buried residue, and Leu is not predicted to interact with surrounding residues, while Gln is predicted to make H-bonds with Q38 and Asp82 in the light chain. Gln is also a germline aligned mutation. Gln is not present in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO:27).

[0373] K45R:Lys is predicted to make H-bonds with S56 and Gly57; the predicted interactions of Arg with adjacent residues are much more extensive, as it is predicted to form a salt bridge with D55, an H-bond with Arg46, and a double H-bond with S56. Arg is also a germline aligned mutation. Arg is not present in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO:27).

[0374] L83V: This is a mutation based on the frequency of residues predicted to be surface exposed. Val is also a germline aligned mutation. Val is a mutation found in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO:27).

[0375] H86Y: Mouse 3D6 VL has a Tyr at this position. Tyr is also the most common residue at this position.

[0376] A100Q: Ala is rare at this position. Ala is predicted to be a surface-exposed residue and is not predicted to interact with surrounding residues. Gln is the most common mutation at this position and is also a germline aligned mutation. Gln is rare at this position in the human germline gene IGKV2-30. * 02 (SEQ ID NO: 27). Gln is predicted to form an H-bond with Ser7 to stabilize the intrachain.

[0377] L106I is a frequency-based and germline aligned mutation. Ile is the most common mutation at this position. Ile is the most common mutation at this position in the human germline gene IGKV2-30. * 02 (SEQ ID NO:27).

[0378] Designs based on these human frameworks are shown below:

[0379] Heavy chain variable region >hu3D6VHvb1 (SEQ ID NO: 76) QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0380] >hu3D6VHvb2 (SEQ ID NO: 77) EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLELSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0381] >hu3D6VHvb3 (SEQ ID NO: 78) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRATITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0382] hu3D6VHvb4 (SEQ ID NO: 79) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0383] >hu3D6VHvb5 (SEQ ID NO: 80) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0384] >hu3D6VHvb6 (SEQ ID NO: 90) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYFCSTLDFWGQGTLVTVSS

[0385] >hu3D6VHvb7 (SEQ ID NO: 91) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0386] Kappa light chain variable region hu3D6VLvb1 (SEQ ID NO: 83) DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCWQGTHFPYTFGAGTKLELKR

[0387] >hu3D6VLvb2 (SEQ ID NO: 84) DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGAGTKLEIKR

[0388] >hu3D6VLvb3 (SEQ ID NO: 85) DVVMTQSPLSLSVTLGEPASISCRSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR

[0389] Humanized sequences are generated using a two-step PCR protocol that allows for the introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682].

[0390] Example 4. Mouse monoclonal antibodies bind to tau in an ELISA assay Methods: Indirect ELISA: 96-well polystyrene plates were coated with capture antibodies anti-6xHis (Figure 3A) or polyclonal anti-tau (Dako #A20024, Figure 3B) suspended in 1xPBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution was removed and the plates were blocked with 1% BSA in 1xPBS for 1 hour and subsequently incubated with human recombinant tau with (Figure 3A) or without (Figure 3B) a polyhistidine tag at the N-terminus of the protein. After washing, the plates were incubated with the indicated antibodies and then with a goat anti-mouse secondary antibody conjugated with HRP. The plates were developed with TMB and incubated for 1 hour at room temperature or 16 hours at 4°C. 450 was measured using a plate reader.

[0391] Sandwich ELISA: 96-well polystyrene plates were coated with anti-mouse antibodies in 1xPBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution was removed and the plates were blocked with 1% BSA in 1xPBS for 1 hour. The plates were then incubated with the same concentrations of antibodies indicated, diluted in 0.1% BSA in 1xPBS. The plates were treated sequentially with human tau, polyclonal rabbit anti-tau (Dako#A0024), and HRP-labeled goat anti-rabbit antibodies, all diluted in 0.1% BSA in PBS with washing between each step. Streptavidin-HRP was added and the plates were developed with TMB and incubated for 1 hour at 4°C. 450 was measured on a plate reader. See Figure 3C.

[0392] Results: A series of hybridoma-produced antibodies were assayed for binding to tau by several different ELISA formats. Detection of tau was confirmed using an indirect format with tau protein immobilized by a polyhistidine tag fused to the N-terminus of tau (Figure 3A). Native, untagged protein was also confirmed (Figure 3B). To assess the nutrient affinity of the various antibodies, a sandwich ELISA format was used in which the hybridoma antibodies tested were used as capture reagents (Figure 3C).

[0393] Example 5. Affinity of Mouse Monoclonal Antibodies to Tau Methods: SPR analysis was performed using a Biacore T200 to measure the binding kinetics of mouse antibodies to recombinant human tau. To prepare the sensor surface, anti-mouse antibodies (GE Life Sciences) were immobilized on a sensor chip CM5 via amine coupling and the antibody was captured at a level ensuring maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 10 to 0.14 nM were passed over the captured ligand at a flow rate of 50 μL / min in running buffer (HBS + 0.05% P-20, 1 mg / mL BSA) for 180 s of binding and 900 s of dissociation. Data were double referenced against both an unrelated sensor without antibody ligand, and a 0 nM analyte concentration to capture dissociation of the ligand from the captured moiety. Data were then analyzed using a global 1:1 fit.

[0394] Results: Several mouse antibodies were selected based on their performance in a series of ELISA assays, and their binding affinities were assessed by SPR. Several sets of antibodies were tested simultaneously, and their binding and dissociation rates were compared to select the best binders to recombinant human tau. The highest binding affinity was observed for antibody clone 3D6. The binding affinities are shown in Figure 4.

[0395] Example 6. Mouse monoclonal antibodies block binding of human tau to the surface of immortalized neuronal cells Methods: Inhibition of tau binding to B103 neuroblastoma cells by anti-tau monoclonal antibodies 1. 5x10 B103 cells 5 Resuspend at a concentration of 1000 cells / mL. Plate 50 μL of cell suspension per well in an MSD high binding plate. This will give 25k cells / well. Cover the plate and allow cells to attach for 2 hours at 37°C, 5% CO2. 2. After cell attachment, remove PBS from wells by inverting the plate and gently tapping to remove excess buffer. 50 μl of 3% MSD Blocker A buffer in PBS or other suitable blocking buffer was added to each well and plates were incubated for 1 hour at room temperature without shaking. 3. During the plate blocking step, co-incubate tau and anti-tau antibodies as follows: Starting with 2 mg / mL anti-tau antibody, seven additional dilutions are made by serial 1:2 dilution in PBS. b. Tau is diluted to 20 nM in PBS, so that the tau concentration is constant in each well. c. Tau and anti-tau antibody are mixed 1:1 for a final tau concentration of 10 nM and a starting anti-tau concentration of 1 mg / mL. d. The mixture is incubated at room temperature with shaking (600 rpm) for approximately 1 hour. 4. After blocking the plate in step 2, remove the blocking buffer from the wells by inverting the plate and gently tapping, then wash the plate twice with PBS using a multichannel pipette. Be sure to completely remove excess buffer. Chill plated cells to 4°C before adding the tau:anti-tau complex. 5. Add 50 μL of chilled complex from step 3 to the plated cells and incubate on ice for 30 min. 6. Wash plates twice with cold PBS as previously described. 7. For detection of cell surface bound tau, add 50 μL of 16B5.SULFO-TAG per well. Incubate on ice for 30 minutes. 8. Wash the plates again twice with cold PBS as previously described. 9. Add 150 μL of 1× detergent-free Read Buffer T (diluted in H2O) per well and read immediately on the MSD SECTOR™ 600 instrument. Avoid introducing air bubbles when adding the Read Buffer. 10. Report the MSD signal vs. Tau concentration.

[0396] The antibodies tested were the anti-tau antibodies 3D6, 16G7, 3H9, 4C5, and 5G8, as well as an isotype control.

[0397] Results: A decrease in sulfo-tag anti-tau signal with increasing numbers of tested antibodies indicates functional blocking of tau binding to neuronal cells. No blocking was observed with isotype control, 16G7, or 3H9. Increasing amounts of functional blocking activity were observed with 4C5, 5G8, and 3D6. 3D6 showed the strongest blocking activity of the antibodies tested. See Figure 5.

[0398] Example 7. Disaggregation activity Methods: Recombinant Tau - Purified recombinant tau with an N-terminal 6xHis tag was mixed with an equimolar amount of low molecular weight heparin in 1x PBS (pH 7.4) and incubated for 96 hours at 37°C on a nutator. Aggregation of the samples was confirmed by binding to Thioflavin T. Incubation with antibodies - Antibodies were incubated with aggregated recombinant tau for 96 hours at 37°C without rotation or nutator. At the end of the experiment, aggregation was measured by incubating samples with 25mM Thioflavin T and measuring the emitted fluorescence (450 / 482 ex / em). Signal was background subtracted to buffer samples.

[0399] Results: 3D6 selectively degrades intact tau fibrils, as shown in Figure 6. Various molar ratios of 3D6 (triangles), isotype control (circles) and 16G7 (squares) were incubated with amyloid-containing tau fibrils for 96 hours. At the end of this period, the degree of aggregation was assessed by binding to Thioflavin T. 3D6 selectively reduces the Thioflavin T signal present in the samples compared to the isotype control antibody as well as 16G7, an anti-tau antibody that binds to a different region of tau.

[0400] Example 8.3D6 and 5G8 immunocapture tau from human disease tissue. Methods: High-salt soluble protein fractions were prepared at 1mg / ml. For each immunoprecipitation, 200μg of sample was used. 10μg of the indicated antibody (isotype control, 3D6, or anti-tau antibody 5G8) was added to the high-salt sample preparation and incubated for 2 hours. Protein G magnetic beads were then added to the mixture and incubated for an additional hour to capture the antibody / antigen complex. Samples were washed thoroughly with 1xPBS and beads were boiled in reducing / denaturing sample buffer to release the captured proteins. Resulting samples were resolved by SDS-PAGE and western blotting was performed using a polyclonal anti-tau antibody (Dako, #A0024).

[0401] Results: As shown in Figure 7, 3D6 and 5G8 immunoprecipitated tau from Alzheimer's disease tissue. The high salt soluble fraction was immunoprecipitated with the indicated antibodies and detected with a polyclonal anti-tau antibody against a region of the tau molecule separated from the binding sites of 3D6 and Tau Antibody A. 3D6 robustly captured tau from this fraction. The input (high salt soluble sample) is shown on the right.

[0402] Example 9. Immunoreactivity of 3D6 by Immunohistochemistry Frontotemporal cortex was obtained from patients without neurodegenerative disease or with confirmed Alzheimer's disease at postmortem evaluation. Immunohistochemistry was performed on 10 um frozen sections mounted on slides and lightly acetone fixed. All staining steps were performed using a Leica BOND Rx automated immunostainer and Leica consumables. Mouse or human 3D6 was incubated with tissue sections, followed by the addition of species-appropriate secondary antibodies conjugated to HRP polymers. To prevent non-specific binding of endogenous immunoglobulins when humanized antibodies are used on human tissues, antibodies were non-covalently labeled in vitro with biotin-conjugated anti-human monovalent Fab fragments prior to tissue incubation. Tissues labeled with primary antibody-biotin Fab fragment complexes were further amplified using an avidin-biotin amplification system (Vector Laboratories, Burlingame, CA). Staining was visualized using DAB chromogen, which produces brown deposits. Negative controls were generated by performing the entire immunohistochemical procedure on adjacent sections using an IgG isotype control antibody.

[0403] The antibodies tested were mouse CD6, chimeric 3D6 (which contained a VH and VL derived from a mouse antibody with human constant regions, heavy chain SEQ ID NO: 72 and light chain SEQ ID NO: 73), and humanized variants hu3D6VHv5 / hu3D6VLv2.

[0404] Staining performed on mouse, chimeric, and humanized forms of 3d6 was qualitatively compared and evaluated for staining intensity and brightness, as well as localization of immunoreactivity. Staining brightness was similar for the chimeric and humanized forms of 3D6 compared to the mouse form of the antibody, with similar localization patterns. Tau was detected in neurofibrillary tangles, fibrils, thread-like structures, and in degenerating axons. Prominent cell body staining was also detected.

[0405] Example 10. Affinity of humanized variants to tau Methods: Indirect ELISA: 96-well polystyrene plates are coated with human recombinant tau suspended in 1xPBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution is removed and the plates are blocked with 1% BSA in 1xPBS for 1 hour. Humanized variant antibody at 1 μg / mL in 0.1% BSA in 1xPBS is added to the plate, followed by a 1 hour hold, washing, and addition of HRP-conjugated goat anti-human antibody. The plates are developed with TMB and A 450 is measured in a plate reader.

[0406] Sandwich ELISA: 96-well polystyrene plates are coated with anti-human antibody in 1xPBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution is removed and the plates are blocked with 1% BSA in 1xPBS for 1 hour. Various concentrations of humanized variant antibodies are added to the plates diluted in 0.1% BSA in 1xPBS and held for 1 hour followed by washing and addition of biotinylated recombinant human tau diluted in 0.1% BSA in 1xPBS. After washing, streptavidin-HRP is added and the plates are developed with TMB and incubated for 1 hour at 4°C for 1 hour. 450 is measured in a plate reader.

[0407] Example 11. Analysis of humanized 3D6 variants The humanized 3D6 variants were analyzed for multiple characteristics, including target binding affinity, activity in cell-based assays, thermostability, expression titer, and the presence of aggregation.

[0408] Plasmids containing DNA encoding heavy chains hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7, and light chains hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3 were generated. Different combinations of heavy and light chains were transiently expressed as complete antibodies in HEK-293 cells, and antibodies were purified from conditioned media using Protein A chromatography. Purified antibodies were analyzed by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to detect the presence of aggregation. The results are shown in Table 9 in the column labeled "% Monomer."

[0409] The purified antibodies were analyzed by differential scanning calorimetry (DSC) to determine the thermal stability of each variant. Thermal stability values ​​were measured using differential scanning calorimetry. All DSC scans were performed using a VP-capillary DSC system (Malvern). All samples were prepared at 0.5mg / mL in 1xPBS and referenced to 1xPBS. Approximately 0.5mL of protein solution and buffer were introduced into the sample and reference cells. Calorimetry scans were performed at a scan rate of 60°C / hr from 25°C to 110°C under constant pressure. Analysis was performed using proprietary software. Values ​​reported are the temperatures at which the maximum heat of the Fab peak is recorded. Results are shown in Table 9 in the column labeled "Thermal Stability (°C)".

[0410] Titers were determined as follows: After expression in 293 suspension cells, the antibody was purified using Protein A chromatography using standard methods. After purification, the antibody was exchanged into 1xPBS and the protein concentration was measured by absorbance at 280 nm. Titers were calculated by dividing the final yield of purified protein by the starting volume of the expression culture and reported in milligrams per liter. The results are shown in Table 9 in the column labeled "Expression Titer (mg / L)".

[0411] The affinity of the purified humanized variants for tau was measured by surface plasmon resonance (SPR). SPR analysis was performed using a Biacore T200 to measure the binding kinetics of the humanized antibodies to recombinant human tau. To prepare the sensor surface, an anti-human antibody (GE Life Sciences) was immobilized on a sensor chip CM3 via amine coupling and the antibody was captured at a level ensuring a maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 50 to 0.62 nM were passed over the captured ligand at a flow rate of 50 μL / min in running buffer (HBS + 0.05% P-20, 1 mg / mL BSA) in one cycle. The data were double referenced both against an unrelated sensor without antibody ligand and against an analyte concentration of 0 nM to capture dissociation of the ligand from the captured moiety. The data were then analyzed using a 1:1 fit. The results are shown in Table 9 as "k on (M -1 s -1 ), k off (s -1 ), and K d The results are shown in the columns labeled "(nM)".

[0412] The ability of purified humanized variants to block tau internalization was measured by a cellular internalization assay. An internalization assay using fluorescence-activated cell sorting (FACS) was performed to evaluate the ability of various antibodies to block neuronal internalization of tau. Antibodies that block internalization are likely to block tau propagation. pHrodo-labeled 4R0N human tau P301L soluble oligomers (1.5 μg / mL final concentration) were pre-incubated with humanized variants (dose titer: 80 μg / mL starting concentration, 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 for 3-4 h at 37 °C in a tissue culture incubator (5% CO2). The cells were then washed three times with medium, then incubated with medium for 10 min, and washed twice with FACS buffer (1% FBS in PBS). The cells were suspended in 100 μL FACS buffer, and the Texas Red mean fluorescence intensity was measured by FACS LSR II. Texas Red fluorescence from pHrodo is activated at low pH, which is associated with the intralysosomal compartment due to internalization. Since FACS detects only the fluorescence of the cells and pHrodo due to internalization, only tau internalized by the cells is detected. The lower the mean fluorescence intensity, the less the amount of tau internalized and the greater the blocking activity of the tested antibodies. The results are shown in Table 9. [Table 9] JPEG2025063136000022.jpg90159

[0413] Array List P10636-8 (SEQ ID NO: 1) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPT REPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHH KPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0414] P10636-7 (SEQ ID NO: 2) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKS PSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0415] P10636-6 (4RON human tau) (SEQ ID NO: 3) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVP MPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0416] P10636-5 (SEQ ID NO: 4) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGT PGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0417] P10636-4 (SEQ ID NO:5) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPP TREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0418] P10636-2 (SEQ ID NO: 6) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0419] SEQ ID NO:7; Mouse 3D6 VH amino acid sequence: EVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0420] SEQ ID NO:8; Kabat / Chothia HCDR1: GFNIKDYYLH

[0421] SEQ ID NO:9; Kabat HCDR2: WIDPENGDTVYDPKFQG

[0422] SEQ ID NO:10; Kabat HCDR3: LDF

[0423] SEQ ID NO:11; Mouse 3D6 VL amino acid sequence: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKR

[0424] SEQ ID NO:12; mouse Kabat LCDR1: KSSQSLLDSDGKTYLN

[0425] SEQ ID NO:13; mouse Kabat LCDR2: LVSKLDS

[0426] SEQ ID NO:14; mouse Kabat LCDR3: WQGTHFPYT

[0427] SEQ ID NO: 15; hu3D6VHv1: EVQLVQSGAEVVRPGALVKVSCKASGFNIKDYYLHWVRQAPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0428] SEQ ID NO: 16; hu3D6VHv2: EVQLVQSGAEVKKPGASVKVSCKVSGFNIKDYYLHWVRQAPEQGLEWMGWIDPENGDTVYDPKFQGRVTITADTSNTAYMELSSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0429] SEQ ID NO: 17; hu3D6VHv1b: EVQLVQSGAEVVRPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0430] SEQ ID NO: 18; hu3D6VHv1bA11: EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0431] SEQ ID NO: 19; hu3D6VHv5: EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0432] SEQ ID NO:20; hu3D6VLv1: DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR

[0433] SEQ ID NO:21; hu3D6VLv2: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR

[0434] SEQ ID NO:22; hu3D6VLv3: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPRRLIYLVSKLDSGVPSRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR

[0435] SEQ ID NO:23; hu3D6VLv4: DIVMTQTPLSLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR

[0436] SEQ ID NO:24; Heavy Chain Variable Receptor Accession Number BAC01986.1 QVQLQQSGAEVKKPGSSVKVSCKASGGTFGSYAISWVRQAPGQGLEWMGRIIPILGIATYAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARGKGEFEGMDVWGQGTTVTVSS

[0437] SEQ ID NO: 25; Heavy Chain Variable Receptor IMGT Accession Number IGHV1-69-2 * 01 EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMGLVDPEDGETIYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCAT

[0438] SEQ ID NO: 26; Heavy chain variable receptor IMGT accession number IGKJ1 * 01 QHWGQGTLVTVSS

[0439] SEQ ID NO: 27; Light chain variable receptor IMGT accession number IGKV2-30 * 02IMGT Accession number IGKV2-30 * 02 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWP

[0440] SEQ ID NO:28; Light chain variable receptor IMGT accession number IGKJ2 * 01 YTFGQGTKLEIK

[0441] SEQ ID NO:29; Light Chain Variable Receptor Accession Number AAZ09048.1 DVVMTQSPLSLTVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYRVSHWDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTYWPLTFGQGTKLEIK

[0442] SEQ ID NO:30; Mouse 3D6 VH nucleic acid sequence: GAGGTTCAGCTGCAGCAGTCTGGGGCTGACCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATTTGCACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGT GATACTGTATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAATACAGCCTACCTGCAGCTCGGCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTTCTACCCTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0443] SEQ ID NO:31; Mouse 3D6 VL nucleic acid sequence: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGT CTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGT

[0444] SEQ ID NO:32; Mouse CDR-H1 Kabat DYYLH

[0445] SEQ ID NO:33; Mouse CDR-H1 Chothia GFNIKDY

[0446] SEQ ID NO:34: Mouse CDR-H2 Chothia DPENGD

[0447] SEQ ID NO:35; Mouse CDR-H2 AbM WIDPENGDTV

[0448] SEQ ID NO: 36: Mouse CDR-L1 Contact KTYLNWL

[0449] SEQ ID NO: 37: Mouse CDR-L2 Contact RLIYLVSKLD

[0450] SEQ ID NO: 38: Mouse CDR-L3 Contact WQGTHFPY

[0451] SEQ ID NO:39: Mouse CDR-H1 Contact KDYYLH

[0452] SEQ ID NO: 40: Mouse CDR-H2 Contact WIGWIDPENGDTV

[0453] SEQ ID NO: 41: Mouse CDR-H3 Contact STLD

[0454] SEQ ID NO:42; Alternative Kabat-Chocid CDR-H1 GFTIKDYYLH

[0455] SEQ ID NO:43; Alternative Kabat CDR-H2 WIDPEDGDTVYAPKFQG

[0456] SEQ ID NO: 44; consensus VH amino acid sequence from Figure 2 of PCT / IB2017 / 052544 EVQLVQSGAEVVXPGALVKISCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGXATITADTSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0457] SEQ ID NO: 45; consensus VL amino acid sequence of FIG. 3 in PCT / IB2017 / 052544 DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTKLEIKR

[0458] SEQ ID NO: 46; hu3D6VHv1bA11B6G2: EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWVDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0459] SEQ ID NO: 47; hu3D6VHv1bA11B6H3: EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGDTVYAPKFQGRATITADTSTDTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0460] SEQ ID NO: 48; hu3D6VHv1c: EVQLVQSGAEVKRPGALVKISCKASGFNFKDYYLHWVRQRPEQGLEWMGWIDPENGDTVYDEKFQGRVTITADTSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0461] SEQ ID NO: 49; hu3D6VHv1d: EVQLVQSGAEVKRPGALVKISCKASGYTFTDYYLHWVRQRPEQGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0462] SEQ ID NO: 50; hu3D6VHv1e: EVQLVQSGADVVKPGALVKISCKASGFTIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYAEKFQGRVTITADTSNTAYLELGSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0463] SEQ ID NO:51; hu3D6VHv1f: EVQLVQSGADVVKPGALVKISCKASGFTIKDYYLHWVRQRPGQGLEWIGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYMELGSLTSEDTAVYFCSTLDYWGQGTTLTVSS

[0464] SEQ ID NO:52; hu3D6VHv3: EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0465] SEQ ID NO:53; hu3D6VHv3b: EVQLVQSGAEVKKPGALVKISCKVSGYNFKDYYLHWVRQAPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0466] SEQ ID NO:54; hu3D6VHv3c: EVQLVQSGAEVKKPGALVKISCKVSGYTFTDYYLHWVRQAPGKGLEWMGWVDPEDGDTVYAEKFQGRVTITADTSTNTAYMELGSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0467] SEQ ID NO:55; hu3D6VHv4: EVQLVQSGAEVVKPGATVKISCKVSGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGKATITADTSTNTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0468] SEQ ID NO:56; hu3D6VHv4b: EVQLVQSGAEVVKPGALVKISCKVSGYNFKDYYLHWVRQRPGKGLEWMGWIDPENGDTVYDEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0469] SEQ ID NO:57; hu3D6VHv4c: EVQLVQSGAEVVKPGALVKISCKVSGYTFTDYYLHWVRQRPGKGLEWWMGWVDPEDGDTVYAEKFQGRVTITADTSTDTAYLELGSLTSEDTAVYYCSTLDFWGQGTLVTVSS

[0470] SEQ ID NO:58; another Kabat-Chothia CDR-H1 (similar to hu3D6VH1c). GFNFKDYYLH

[0471] SEQ ID NO:59; another Kabat-Chothia CDR-H1 (similar to hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c). GYTFTDYYLH

[0472] SEQ ID NO:60; Alternative Kabat-Chothia CDR-H1 (similar to hu3D6VHv3b and hu3D6VHv4b) GYNFKDYYLH

[0473] SEQ ID NO:61; another Kabat CDR-H2 (similar to hu3D6VHv1bA11B6G2). WVDPEDGDTVYAPKFQG

[0474] SEQ ID NO:62; another Kabat CDR-H2 (similar to hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b). WIDPENGDTVYDEKFQG

[0475] SEQ ID NO:63; another Kabat CDR-H2 (similar to hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c) WVDPEDGDTVYAEKFQG

[0476] SEQ ID NO:64; another Kabat CDR-H2 (similar to hu3D6VHv1e). WIDPENGDTVYAEKFQG

[0477] SEQ ID NO:65; another Kabat CDR-H3 (similar to hu3D6VHv1f) L.D.Y.

[0478] SEQ ID NO:66; heavy chain variable region of mouse 6A10 antibody EVQLQQSGAELVRSGASVKLSCTASGLNIKDYYIHWVKQRPEQGLEWIGWIDPENDDTEYAPKFQGRATLTTDTSSNTAYLQLSSLTSEDTAVYYCTPLDYWGQGTSVTVSS

[0479] SEQ ID NO:67; Kabat / Chothia combination CDR-H1 of mouse 6A10 antibody GLNIKDYYIH

[0480] SEQ ID NO:68; Kabat CDR-H2 of mouse 6A10 antibody WIDPENDDTEYAPKFQG

[0481] SEQ ID NO:69; Kabat CDR-H3 of mouse 6A10 antibody L.D.Y.

[0482] SEQ ID NO: 70; Mus VH structural template (PDB number 1CR9_H) KVKLQQSGAELVRSGASVKLSCTASGFNIKDYYIQWVKQRPEQGLEWIGWIDPENGNSEYAPRFQGKATMTADTLSNTAYLQLSSLTSEDTAVYYCNADLHDYWGQGTTLTVSS

[0483] SEQ ID NO: 71; consensus VH amino acid sequence from Figures 4A and 4B of PCT / IB2017 / 052544 EVQLVQSGAEVVKPGALVKISCKASGFNIKDYYLHWVRQRPGQGLEWIGWIDPENGDTVYDPKFQGRVTITADTSNTAYLELGSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0484] SEQ ID NO:72; heavy chain of chimeric 3D6 antibody EVQLQQSGADLVRPGALVKLSCKASGFNIKDYYLHWVRQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLGSLTSEDTAVYFCSTLDFWGQGTTLTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0485] SEQ ID NO:73; light chain of chimeric 3D6 antibody DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0486] SEQ ID NO: 74: Amino acid sequence of heavy chain variable structure model receptor accession number 5MYX-VH_mSt EVQLQQSGAELVRPGSSVKISCKASGYIFNNYWINWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAREGYIVYWGQGTLVTVSA

[0487] SEQ ID NO: 75; Amino acid sequence of heavy chain variable receptor accession number 2RCS-VH_huFrwk QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCASYYGIYWGQGTTLTVSS

[0488] SEQ ID NO: 76: Amino acid sequence of the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0489] SEQ ID NO: 77: Amino acid sequence of the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLELSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0490] SEQ ID NO: 78: Amino acid sequence of the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRATITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0491] SEQ ID NO: 79; Amino acid sequence of the heavy chain variable region hu3D6VHvb4 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0492] SEQ ID NO: 80: Amino acid sequence of the heavy chain variable region hu3D6VHvb5 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0493] SEQ ID NO: 81; Amino acid sequence of light chain variable structure model accession number 5MYX-VL_mSt DVVLTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYVVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIK

[0494] SEQ ID NO: 82; Amino acid sequence of light chain variable receptor accession number ARX71335-VL_huFrwk DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCEQGTHFPLTFGAGTKLELK

[0495] SEQ ID NO: 83: Amino acid sequence of the light chain variable region hu3D6VLvb1 of the humanized 3D6 antibody DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCWQGTHFPYTFGAGTKLELKR

[0496] SEQ ID NO: 84: Amino acid sequence of the light chain variable region hu3D6VLvb2 of the humanized 3D6 antibody DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGAGTKLEIKR

[0497] SEQ ID NO: 85: Amino acid sequence of the light chain variable region hu3D6VLvb3 of the humanized 3D6 antibody DVVMTQSPLSLSVTLGEPASISCRSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR

[0498] SEQ ID NO:86; Amino acid sequence of another Kabat-Chothia combination CDR-H1 of humanized 3D6 antibody (similar to hu3D6VHvb4 and hu3D6VHvb5) GFTIKDYYLH

[0499] SEQ ID NO:87; Amino acid sequence of another Kabat CDR-H2 of humanized 3D6 antibody (similar to hu3D6VHvb3 and hu3D6VHvb4) WIDPENGDTIYDPKFQG

[0500] SEQ ID NO:88; Amino acid sequence of another Kabat CDR-H2 of humanized 3D6 antibody (similar to hu3D6VHvb5) WIDPEDGETIYDPKFQG

[0501] SEQ ID NO:89; Amino acid sequence of another Kabat CDR-L1 of humanized 3D6 antibody (similar to hu3D6VLvb3) RSSQSLLDSDGKTYLN

[0502] SEQ ID NO: 90: Amino acid sequence of the heavy chain variable region hu3D6VHvb6 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYFCSTLDFWGQGTLVTVSS

[0503] SEQ ID NO: 91: Amino acid sequence of the heavy chain variable region hu3D6VHvb7 of the humanized 3D6 antibody EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0504] SEQ ID NO:92; Amino acid sequence of another Kabat CDR-H2 of humanized 3D6 antibody (similar to hu3D6VHvb6 and hu3D6VHvb7) WIDPEDGETVYDPKFQG

[0505] SEQ ID NO:93; Nucleic acid sequence encoding the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody CAAGTGCAGCTGCAGCAGAGCGGGGCAGAATTGGTCAAGCCCGGAGCGTCAGTGAAGCTGAGCTGCACCGCCTCCGGCTTCAACATCAAAGACTACTATCTTCACTGGGTCAAGCAACGGCCTGAACAGGGCCTGGAGTGGATTGGTTGGATCGACCCAGAAAACGGCGACACCGTGTACGATCCGAAGTTTCAGGGGAAGGCCACCATC ACTGCTGATACGTCCTCGAACACCGCCTACCTCCAACTGAGCTCCCTGACTTCGGAGGACACTGCCGTGTACTTCTGTTCCACCCTGGACTTCTGGGACAGGGAACTACCCTCACCGTGTCCTCGGCCAGACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0506] SEQ ID NO: 94; Nucleic acid sequence encoding the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody GAAGTGCAGCTCGTGCAGTCCGGTGCCGAAGTCGTGAAACCGGGAGCCAGCGTGAAGATTAGCTGCAAGGCCTCAGGGTTCAACATCAAGGACTATTACCTTCACTGGGTCAGACAGCGGCCTGGAAAGGGCTTGGAGTGGATCGGATGGATTGACCCCGAGAACGGCGACACCGTGTACGATCCGAAGTTTCAGGGCCGCGCAACCATCACTGCTGACACCTCCACCGATACCGCGTACCTGGAACTCTCGAGCCTGACTTCCGAGGATACGGCCGTGTACTTCTGTTCCACCCTGGACTTCTGGGGACAAGGGACTCTGGTCACCGTGTCCTCGGCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0507] SEQ ID NO: 95; Nucleic acid sequence encoding the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody GAGGTGCAACTGGTGCAGTCCGGAGCCGAAGTCGTGAAGCCGGGAGCCACCGTGAAGATTTCGTGCAAAGCGTCAGGGTTTAACATCAAGGACTACTATCTGCACTGGGTCCGCCAGAGGCCCGGGAAGGGCCTCGAGTGGATCGGTTGGATCGACCCTGAAAACGGCGACACCATCTACGATCCAAAGTTCCAGGGCAGAGCCACTATTACCGCTGACACGAGCACCGATACTGCATACATGGAATTGTCCTCCCTGCGGTCCGAGGATACTGCCGTGTACTACTGTAGCACCCTGGACTTCTGGGGACAGGGAACCCTTGTGACCGTGTCGTCCGCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0508] SEQ ID NO:96; Nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb4 GAGGTGCAGCTCGTGCAGTCCGGTGCTGAAGTCGTGAAGCCCGGCGCAACTGTGAAGATTAGCTGCAAGGCCTCAGGGTTCACGATCAAGGACTACTATCTGCACTGGGTCCGCCAACGGCCAGGAAAGGGACTGGAGTGGATCGGATGGATCGATCCTGAAAACGGCGACACCATCTACGACCCGAAATTTCAGGGGAGAGTGACCATT ACCGCCGATACCTCCACCGACACTGCGTACATGGAACTGTCCAGCCTTCGGTCCGAGGACACCGCCGTGTACTACTGTTCGACCCTGGATTTCTGGGGACAGGGCACTCTCGTGACTGTGTCGTCCGCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0509] SEQ ID NO: 97; Nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb5 GAAGTGCAACTGGTGCAGTCCGGCGCAGAAGTCGTGAAGCCTGGAGCCACCGTGAAGATCAGCTGCAAGGCCTCCGGCTTCACCATCAAAGACTACTACTTGCACTGGGTCAGACAGCGCCCAGGAAAGGGTCTGGAATGGATTGGATGGATTGACCCCGAGGACGGGGAGACTATCTACGATCCGAAGTTTCAGGGCCGGGTCACCATCACGGCTGATACCTCGACCGACACTGCGTACATGGAACTTTCCTCGCTGCGGTCCGAGGACACCGCCGTGTATTACTGTTCCACCCTGGATTTCTGGGGACAGGGGACTCTCGTGACTGTGTCAAGCGCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0510] Accession No. 98; Nucleic acid sequence encoding the heavy chain variable region hu3D6VHvb6 of the humanized 3D6 antibody GAAGTGCAACTGGTGCAGTCCGGCGCAGAAGTCGTGAAGCCTGGAGCCACCGTGAAGATCAGCTGCAAGGCCTCCGGCTTCACCATCAAAGACTACTACTTGCACTGGGTCAGACAGCGCCCAGGAAAGGGTCTGGAATGGATTGGATGGATTGACCCCGAGGACGGGGAGACTGTGTACGATCCGAAGTTTCAGGGCCGGGTCACCATCACGGCTGATACCTCGACCGACACTGCGTACATGGAACTTTCCTCGCTGCGGTCCGAGGACACCGCCGTGTATTTCTGTTCCACCCTGGATTTCTGGGGACAGGGGACTCTCGTGACTGTGTCAAGCGCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0511] SEQ ID NO:99; Nucleic acid sequence encoding the heavy chain variable region of the humanized 3D6 antibody, hu3D6VHvb7 GAAGTGCAACTGGTGCAGTCCGGCGCAGAAGTCGTGAAGCCTGGAGCCACCGTGAAGATCAGCTGCAAGGCCTCCGGCTTCACCATCAAAGACTACTACTTGCACTGGGTCAGACAGCGCCCAGAAAGGGTCTGGAATGGATTGGATGGATTGACCCCGAGGACGGGGAGACTGTGTACGATCCGAAGTTTCAGGGCCGGGTCACCATC ACGGCTGATACCTCGACCGACACTGCGTACATGGAACTTTCCTCGCTGCGGTCCGAGGACACCGCCGTGTATTACTGTTCCACCCTGGATTTCTGGGACAGGGGACTCTCGTGACTGTGTCAAGCGCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCG

[0512] SEQ ID NO:100; Nucleic acid sequence encoding the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb1 GATGTCGTGATGACCCAGACGCCGCTGACCCTGTCCGTGACTATCGGCCAGCCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTGGACTCCGACGGAAAGACCTACCTGAACTGGCTGTTGCAACGGCCGGGACAGTCACCCAAGCGCCTCATCTATCTGGTG TCCAAGCTCGACTCGGGAGTGCCTGATAGGTTTTCGGGATCCGGCAGCGGGACCGACTTCACCCTGAAAATCTCAAGAGTGGAAGCCGAGGACCTTGGTGTCCATTACTGTTGGCAGGGTACCCACTTCCCATACACTTTCGGGGCCGGCACTAAGCTCGAACTGAAG

[0513] SEQ ID NO:101; Nucleic acid sequence encoding the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb2 GATGTCGTGATGACCCAGTCGCCGCTGTCCCTGTCCGTGACCCTGGGACAGCCAGCCTCCATTAGCTGCAAGAGCAGCCAGTCCTTGCTGGACTCAGACGGAAAGACCTATCTGAACTGGCTGCTGCAAAGGCCCGGCCAGTCCCCGAAGAGACTCATCTACCTCGTG TCGAAGCTGGACTCCGGCGTGCCTGATCGCTTCTCGGGTTCCGGGTCTGGAACTGACTTCACCCTCAAAAATCTCACGGGTCGAAGCCGAGGACGTGGGCGTGTACTACTGTTGGCAGGGTACCCACTTTCCCTACACTTTCGGGGCGGGAACTAAGCTTGAGATCAAG

[0514] SEQ ID NO:102; Nucleic acid sequence encoding the light chain variable region of the humanized 3D6 antibody, hu3D6VLvb3 GATGTCGTGATGACCCAGAGCCCCCTGTCCCTGAGCGTGACTCTGGGGGAACCGGCCAGCATTTCATGCCGGTCCTCACAATCGCTGCTCGACTCCGACGGAAAGACCTATTTGAACTGGCTGCAGCAAAGACCAGGACAGTCCCCTCGCCGGCTCATCTACCTGGTG TCCAAGCTTGACTCGGGCGTGCCGGATAGGTTCTCCGGGTCCGGAAGCGGCACCGACTTCACTCTGAAAATCTCGCGCGTGGAAGCCGAGGACGTGGGAGTCTACTACTGTTGGCAGGGTACCCACTTCCCCTACACGTTTGGCCAGGGTACCAAGCTCGAGATCAAG

[0515] SEQ ID NO: 103: Representative amino acid sequence of an IgG1 heavy chain constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0516] SEQ ID NO:104: Representative amino acid sequence of a kappa light chain constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0517] SEQ ID NO: 105; a nucleic acid sequence encoding a representative IgG1 heavy chain constant region GCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCGTGAAGGACTACTTCCCCGAGCCGGTCACCGTGTCCTGGAACTCGGGAGCCCTGACCTCCGGAGTGCATACTTTCCCTGCGGTGCTGCAGTCCTCCGGGCTCTACTCGCTGTCAAGCGTGGTCACCGTCCCGAGCTCATCCCTGGGTACTCAGACCTACATTTGCAACGTGAACCACAAACCTTCCAACACCAAGGTCGACAAGAAAGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCCCCGTGTCCCGCCCCTGAGCTGCTGGGCGGCCCCAGCGTGTTCCTCTTCCCGCCTAAGCCGAAGGACACTCTGATGATCTCGAGAACCCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGATCCGGAAGTGAAGTTCAATTGGTACGTGGACGGAGTGGAAGTCCATAACGCCAAGACCAAGCCCCGCGAGGAACAGTACAACTCAACTTACCGGGTGGTGTCAGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTCTCCAACAAGGCGCTGCCGGCCCCCATTGAAAAGACCATCAGCAAGGCTAAGGGCCAGCCCCGGGAACCACAGGTCTACACCTTGCCCCCTTCCCGGGAGGAAATGACCAAGAACCAAGTGTCGCTGACGTGCCTGGTCAAGGGCTTTTATCCATCTGACATCGCCGTGGAGTGGGAAAGCAACGGCCAGCCGGAAAACAACTACAAGACTACCCCGCCTGTGCTGGACTCCGACGGCTCGTTCTTCCTGTATTCCAAGCTCACCGTGGATAAGTCCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCATGAGGCCCTGCACAACCACTACACTCAGAAATCACTGTCCCTTTCCCCCGGAAAGTAA

[0518] SEQ ID NO:106; A nucleic acid sequence encoding a representative kappa light chain constant region CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA

Claims

1. a mature heavy chain variable region comprising a CDR-H1 comprising SEQ ID NO:8, a CDR-H2 comprising SEQ ID NO:9, and a CDR-H3 comprising the sequence of LDF, except that Kabat position H28 may be occupied by N or T, Kabat position H54 may be occupied by N or D, Kabat position H56 may be occupied by D or E, and Kabat position H58 is occupied by V or I; a mature light chain variable region comprising CDR-L1 comprising SEQ ID NO: 12, CDR-L2 comprising SEQ ID NO: 13, and CDR-L3 comprising SEQ ID NO: 14, except that Kabat position L24 may be occupied by K or R; wherein at least one of the following positions is occupied by amino acids as specified: Kabat position H1 is occupied by a Q, Kabat position H5 is occupied by a Q, Kabat position H11 is occupied by an L, Kabat position H20 is occupied by an L, Kabat position H23 is occupied by a T, Kabat position H38 is occupied by a K, Kabat position H75 is occupied by an S, Kabat position H56 is occupied by an E, Kabat position H58 is occupied by an I, Kabat position L10 is occupied by a T, Kabat position L17 is occupied by an E, Kabat position L24 is occupied by an R, Kabat position L37 is occupied by a Q, Kabat position L83 is occupied by an L, Kabat position L86 is occupied by an H, Kabat position L100 is occupied by an A or a Q, Kabat position L106 is occupied by an L, An antibody or an antigen-binding antibody fragment.

2. 2. The antibody or antigen-binding antibody fragment of claim 1 which is a humanized antibody, a humanized antigen-binding antibody fragment, a veneered antibody, a veneered antigen-binding antibody fragment, a chimeric antibody, or a chimeric antigen-binding antibody fragment.

3. 3. The antibody or antigen-binding antibody fragment of claim 2, which is a humanized antibody or humanized antigen-binding antibody fragment comprising a humanized mature heavy chain variable region and a humanized mature light chain variable region.

4. At least one of the following positions is occupied by an amino acid as specified: Kabat position H1 is occupied by Q, Kabat position H5 is occupied by Q or V, Kabat position H11 is occupied by L or V, Kabat position H17 is occupied by S or T, Kabat position H20 is occupied by L or I, Kabat position H23 is occupied by T or K, Kabat position H28 is occupied by N or T, Kabat position H38 is occupied by K or R, Kabat position H42 is occupied by E or G, Kabat position H43 is occupied by Q or K, Kabat position H54 is occupied by N or D, Kabat position H56 is occupied by D or E, Kabat position H58 is occupied by V or I, Kabat position H66 is occupied by K or R, Kabat position H67 is occupied by A or V, Kabat position H75 is occupied by S or T, Kabat position H76 is occupied by N or D, Kabat position H80 is occupied by L or M, Kabat position H81 is occupied by Q or E, Kabat position H83 is occupied by T or R, Kabat position H91 is occupied by F or Y, Kabat position H93 is occupied by S, Kabat position H94 is occupied by T, Kabat position H108 is occupied by T or L, Kabat position 109 is occupied by L or V, The antibody or antigen-binding antibody fragment of claim 3.

5. 5. The antibody or antigen-binding antibody fragment of claim 4, wherein Kabat positions H1, H5, H11, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and H109 are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T, L, and V, respectively.

6. At least one of the following positions is occupied by an amino acid as specified: Kabat position L7 is occupied by T or S, Kabat position L10 is occupied by T or S, Kabat position L15 is occupied by I or L, Kabat position L17 is occupied by Q or E, Kabat position L24 is occupied by K or R, Kabat position L37 is occupied by L or Q, Kabat position L45 is occupied by K or R, Kabat position L83 is occupied by L or V, Kabat position L86 is occupied by H or Y, Kabat position L100 is occupied by A or Q, Kabat position L106 is occupied by L or I, The antibody or antigen-binding antibody fragment of claim 3.

7. 7. The antibody or antigen-binding antibody fragment of claim 6, wherein Kabat positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and I, respectively.

8. 7. The antibody or antigen-binding antibody fragment of claim 6, wherein Kabat positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100, and L106 are occupied by S, S, L, E, R, Q, R, V, Y, Q, and I, respectively.

9. the humanized mature heavy chain variable region comprises SEQ ID NO:76, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:76, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:76, and the humanized mature light chain variable region comprises SEQ ID NO:85; the humanized mature heavy chain variable region comprises SEQ ID NO:77, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:77, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:77, and the humanized mature light chain variable region comprises SEQ ID NO:85; the humanized mature heavy chain variable region comprises SEQ ID NO:78, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:78, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:78, and the humanized mature light chain variable region comprises SEQ ID NO:85; the humanized mature heavy chain variable region comprises SEQ ID NO:79, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:79, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:79, and the humanized mature light chain variable region comprises SEQ ID NO:85; the humanized mature heavy chain variable region comprises SEQ ID NO:80, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:80, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:80, and the humanized mature light chain variable region comprises SEQ ID NO:85; the humanized mature heavy chain variable region comprises SEQ ID NO:90, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:90, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:90 and the humanized mature light chain variable region comprises SEQ ID NO:85; the humanized mature heavy chain variable region comprises SEQ ID NO:91, and the humanized mature light chain variable region comprises SEQ ID NO:83; the humanized mature heavy chain variable region comprises SEQ ID NO:91, and the humanized mature light chain variable region comprises SEQ ID NO:84; the humanized mature heavy chain variable region comprises SEQ ID NO:91, and the humanized mature light chain variable region comprises SEQ ID NO:85; The antibody or antigen-binding antibody fragment of claim 3.

10. The antibody or antigen-binding antibody fragment of any one of claims 1 to 9 which is an antibody.

11. The antibody of any one of claims 1 to 9, which is an antigen-binding antibody fragment.

12. The antibody or antigen-binding antibody fragment of claim 10, wherein the antibody has an isotype of human IgG1.

13. 10. The antibody or antigen-binding antibody fragment of any one of claims 1 to 9, wherein the mature light chain variable region is fused to a light chain constant region and the mature heavy chain variable region is fused to a heavy chain constant region.

14. The antibody or antigen-binding antibody fragment of claim 13, wherein the heavy chain constant region is a variant of a native human heavy chain constant region that has reduced binding to Fcγ receptors compared to the native human heavy chain constant region.

15. 15. The antibody or antigen-binding antibody fragment of claim 14, wherein the human heavy chain constant region is of the human IgG1 isotype.

16. The antibody or antigen-binding antibody fragment of claim 13, wherein the mature heavy chain variable region is fused to a heavy chain constant region having the sequence of SEQ ID NO: 103, with or without a C-terminal lysine, and / or the mature light chain variable region is fused to a light chain constant region having the sequence of SEQ ID NO:

104.

17. The antibody or antigen-binding antibody fragment of claim 13, having at least one mutation in the heavy chain constant region.

18. The antibody of claim 17, wherein the at least one mutation in the heavy chain constant region reduces binding or activation of complement by the heavy chain constant region.

19. The antibody or antigen-binding antibody fragment of claim 10, wherein the antibody has a human IgG2 or IgG4 isotype.

20. A pharmaceutical composition comprising an antibody or antigen-binding antibody fragment as defined in any one of claims 1 to 19 and a pharma- ceutically acceptable carrier.

21. A nucleic acid encoding the heavy and light chains of an antibody according to any one of claims 1 to 10 and 12 to 19.

22. A vector comprising the nucleic acid of claim 21.

23. 23. A method for expressing an antibody in a mammalian cell, comprising introducing into said mammalian cell in vitro the vector described in claim 22, whereby said antibody is expressed.

24. A first and a second vector comprising nucleic acid encoding the heavy and light chains of the antibody according to any one of claims 1 to 10 and 12 to 19, each operably linked to one or more regulatory sequences for effecting expression of the antibody in a mammalian cell.

25. 25. A method for expressing an antibody in a mammalian cell, comprising introducing into said mammalian cell in vitro the vector described in claim 24, whereby said antibody is expressed.

26. 3. A method for producing a humanized, chimeric or veneered antibody according to claim 2, comprising: (a) culturing in a cell culture medium a cell transformed with nucleic acid encoding the heavy and light chains of the humanized, chimeric, or veneered antibody, such that the cell secretes the humanized, chimeric, or veneered antibody; and (b) purifying the humanized, chimeric, or veneered antibody from the cell culture medium. A method comprising:

27. A method for producing a cell line producing the humanized, chimeric or veneered antibody of claim 2, comprising the steps of: (a) introducing into a cell in vitro a vector encoding the heavy and light chains of the humanized, chimeric, or veneered antibody and a selectable marker; (b) growing the cells under conditions that select for cells having increased copy numbers of the vector; (c) isolating single cells from the selected cells; and (d) storing the cloned cells from the single cells selected based on the yield of said humanized, chimeric, or veneered antibody. A method comprising:

28. 20. Use of the antibody or antigen-binding antibody fragment of any one of claims 1 to 19 in the manufacture of a medicament for inhibiting or reducing tau aggregation in a subject having or at risk of developing tau-mediated amyloidosis.

29. 20. Use of the antibody or antigen-binding antibody fragment of any one of claims 1 to 19 in the manufacture of a medicament for treating or preventing a tau-related disease in a subject.

30. 30. The use of claim 29, wherein the tau-related disease is 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

31. 20. An in vitro method for detecting tau protein deposits in a subject having or at risk for a disease associated with tau aggregation or deposition, comprising detecting an antibody or antigen-binding antibody fragment of any one of claims 1 to 19 bound to tau in a biological sample obtained from the subject.

32. 32. The in vitro method of claim 31, wherein the disease associated with tau aggregation or deposition is 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-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

Images