Tau-recognizing antibody

Abstract

To provide antibodies that specifically bind tau and methods for treating or preventing tau-associated diseases.SOLUTION: Provided is an antibody or antigen-binding antibody fragment specifically binding to human tau, comprising: a mature heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:8, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising the amino acid sequence LDF; and a mature light chain variable region comprising CDR-L1 comprising SEQ ID NO:12, CDR-L2 comprising SEQ ID NO:168, and CDR-L3 comprising SEQ ID NO:14.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 813,126, filed March 3, 2019, U.S. Provisional Patent Application No. 62 / 813,137, filed March 3, 2019, and U.S. Provisional Patent Application No. 62 / 838,159, filed April 24, 2019. Each of these provisional patents is incorporated in its entirety by reference in this application.

[0002] Reference to array list This application was prepared on February 24, 2020, and includes an electronic sequence list filenamed 536323WO_ST25.TXT, containing 168,875 bytes. This sequence list is incorporated in its entirety by reference in this application. [Background technology]

[0003] Tau is a well-known human protein that can exist in a phosphorylated form (see, for example, 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 microtubule stabilization, particularly in the central nervous system. Total tau (t-tau, i.e., phosphorylated and unphosphorylated forms) and phosphorylated tau (phospho-tau) (p-tau, i.e., phosphorylated tau) are released by the brain in response to nerve damage and neurodegeneration, and have been reported to be at increased levels 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 a major component of neurofibrillary hierarchies and, along with plaques, is a prominent feature of Alzheimer's disease. These hierarchies consist of abnormal fibrils, 10 nm in diameter, that form pairs spirally wound with a regular 80 nm period. Tau within neurofibrillary hierarchies is abnormally phosphorylated (hyperphosphorylated) with phosphate groups bound to specific sites on the molecule. The strong involvement of neurofibrillary hierarchies in Alzheimer's disease is found in entorhinal cortical layer II neurons, the CA1 and hippocampal regions of the hippocampus, the amygdala, and deeper layers of the neocortex (layers III, V, and surface VI). Hyperphosphorylated tau has also been reported to interfere with microtubule assembly, which may promote the breakdown of neural networks.

[0005] Tau inclusions are a characteristic neuropathological feature of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and Pick's disease. [Overview of the project]

[0006] In one embodiment, the present invention provides an isolated antibody that specifically binds to human tau, comprising a mature heavy chain variable region that is at least 90% identical to SEQ ID NO: 18, comprising CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9 or SEQ ID NO: 149, and CDR-H3 comprising SEQ ID NO: 10, and CDR-L2 comprising SEQ ID NO: 150, 151, 153, 156, 158, 159, 160, 163, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 174, and CDR-L3 comprising SEQ ID NO: 14, and a mature light chain variable region that is at least 90% identical to SEQ ID NO: 122.

[0007] In some antibodies, at least one of positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81, and H91 may be occupied by V, K, T, A, R, Q, I, R, A, D, L, Q, and F, respectively, and at least one of positions L2, L12, L15, L37, L39, L45, L60, and L100 may be occupied by V, P, L, Q, R, R, D, and Q, respectively.

[0008] In some antibodies, CDR-L2 includes SEQ ID NOs: 150, 151, 163, 167, 168, or 169. In some antibodies, the heavy chain variable region includes SEQ ID NO: 18, and the light chain variable region includes SEQ ID NOs: 110, 121, 122, or 123.

[0009] In some antibodies, the light chain variable region includes SEQ ID NO: 110. In some antibodies, the light chain variable region includes SEQ ID NO: 121. In some antibodies, the light chain variable region includes SEQ ID NO: 122. In some antibodies, the light chain variable region includes SEQ ID NO: 123.

[0010] In some antibodies, the heavy chain variable region contains SEQ ID NO: 146, and the light chain variable region contains SEQ ID NO: 94 or 122. In some antibodies, the light chain variable region contains SEQ ID NO: 94. In some antibodies, the light chain variable region contains SEQ ID NO: 122.

[0011] In some antibodies, the heavy chain variable region includes SEQ ID NO: 18 or 146, and the light chain variable region includes SEQ ID NO: 122.

[0012] In another embodiment, the present invention includes a mature heavy chain variable region containing CDR H1, H2, and H3, each containing Sequence IDs 8, 9, and 10, 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, position H58 may be occupied by V or I, and position H60 may be indicated by D or E; and a CDR containing Sequence IDs 12, 13, and 14, each containing Sequence IDs 12, 13, and 14, except that position L24 may be occupied by K or R, position L50 may be occupied by L, E, D, G, or V, position L52 may be occupied by S or G, and position L54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S. The present invention provides an antibody that specifically binds to human tau, comprising a mature light chain variable region including L1, L2, and L3, wherein at least one of the following positions is occupied by a specified amino acid: H1 is occupied by Q, H5 by Q, H11 by L, H20 by L, H23 by T, H38 by K, H75 by S, H56 by E, H58 by I, H60 by E, H82 by V, and L10 by T. L17 is occupied by E, L24 by R, L37 by Q, L47 by G, N, D, E, P, T, S, or A, L48 by G or D, L49 by E, L50 by E, D, G, or V, L52 by G, L54 by D, G, N, E, Q, K, R, T, V, or S, L83 by L, L86 by H, L100 by Q, and L106 by L.

[0013] In another embodiment, the present invention provides an isolated monoclonal antibody conjugating human tau, comprising three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein position H27 may be occupied by F or Y, position H28 may be occupied by N or T, position H29 may be occupied by I or F, position H30 may be occupied by K or T, position H51 may be occupied by I or V, position H54 may be occupied by N or D, position H60 may be occupied by D, A or E, position H61 may be occupied by P or E, position H102 may be occupied by F or Y, position L50 may be occupied by L, E, D, G or V, and position L52 may be occupied by S or G. Except that position L54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S, 3D6 is a mouse antibody characterized by a heavy chain variable region having an amino acid sequence including SEQ ID NO: 7 and a light chain variable region having an amino acid sequence including SEQ ID NO: 11, wherein at least one of the following positions is occupied by a specified amino acid: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L100 is occupied by Q, H60 is occupied by E, and H82 is occupied by V.

[0014] In some antibodies, CDR-H1 has an amino acid sequence containing SEQ ID NO: 86. In some antibodies, CDR-H2 has an amino acid sequence containing SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 92, or SEQ ID NO: 149. In some antibodies, CDR-L2 has an amino acid sequence containing any of SEQ ID NOs: 150-175. In some antibodies, CDR-L1 has an amino acid sequence containing SEQ ID NO: 89. In some antibodies, CDR-H1 has an amino acid sequence containing SEQ ID NO: 86, and CDR-H2 has an amino acid sequence containing SEQ ID NO: 87. In some antibodies, CDR-H1 has an amino acid sequence containing SEQ ID NO: 86, and CDR-H2 has an amino acid sequence containing SEQ ID NO: 88. In some antibodies, CDR-H1 has an amino acid sequence containing SEQ ID NO: 86, and CDR-H2 has an amino acid sequence containing SEQ ID NO: 92. In some antibodies, CDR-H1 has an amino acid sequence containing SEQ ID NO: 42, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60. In some antibodies, CDR-H2 has an amino acid sequence containing SEQ ID NOs. 43, 61, 62, 63, 64, or 149. In some antibodies, CDR-H3 has an amino acid sequence containing SEQ ID NOs. 65. In some antibodies, CDR-L2 has an amino acid sequence containing any of SEQ ID NOs. 150-175.

[0015] Some antibodies are humanized antibodies, veneered antibodies, or chimeric antibodies.

[0016] Some antibodies include a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to one of SEQ ID NOs. 76-80 and SEQ ID NOs. 90-91, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to one of SEQ ID NOs. 83-85.

[0017] Some antibodies include a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to one of SEQ ID NOs. 18 and SEQ ID NOs. 146-148, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to one of SEQ ID NOs. 93-145.

[0018] In some antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H93 is occupied by S, and H94 is occupied by T. In some antibodies, positions H93 and H94 are occupied by S and T, respectively. In some antibodies, position H91 in the VH region is occupied by F.

[0019] In some 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 by V, H11 by V, H20 by I, H23 by K, H38 by R, H42 by G, H43 by K, H66 by R, H75 by T, H76 by D, H81 by E, H108 by L, and H109 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.

[0020] In some antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H17 is occupied by T, H80 by M, and H83 by R. In some antibodies, positions H17, H80, and H83 in the VH region are occupied by T, M, and R, respectively.

[0021] In some antibodies, position H58 in the VH region is occupied by I.

[0022] In some antibodies, at least one of the following positions in the VH region is occupied by a 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.

[0023] In some antibodies, at least one of the following positions in the VH region is occupied by a 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.

[0024] In some 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 by Q or V, H11 by L or V, H17 by S or T, H20 by L or I, H23 by T or K, H28 by N or T, H38 by K or R, H42 by E or G, H43 by Q or K, H54 by N or D, H56 by D or E, H5 8 is occupied by V or I, H60 by D or E, H66 by K or R, H67 by A or V, H75 by S or T, H76 by N or D, H80 by L or M, H81 by Q or E, H82c by L or V, H83 by T or R, H91 by F or Y, H93 by S, H94 by T, H108 by T or L, and H109 by L or V.

[0025] In some antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H10 is occupied by E or D, H12 by K or V, H13 by K or R, H17 by T, L or S, H24 by V or A, H27 by F or Y, H28 by N or T, H29 by I or F, H30 by K or T, H38 by Q or R, H40 by A or R, H42 by G or E, H43 by K or Q, H48 by M or I, and H51 by V H54 is occupied by I, H60 by D, A, or E, H61 by P or E, H66 by R or K, H67 by V or A, H76 by D or N, H80 by M or L, H81 by E or Q, H82 by S or G, H82c by L or V, H83 by T or R, H91 by Y or F, H93 by A or S, H102 by F or Y, H108 by T or L, and H109 by L or V.

[0026] In some antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively.

[0027] 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 by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T, L, and V, respectively. 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.

[0028] 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.

[0029] In some antibodies, position H60 is occupied by E. In some antibodies, position H82C is occupied by V. In some antibodies, positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, respectively.

[0030] In some 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 by S, L15 by L, L83 by V, L86 by Y, and L106 by I. In some antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.

[0031] In some 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 by T or S, L15 by I or L, L17 by Q or E, L24 by K or R, L37 by L or Q, L45 by K or R, and L47 by L, G, N, D, E, P, T, S, or A. L48 is occupied by I, G, or D; L49 is occupied by Y or E; L50 is occupied by L, E, D, G, or V; L52 is occupied by S or G; L54 is occupied by L, D, G, N, E, Q, K, R, T, V, or S; 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.

[0032] In some antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L2 is occupied by V or I, L7 by S or T, L12 by P or S, L15 by L or I, L36 by L, L37 by L or Q, L45 by R or K, L47 by L, G, N, D, E, P, T, S, or A, L48 by I, G, or D, L49 by Y or E, L50 by L, E, D, G, or V, L52 by S or G, L54 by L, D, G, N, E, Q, K, R, T, V, L60 by D or S, and L100 by G or Q.

[0033] 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.

[0034] In some antibodies, position L54 is occupied by D. In some antibodies, position L54 is occupied by G. In some antibodies, position L54 is occupied by N. In some antibodies, position L54 is occupied by E. In some antibodies, position L50 is occupied by E. In some antibodies, position L54 is occupied by Q. In some antibodies, position L50 is occupied by D. In some antibodies, position L54 is occupied by K. In some antibodies, position L54 is occupied by R. In some antibodies, position L54 is occupied by T. In some antibodies, position L50 is occupied by G. In some antibodies, position L48 is occupied by G. In some antibodies, position L48 is occupied by D. In some antibodies, position L47 is occupied by G. In some antibodies, position L49 is occupied by E. In some antibodies, position L54 is occupied by V. In some antibodies, position L54 is occupied by S.

[0035] In some antibodies, position L52 is occupied by G. In some antibodies, position L47 is occupied by N. In some antibodies, position L47 is occupied by D. In some antibodies, position L47 is occupied by E. In some antibodies, position L47 is occupied by P. In some antibodies, position L47 is occupied by T. In some antibodies, position L47 is occupied by S. In some antibodies, position L47 is occupied by A. In some antibodies, position L50 is occupied by V.

[0036] In some antibodies, positions L37, L50, and L54 are occupied by Q, G, and R, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, G, and G, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and G, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and R, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and T, respectively. In some antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively. In some antibodies, L37 and L54 are occupied by Q and R, respectively.

[0037] In some antibodies, positions L37 and L54 are occupied by Q and G, respectively. In some antibodies, positions L37 and L54 are occupied by Q and D, respectively. In some antibodies, positions L37 and L50 are occupied by Q and G, respectively. In some antibodies, positions L37 and L50 are occupied by Q and D, respectively. In some antibodies, positions L37 and L54 are occupied by Q and T, respectively. In some antibodies, positions L37 and L52 are occupied by Q and G, respectively. In some antibodies, positions L37 and L54 are occupied by Q and E, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, D, and G, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, D, and R, respectively.

[0038] In some antibodies, positions L37, L50, and L54 are occupied by Q, E, and G, respectively. In some antibodies, positions L37, L50, and L54 are occupied by Q, E, and R, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively. In some antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, R, and Q, respectively. In some antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, D, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, G, and Q, respectively. In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, R, and Q, respectively.

[0039] In some antibodies, positions L37, L50, L54, and L100 are occupied by Q, V, D, and Q, respectively. In some antibodies, position L37 is occupied by Q. In some antibodies, position L100 is occupied by Q.

[0040] In some antibodies, the mature heavy chain variable region has one amino acid sequence from SEQ ID NOs. 76-80 and SEQ ID NOs. 90-91, and the mature light chain variable region has one amino acid sequence from SEQ ID NOs. 83-85. In some antibodies, the mature heavy chain variable region has one amino acid sequence from SEQ ID NOs. 18 and SEQ ID NOs. 146-148, and the mature light chain variable region has one amino acid sequence from SEQ ID NOs. 93-145.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 122. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 123.

[0049] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 146, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 122.

[0050] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 121. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 110.

[0051] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 146, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 94.

[0052] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 103.

[0053] For example, the antibody may be a chimeric antibody, a benilla antibody, or a humanized antibody.

[0054] The antibodies may be fully chimeric antibodies, benya antibodies or humanized antibodies or binding fragments, single-chain antibody Fab fragments, Fab'2 fragments, or single-chain Fv. Some antibodies may have the human IgG1 isotype, while others may have the human IgG2 or IgG4 isotype. Some antibodies have a mature light chain variable region fused to the light chain constant region and a mature heavy chain variable region fused to the heavy chain constant region. The heavy chain constant region of some antibodies is a variant of the native human constant region with reduced binding to the Fcγ receptor compared to the native human constant region.

[0055] In some antibodies, the heavy chain constant region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region fused to the heavy chain constant region has the amino acid sequence of SEQ ID NO: 178. In some antibodies, the heavy chain further contains a single peptide fused to the mature heavy chain and / or light chain variable region. In some antibodies, the heavy chain has the amino acid sequence of SEQ ID NO: 180. In some antibodies, the light chain constant region has the amino acid sequence of SEQ ID NO: 177. In some antibodies, the mature light chain variable region fused to the light chain constant region has the amino acid sequence of SEQ ID NO: 179. In some antibodies, the light chain has the amino acid sequence of SEQ ID NO: 181. In some antibodies, the heavy chain has the amino acid sequence of SEQ ID NO: 178 and the light chain has the amino acid sequence of SEQ ID NO: 179. In some antibodies, the heavy chain has the amino acid sequence of SEQ ID NO: 180 and the light chain has the amino acid sequence of SEQ ID NO: 181.

[0056] Some antibodies may have at least one mutation in the constant region, for example, a mutation that reduces complement binding or activation by the constant region, e.g., a mutation at one or more positions in EU numbering, such as positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331. Some antibodies have alanine at positions 318, 320, and 322. Some antibodies may have a purity of at least 95% w / w. Antibodies can be conjugated into therapeutic agents, cytotoxic agents, cell division arresters, neurotrophic agents, or neuroprotective agents.

[0057] In another embodiment, the present invention provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharmaceutically acceptable carrier.

[0058] In another aspect, the present invention provides nucleic acids encoding the heavy and / or light chains of any of the antibodies disclosed herein, a recombinant expression vector comprising the nucleic acids, and host cells transformed with the recombinant expression vector.

[0059] In some nucleic acids, the heavy chain is encoded by the sequence containing sequence number 182, and the light chain is encoded by the sequence containing sequence number 183.

[0060] Methods for producing such humanized, chimeric, or veneer antibodies, such as humanized, chimeric, or veneer types of 3D6, are also provided. In such a method, cells transformed with nucleic acids encoding the heavy and light chains of the antibody are cultured to secrete the antibody. The antibody can then be purified from the cell medium.

[0061] Cell lines that produce any of the antibodies disclosed herein may be produced by introducing vectors encoding the heavy and light chains of the antibody and a selection marker into cells, growing the cells under conditions of selection for cells with increased copy numbers of the vector, isolating single cells from the selected cells, and banking cells cloned from the single cells selected based on antibody yield.

[0062] In another embodiment, the present invention includes 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 performing the expression of any of the antibodies disclosed herein in mammalian cells. In some vectors, the antibody to be expressed is an scFv or Fab fragment. In some vectors, one or more regulatory sequences include one or more promoters, enhancers, ribosome binding sites, and transcription termination signals. In some vectors, the nucleic acid further encodes a signal peptide fused to the mature heavy chain and light chain variable regions. In some vectors, the nucleic acid is codon-optimized for expression in host cells. In some vectors, one or more regulatory sequences include a eukaryotic promoter. In some vectors, the nucleic acid further encodes a selectable gene.

[0063] In yet another aspect, the present invention provides a method for expressing an antibody in mammalian cells, comprising incorporating the nucleic acids disclosed herein into the genome of a genetically modified animal in which the antibody is expressed.

[0064] In yet another embodiment, the present invention provides first and second vectors, each comprising a nucleic acid encoding a mature heavy chain variable region and a mature light chain variable region, respectively, which are operably linked to one or more regulatory sequences to perform the expression of any of the antibodies disclosed herein, and a host cell comprising the nucleic acid. In some first and second vectors, the nucleic acid further encodes 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 first and second vectors, the heavy chain constant region has the sequence of SEQ ID NO: 176, with or without C-terminal lysine, and the light chain constant region has the sequence of SEQ ID NO: 177.

[0065] In yet another aspect, the present invention provides a method for expressing an antibody in a mammalian cell, comprising incorporating one of the nucleic acids disclosed herein into the genome of a genetically modified animal in which the antibody is expressed.

[0066] Methods for generating antibodies such as humanized, chimeric, or benya antibodies are also provided. In such methods, cells transformed with nucleic acids encoding the heavy and light chains of any of the antibodies disclosed herein are cultured to secrete the antibodies. The antibodies can then be purified from the cell medium.

[0067] A cell line that produces any of the antibodies disclosed herein may be produced by introducing a vector encoding the heavy and light chains of the antibody of claim 1 and a selection marker into cells, growing the cells under conditions of selection for cells having an increased copy number of the vector, isolating single cells from the selected cells, and banking cells cloned from single cells selected based on antibody yield.

[0068] Some cells were amplified under selective conditions to express spontaneously, at least 100 mg / L / 10 6 Cell lines secreting 24-hour cells can be screened. Single cells can be isolated from selected cells. Cells cloned from single cells can then be stored. Single cells can be selected based on desirable characteristics such as antibody yield. Representative cell lines are those expressing 3D6 or a humanized version of 3D6.

[0069] The present invention also provides a method for inhibiting or reducing tau aggregation in subjects at risk of developing tau-mediated amyloidosis, the method comprising administering to the subject an effective dosing regimen of the antibodies disclosed herein, thereby inhibiting or reducing tau aggregation in the subject. Representative antibodies include humanized forms of 3D6.

[0070] Methods for treating or preventing target tau-related diseases are also provided, the methods comprising administering an effective dosing regimen of the antibodies disclosed herein to thereby treat or prevent the disease. Examples of such diseases include Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some ways, tau-related disorders are Alzheimer's disease. In some ways, patients are ApoE4 carriers.

[0071] Furthermore, methods for reducing the abnormal transmission of tau are also provided, the methods comprising administering an effective administration regimen of the antibodies disclosed herein, thereby reducing the transmission of tau.

[0072] Furthermore, a method for inducing tau phagocytosis is also provided, which comprises administering an effective administration regimen of the antibody disclosed herein, thereby inducing tau phagocytosis.

[0073] Furthermore, methods for inhibiting tau aggregation or deposition are also provided, the methods comprising administering an effective dosage regimen of the antibody disclosed herein, thereby inhibiting tau aggregation or deposition.

[0074] Furthermore, a method for suppressing the formation of tau concentrates is also provided, the method comprising administering an effective dosage regimen of the antibody disclosed herein.

[0075] The present invention also provides a method for detecting tau protein deposits in subjects with diseases associated with tau aggregation or deposition, or subjects at risk of such diseases, the method comprising administering an antibody disclosed herein to a subject and detecting the antibody bound to tau in the subject. Examples of such diseases include Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spherical glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spherical 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 creating a hole through the subject's skull. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled by fluorescent, paramagnetic, or radioactive labeling. In some embodiments, the radioactive labeling is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0076] The present invention also provides a method for measuring the efficacy of a treatment for a subject being treated for a disease related to tau aggregation or deposition, the method comprising measuring a first level of tau protein deposition in the subject before treatment by administering the antibody disclosed herein to the subject, measuring a first amount of antibody bound to the subject's tau, administering the treatment to the subject, measuring a second level of tau protein deposition in the subject after treatment by administering the antibody to the subject, and detecting antibody bound to the subject's tau, wherein a decrease in the level of tau protein deposition indicates a positive response to the treatment.

[0077] The present invention also provides a method for measuring the efficacy of a treatment to a subject being treated for a disease associated with tau aggregation or deposition, the method comprising: measuring a first level of tau protein deposition in the subject before treatment by administering the antibody disclosed herein to the subject, and measuring a first amount of antibody bound to the subject's tau, administering the treatment to the subject, measuring a second level of tau protein deposition in the subject after treatment by administering the antibody to the subject, and detecting a second amount of antibody bound to the subject's tau, wherein no change in the level of tau protein deposition or a small increase in tau protein deposition indicates a positive response to the treatment.

[0078] In another aspect, the present invention provides a method for generating an antibody that specifically binds to human tau at an epitope within the motif of formula KXXSXXNX(K / H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194), comprising immunizing an animal with human tau or a fragment thereof to generate the antibody, and screening for one of the generated antibodies that specifically binds within the motif. In another aspect, the present invention provides a method for generating an antibody that specifically binds to a peptide consisting of residues KXXSXXNX(K / H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194), comprising immunizing an animal with human tau or a fragment thereof to generate the antibody, and screening for one of the antibodies that specifically binds to this peptide. In another aspect, the present invention provides a method for generating an antibody that specifically binds to an epitope containing KXXSXXNX(K / H)H (SEQ ID NO: 191), comprising immunizing an animal with tau or a fragment thereof, and screening for an antibody that specifically binds to this epitope.

[0079] In some methods, animals are immunized with 383 amino acid human tau (4R0N). In some methods, human tau contains the P301S mutation. In some methods, human tau is recombinantly N-terminally His-tagged.

[0080] In some methods, screening determines specific binding between an antibody and one or more peptides of 15 amino acids or less, each containing KIGSTENLKH (SEQ ID NO: 188), KCGSKDNIKH (SEQ ID NO: 192), KCGSLGNIHH (SEQ ID NO: 193), or any other consensus motif represented by KXXSXXNX(K / H)H (SEQ ID NO: 191). In some methods, one or more peptides contain KIGSTENLKH (SEQ ID NO: 188), KCGSKDNIKH (SEQ ID NO: 192), or KCGSLGNIHH (SEQ ID NO: 193), each. In some methods, animals are immunized with a tau fragment of 15 amino acids or less containing KXXSXXNX(K / H)H (SEQ ID NO: 191) linked to a carrier. In some methods, the peptide is KIGSTENLKH (SEQ ID NO: 188), KCGSKDNIKH (SEQ ID NO: 192), or KCGSLGNIHH (SEQ ID NO: 193). [Brief explanation of the drawing]

[0081] [Figure 1] The results of experiments designed to map epitopes (single or multiple) conjugated with mouse 3D6 monoclonal antibodies are shown. [Figure 2]This shows a sequence comparison of the heavy chain variable region of the mouse 3D6 antibody (SEQ ID NO: 7) and the heavy chain variable regions of the humanized 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 (SEQ ID NO: 25) and the human receptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO: 75). hu3D6VHvb1 is sequence number 76, hu3D6VHvb2 is sequence number 77, hu3D6VHvb3 is sequence number 78, hu3D6VHvb4 is sequence number 79, hu3D6VHvb5 is sequence number 80, hu3D6VHvb6 is sequence number 90, and hu3D6VHvb7 is sequence number 91. CDRs defined by Kabat / Cotia combinations are shown in bold. [Figure 3] This document shows sequence comparisons of the light chain variable regions of the mouse 3D6 antibody (SEQ ID NO: 11) and the humanized versions 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. CDRs as defined by Kabat are shown in bold. [Figure 4A] The results of ELISA screening for selected mouse monoclonal anti-tau antibodies are shown. [Figure 4B] The results of ELISA screening for selected mouse monoclonal anti-tau antibodies are shown. [Figure 4C] The results of ELISA screening for selected mouse monoclonal anti-tau antibodies are shown. [Figure 5] The binding kinetics of selected mouse monoclonal anti-tau antibodies to recombinant human tau are shown. [Figure 6]The results of a functional blocking assay against a selected mouse monoclonal anti-tau antibody are shown. [Figure 7] The results of a deaggregation assay against selected mouse monoclonal anti-tau antibodies are shown. [Figure 8] We present experimental results showing that 3D6 and 5G8 immunocapture tau derived from human Alzheimer's disease tissue. [Figure 9A] This shows a sequence comparison between the mouse 3D6 heavy chain variable region (SEQ ID NO: 7) and the heavy chain variable regions of humanized 3D6 antibodies (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHv1bA11, h3D6VHvb8, and h3D6VHvb9) and the human germline heavy chain variable region sequence IGHV1-69-2*01 (SEQ ID NO: 25) and the human receptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO: 75). hu3D6VHvb1 is sequence number 76, hu3D6VHvb2 is sequence number 77, hu3D6VHvb3 is sequence number 78, hu3D6VHvb4 is sequence number 79, hu3D6VHvb5 is sequence number 80, hu3D6VHvb6 is sequence number 90, hu3D6VHvb7 is sequence number 91, hu3D6VHv1bA11 is sequence number 18, h3D6VHvb8 is sequence number 146, and h3D6VHvb9 is sequence number 148. Residues identical to those in the heavy chain variable region of mouse 3D6 (sequence number 7) are noted with ".". CDRs defined by the Kabat / Cotia combination are shown in bold. [Figure 9B]This shows a sequence comparison between the mouse 3D6 heavy chain variable region (SEQ ID NO: 7) and the heavy chain variable regions of humanized 3D6 antibodies (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHv1bA11, h3D6VHvb8, and h3D6VHvb9) and the human germline heavy chain variable region sequence IGHV1-69-2*01 (SEQ ID NO: 25) and the human receptor heavy chain variable region sequence 2RCS VH hFrwk (SEQ ID NO: 75). hu3D6VHvb1 is sequence number 76, hu3D6VHvb2 is sequence number 77, hu3D6VHvb3 is sequence number 78, hu3D6VHvb4 is sequence number 79, hu3D6VHvb5 is sequence number 80, hu3D6VHvb6 is sequence number 90, hu3D6VHvb7 is sequence number 91, hu3D6VHv1bA11 is sequence number 18, h3D6VHvb8 is sequence number 146, and h3D6VHvb9 is sequence number 148. Residues identical to those in the heavy chain variable region (sequence number 7) of mouse 3D6 are noted with ".". CDRs defined by Kabat / Cotia combinations are shown in bold. [Figure 10A]3D6 antibodies: hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50D_L54G (Sequence ID 132), hu3D6VLv2 L37Q_S52G_L54D (Sequence ID 124), hu3D6VLv2 L37Q_S52G_L54G (Sequence ID 121), hu3D6VLv2 L37Q_S52G_L54T (Sequence ID 123), hu3D6VLv2 L37Q_S52G_L54R (Sequence ID 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (Sequence ID 141), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2This shows a sequence comparison of the light chain variable region of the humanized L37Q_S52G_L54R_G100Q (SEQ ID NO: 138). Residues identical to those in the light chain variable region of hu3D6VLv2 (SEQ ID NO: 21) are noted with a ".". The CDR as defined by Kabat is shown in bold. [Figure 10B]3D6 antibodies: hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50D_L54G (Sequence ID 132), hu3D6VLv2 L37Q_S52G_L54D (Sequence ID 124), hu3D6VLv2 L37Q_S52G_L54G (Sequence ID 121), hu3D6VLv2 L37Q_S52G_L54T (Sequence ID 123), hu3D6VLv2 L37Q_S52G_L54R (Sequence ID 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (Sequence ID 141), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2This shows a sequence comparison of the light chain variable region of the humanized L37Q_S52G_L54R_G100Q (SEQ ID NO: 138). Residues identical to those in the light chain variable region of hu3D6VLv2 (SEQ ID NO: 21) are noted with a ".". The CDR as defined by Kabat is shown in bold. [Figure 10C]3D6 antibodies: hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50D_L54G (Sequence ID 132), hu3D6VLv2 L37Q_S52G_L54D (Sequence ID 124), hu3D6VLv2 L37Q_S52G_L54G (Sequence ID 121), hu3D6VLv2 L37Q_S52G_L54T (Sequence ID 123), hu3D6VLv2 L37Q_S52G_L54R (Sequence ID 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (Sequence ID 141), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2This shows a sequence comparison of the light chain variable region of the humanized L37Q_S52G_L54R_G100Q (SEQ ID NO: 138). Residues identical to those in the light chain variable region of hu3D6VLv2 (SEQ ID NO: 21) are noted with a ".". The CDR as defined by Kabat is shown in bold. [Figure 10D]3D6 antibodies: hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50D_L54G (Sequence ID 132), hu3D6VLv2 L37Q_S52G_L54D (Sequence ID 124), hu3D6VLv2 L37Q_S52G_L54G (Sequence ID 121), hu3D6VLv2 L37Q_S52G_L54T (Sequence ID 123), hu3D6VLv2 L37Q_S52G_L54R (Sequence ID 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (Sequence ID 141), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2This shows a sequence comparison of the light chain variable region of the humanized L37Q_S52G_L54R_G100Q (SEQ ID NO: 138). Residues identical to those in the light chain variable region of hu3D6VLv2 (SEQ ID NO: 21) are noted with a ".". The CDR as defined by Kabat is shown in bold. [Figure 11] The results of the tau endogenization assay for selected humanized 3D6 variants are shown. [Figure 12A] The results of substitutions mapping microarray experiments are shown. Figure 12A shows a plot of substitution effects, and Figure 12B shows a partial sequence comparison of tau microtubule-binding repeats, highlighting key residues for binding to 3D6. aa255~271 is sequence number 184, aa286~302 is sequence number 185, aa317~333 is sequence number 186, and aa349~365 is sequence number 187. [Figure 12B] The results of substitutions mapping microarray experiments are shown. Figure 12A shows a plot of substitution effects, and Figure 12B shows a partial sequence comparison of tau microtubule-binding repeats, highlighting key residues for binding to 3D6. aa255~271 is sequence number 184, aa286~302 is sequence number 185, aa317~333 is sequence number 186, and aa349~365 is sequence number 187. [Figure 13] This image shows the results of immunohistochemical experiments demonstrating that 3D6 binds to tau in normal tissue (top panel) and Alzheimer's disease tissue (middle and bottom panels). [Figure 14] The results of mass spectrometry experiments evaluating the bond stoichiometry of 3D6 are shown. [Figure 15] This study demonstrates that 3D6 disrupts tau seeding in an in vivo disease model of Alzheimer's disease. [Figure 16] We demonstrate that the 3D6 humanized variants hu3D6VHv1bA11 / hu3D6VLv2 and hu3D6VHv1bA11 / L2-DIM4 block the interaction between tau and heparin. [Figure 17]This study demonstrates that the 3D6 humanized variants hu3D6VHv1bA11 / hu3D6VLv2 and hu3D6VHv1bA11 / L2-DIM4 bind to the fibril form of tau. [Modes for carrying out the invention]

[0082] A brief explanation of arrays Sequence ID 1 shows the amino acid sequence of a human tau isoform (Swiss plot P10636-8).

[0083] Sequence ID 2 shows the amino acid sequence of a human tau isoform (Swiss plot P10636-7).

[0084] Sequence ID 3 shows the amino acid sequences of the human tau isoform (Swissplot P10636-6), (4R0N human tau).

[0085] Sequence ID 4 shows the amino acid sequence of a human tau isoform (Swiss plot P10636-5).

[0086] Sequence ID 5 shows the amino acid sequence of the human tau isoform (Swiss plot P10636-4).

[0087] Sequence ID 6 shows the amino acid sequence of the human tau isoform (Swiss plot P10636-2).

[0088] Sequence ID 7 shows the amino acid sequence of the heavy chain variable region of the mouse 3D6 antibody.

[0089] Sequence ID 8 shows the amino acid sequence of the mouse 3D6 antibody Kabat / Cotia combination CDR-H1.

[0090] Sequence ID 9 shows the amino acid sequence of the mouse 3D6 antibody Kabat-CDR-H2.

[0091] Sequence ID 10 shows the amino acid sequence of the mouse 3D6 antibody Kabat-CDR-H3.

[0092] Sequence ID 11 shows the amino acid sequences of the light chain variable regions of mouse 3D6 antibody and mouse 6A10 antibody.

[0093] Sequence ID 12 shows the amino acid sequences of Kabat-CDR-L1 for mouse 3D6 antibody and mouse 6A10 antibody.

[0094] Sequence ID 13 shows the amino acid sequences of Kabat-CDR-L2 for mouse 3D6 antibody and mouse 6A10 antibody.

[0095] Sequence ID No. 14 shows the amino acid sequences of Kabat-CDR-L3 for mouse 3D6 antibody and mouse 6A10 antibody.

[0096] Sequence ID 15 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1.

[0097] Sequence ID 16 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv2.

[0098] Sequence ID 17 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1b.

[0099] Sequence ID 18 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1bA11.

[0100] Sequence ID 19 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv5.

[0101] Sequence ID 20 shows the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv1.

[0102] Sequence ID 21 shows the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv2.

[0103] Sequence ID 22 shows the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv3.

[0104] Sequence ID 23 shows the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLv4.

[0105] Sequence ID 24 shows the amino acid sequence of heavy chain variable receptor acceptor number BAC01986.1.

[0106] Sequence ID 25 is for the heavy chain variable receptor IMGT, acceptance number IGHV1-69-2. * The amino acid sequence of 01 is shown.

[0107] Sequence ID 26 is for the heavy chain variable receptor IMGT, acceptance number IGKJ1. * The amino acid sequence of 01 is shown.

[0108] Sequence ID 27 is for the light chain variable receptor IMGT with acceptance number IGKV2-30. * The amino acid sequence of O2 is shown.

[0109] Sequence ID 28 is for the light chain variable receptor IMGT, with acceptance number IGKJ2. * The amino acid sequence of 01 is shown.

[0110] Sequence ID 29 shows the amino acid sequence of light chain variable receptor acceptor number AAZ09048.1.

[0111] Sequence ID 30 shows the nucleic acid sequence encoding the heavy chain variable region of the mouse 3D6 antibody.

[0112] Sequence ID 31 shows the nucleic acid sequence encoding the light chain variable region of the mouse 3D6 antibody.

[0113] Sequence ID 32 shows the amino acid sequence of the mouse 3D6 antibody Kabat-CDR-H1.

[0114] Sequence ID 33 shows the amino acid sequence of the mouse 3D6 antibody Kotia CDR-H1.

[0115] Sequence ID 34 shows the amino acid sequence of the mouse 3D6 antibody, Cotia CDR-H2.

[0116] Sequence ID 35 shows the amino acid sequence of the mouse 3D6 antibody AbM CDR-H2.

[0117] Sequence ID 36 shows the amino acid sequence of Contact CDR-L1, a mouse 3D6 antibody.

[0118] Sequence ID 37 shows the amino acid sequence of Contact CDR-L2 of the mouse 3D6 antibody.

[0119] Sequence ID 38 shows the amino acid sequence of Contact CDR-L3 of the mouse 3D6 antibody.

[0120] Sequence ID 39 shows the amino acid sequence of the mouse 3D6 antibody Contact CDR-H1.

[0121] Sequence ID 40 shows the amino acid sequence of the mouse 3D6 antibody Contact CDR-H2.

[0122] Sequence ID 41 shows the amino acid sequence of Contact CDR-H3, a mouse 3D6 antibody.

[0123] Sequence ID 42 shows the amino acid sequence of another Kabat-Cotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3, hu3D6VHv1e, and hu3D6VHv1f).

[0124] Sequence ID 43 shows the amino acid sequence of another Kabat CDR-H2 humanized 3D6 antibody (similar to hu3D6VHv5 and hu3D6VHv1bA11B6H3).

[0125] Sequence ID 44 shows the consensus amino acid sequence in the heavy chain variable region of mouse 3D6 and selected humanized 3D6 antibodies (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) (labeled "Majority" in Figure 2 of PCT / IB2017 / 052544).

[0126] Sequence ID 45 shows the consensus amino acid sequence between the light chain variable regions of mouse 3D6 and a selected humanized 3D6 antibody (labeled "Majority" in Figure 3 of PCT / IB2017 / 052544).

[0127] Sequence ID 46 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1bA11B6G2.

[0128] Sequence ID 47 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1bA11B6H3.

[0129] Sequence ID 48 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1c.

[0130] Sequence ID 49 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1d.

[0131] Sequence ID 50 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1e.

[0132] Sequence ID 51 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv1f.

[0133] Sequence ID 52 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3.

[0134] Sequence ID 53 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3b.

[0135] Sequence ID 54 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv3c.

[0136] Sequence ID 55 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4.

[0137] Sequence ID 56 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4b.

[0138] Sequence ID 57 shows the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHv4c.

[0139] Sequence ID 58 shows the amino acid sequence of another Kabat-Cotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VH1c).

[0140] Sequence ID 59 shows the amino acid sequence of another Kabat-Cotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c).

[0141] Sequence ID 60 shows the amino acid sequence of another Kabat-Cotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv3b and hu3D6VHv4b).

[0142] Sequence ID 61 shows the amino acid sequence of another Kabat-CDR-H2 humanized 3D6 antibody (similar to hu3D6VHv1bA11B6G2).

[0143] Sequence ID 62 shows the amino acid sequence of another Kabat CDR-H2 humanized 3D6 antibody (similar to hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b).

[0144] Sequence ID 63 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c).

[0145] Sequence ID 64 shows the amino acid sequence of another Kabat CDR-H2 humanized 3D6 antibody (similar to hu3D6VHv1e).

[0146] Sequence ID 65 shows the amino acid sequence of another Kabat-CDR-H3 of the humanized 3D6 antibody (similar to hu3D6VHv1f).

[0147] Sequence ID 66 shows the amino acid sequence of the heavy chain variable region of the mouse 6A10 antibody.

[0148] Sequence ID 67 shows the amino acid sequence of the mouse 6A10 antibody Kabat / Cotia combination CDR-H1.

[0149] Sequence ID 68 shows the amino acid sequence of the mouse 6A10 antibody Kabat-CDR-H2.

[0150] Sequence ID 69 shows the amino acid sequence of the mouse 6A10 antibody Kabat-CDR-H3.

[0151] Sequence ID 70 shows the amino acid sequence of the VH region of the mouse antibody (pdb code 1CR9) used as a structural template for heavy chain humanization.

[0152] Sequence ID 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).

[0153] Sequence ID 72 shows the amino acid sequence of the heavy chain of the chimeric 3D6 antibody.

[0154] Sequence ID 73 shows the amino acid sequence of the light chain of the chimeric 3D6 antibody.

[0155] Sequence ID 74 shows the amino acid sequence of heavy chain variable structure model acceptance number 5MYX-VH_mSt.

[0156] Sequence ID 75 shows the amino acid sequence of heavy chain variable receptor acceptor number 2RCS-VH_huFrwk.

[0157] Sequence ID 76 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody.

[0158] Sequence ID 77 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody.

[0159] Sequence ID 78 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody.

[0160] Sequence ID 79 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb4 of the humanized 3D6 antibody.

[0161] Sequence ID 80 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb5 of the humanized 3D6 antibody.

[0162] Sequence ID 81 shows the amino acid sequence of light chain variable structure model acceptance number 5MYX-VL_mSt.

[0163] Sequence ID 82 shows the amino acid sequence of the light chain variable receptor acceptor number ARX71335-VL_huFrwk.

[0164] Sequence ID 83 shows the amino acid sequence of the light chain variable region hu3D6VLvb1 of the humanized 3D6 antibody.

[0165] Sequence ID 84 shows the amino acid sequence of the light chain variable region hu3D6VLvb2 of the humanized 3D6 antibody.

[0166] Sequence ID 85 shows the amino acid sequence of the light chain variable region hu3D6VLvb3 of the humanized 3D6 antibody.

[0167] Sequence ID 86 shows the amino acid sequence of another Kabat-Cotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHvb4 and hu3D6VHvb5).

[0168] Sequence ID 87 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb3 and hu3D6VHvb4).

[0169] Sequence ID 88 shows the amino acid sequence of another Kabat CDR-H2 humanized 3D6 antibody (similar to hu3D6VHvb5).

[0170] Sequence ID 89 shows the amino acid sequence of another Kabat CDR-L1 humanized 3D6 antibody (similar to hu3D6VLvb3).

[0171] Sequence ID 90 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb6 of the humanized 3D6 antibody.

[0172] Sequence ID 91 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb7 of the humanized 3D6 antibody.

[0173] Sequence ID 92 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb6 and hu3D6VHvb7).

[0174] Sequence ID 93 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54D.

[0175] Sequence ID 94 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54G.

[0176] Sequence ID 95 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L45N.

[0177] Sequence ID 96 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54E.

[0178] Sequence ID 97 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L50E.

[0179] Sequence ID 98 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54Q.

[0180] Sequence ID 99 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L50D.

[0181] Sequence ID 100 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54K.

[0182] Sequence ID 101 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54R.

[0183] Sequence ID 102 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54T.

[0184] Sequence ID 103 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L50G.

[0185] Sequence ID 104 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant I48G.

[0186] Sequence ID 105 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant I48D.

[0187] Sequence ID 106 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47G.

[0188] Sequence ID 107 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant Y49E.

[0189] Sequence ID 108 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54V.

[0190] Sequence ID 109 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L54S.

[0191] Sequence ID 110 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant S52G.

[0192] Sequence ID 111 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47N.

[0193] Sequence ID 112 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47D.

[0194] Sequence ID 113 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47E.

[0195] Sequence ID 114 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47P.

[0196] Sequence ID 115 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47T.

[0197] Sequence ID 116 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47S.

[0198] Sequence ID 117 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L47A.

[0199] Sequence ID 118 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L50V.

[0200] Sequence ID 119 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50G_L54R.

[0201] Sequence ID 120 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50G_L54G.

[0202] Sequence ID 121 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G_L54G.

[0203] Sequence ID 122 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G_L54R.

[0204] Sequence ID 123 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G_L54T.

[0205] Sequence ID 124 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G_L54D.

[0206] Sequence ID 125 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L54R.

[0207] Sequence ID 126 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L54G.

[0208] Sequence ID 127 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L54D.

[0209] Sequence ID 128 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50G.

[0210] Sequence ID 129 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50D.

[0211] Sequence ID 130 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L54T.

[0212] Sequence ID 131 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G.

[0213] Sequence ID 132 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50D_L54G.

[0214] Sequence ID 133 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50D_L54R.

[0215] Sequence ID 134 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50E_L54G.

[0216] Sequence ID 135 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50E_L54R.

[0217] Sequence ID 136 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50G_L54R_G100Q.

[0218] Sequence ID 137 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_L50G_L54G_G100Q.

[0219] Sequence ID 138 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G_L54R_G100Q.

[0220] Sequence ID 139 shows the amino acid sequence of the light chain variable region of the hu3D6VLv2 variant L37Q_S52G_L54D_G100Q.

[0221] Sequence ID 140 shows the amino acid sequence of the light chain variable region of the Hu3D6VLv2 variant L37Q_L50D_L54G_G100Q.

[0222] Sequence ID 141 shows the amino acid sequence of the light chain variable region of the Hu3D6VLv2 variant L37Q_L50D_L54R_G100Q.

[0223] Sequence ID 142 shows the amino acid sequence of the light chain variable region of the Hu3D6VLv2 variant L37Q_L50V_L54D_G100Q.

[0224] Sequence ID 143 shows the amino acid sequence of the light chain variable region of the Hu3D6VLv2 variant L37Q.

[0225] Sequence ID 144 shows the amino acid sequence of the light chain variable region of the Hu3D6VLv2 variant G100Q.

[0226] Sequence ID 145 shows the amino acid sequence of the light chain variable region of the Hu3D6VLv2 variant L37Q_L54E.

[0227] Sequence ID 146 shows the amino acid sequence of the heavy chain variable region of the hu3D6VHv1bA11 variant D60E, also known as h3D6VHvb8.

[0228] Sequence ID 147 shows the amino acid sequence of the heavy chain variable region of the hu3D6VHv1bA11 variant L82cV.

[0229] Sequence ID 148 shows the amino acid sequence of the heavy chain variable region of the hu3D6VHv1bA11 variant D60E_L80M_Q81E_L82cV_T83R, also known as h3D6VHvb9.

[0230] Sequence ID 149 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to h3D6VHvb8 and h3D6VHvb9).

[0231] Sequence ID 150 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2L54D and hu3D6VLv2L37Q_L54D).

[0232] Sequence ID 151 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2L54G and hu3D6VLv2L37Q_L54G).

[0233] Sequence number 152 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54N).

[0234] Sequence number 153 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54E and hu3D6VLv2L37Q_L54E).

[0235] Sequence number 154 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50E).

[0236] Sequence number 155 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54Q).

[0237] Sequence number 156 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50D and hu3D6VLv2L37Q_L50D).

[0238] Sequence number 157 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54K).

[0239] Sequence number 158 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54R and hu3D6VLv2L37Q_L54R).

[0240] Sequence number 159 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54T and hu3D6VLv2L37Q_L54T).

[0241] Sequence number 160 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50G and hu3D6VLv2L37Q_L50G).

[0242] Sequence ID 161 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2L54V).

[0243] Sequence ID 162 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2L54S).

[0244] Sequence ID 163 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2S52G and hu3D6VLv2L37Q_S52G).

[0245] Sequence ID 164 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2L50V).

[0246] Sequence ID 165 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50G_L54R and hu3D6VLv2 L37Q_L50G_L54R_G100Q).

[0247] Sequence ID 166 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50G_L54G and hu3D6VLv2 L37Q_L50G_L54G_G100Q).

[0248] Sequence ID 167 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54G).

[0249] Sequence ID 168 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54R and hu3D6VLv2 L37Q_S52G_L54R_G100Q).

[0250] Sequence ID 169 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54T).

[0251] Sequence ID 170 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54D and hu3D6VLv2 L37Q_S52G_L54D_G100Q).

[0252] Sequence ID 171 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50D_L54G and hu3D6VLv2 L37Q_L50D_L54G_G100Q).

[0253] Sequence ID 172 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50D_L54R and hu3D6VLv2 L37Q_L50D_L54R_G100Q).

[0254] Sequence ID 173 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50E_L54G).

[0255] Sequence ID 174 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50E_L54R).

[0256] Sequence ID 175 shows the amino acid sequence of another Kabat CDR-L2 humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50V_L54D_G100Q).

[0257] Sequence ID 176 shows the amino acid sequence of the heavy chain constant region (IgG1: allotype G1m17,1).

[0258] Sequence ID 177 shows the amino acid sequence of the light chain constant region (Kappa).

[0259] Accession number 178 shows the amino acid sequence of the mature heavy chain of the 3D6 humanized variant (hu3D6 VH v1bA11 IgG1 G1m17 allotype).

[0260] Accession number 179 shows the amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6 VL v2 variant L37Q_S52G_L54R, L2-DIM4 kappa).

[0261] Accession number 180 shows the amino acid sequence of the heavy chain of the 3D6 humanized variant having a bovine alpha-lactalbumin signal peptide at the N-terminus (hu3D6 VH v1bA11 IgG1 G1m17 allotype).

[0262] Accession number 181 shows the amino acid sequence of the light chain of the 3D6 humanized variant having a bovine alpha-lactalbumin signal peptide at the N-terminus (hu3D6 VL v2 variant L37Q_S52G_L54R, L2-DIM4 kappa).

[0263] Accession number 182 shows the nucleotide sequence encoding the heavy chain of the 3D6 humanized variant having a bovine alpha-lactalbumin signal peptide at the N-terminus (hu3D6 VH v1bA11 IgG1 G1m17 allotype).

[0264] Accession number 183 shows the nucleotide sequence encoding the light chain of the 3D6 humanized variant having a bovine alpha-lactalbumin signal peptide at the N-terminus (hu3D6 VL v2 variant L37Q_S52G_L54R, L2-DIM4 kappa).

[0265] Accession number 184 shows the amino acid sequence of the region of tau microtubule binding repeat 1 (amino acid residues 255-271 of SEQ ID NO: 1).

[0266] Accession number 185 shows the amino acid sequence of the region of tau microtubule binding repeat 2 (amino acid residues 286-302 of SEQ ID NO: 1).

[0267] Sequence ID 186 shows the amino acid sequence of the tau microtubule-binding repeat 3 region (amino acid residues 317-333 of Sequence ID 1).

[0268] Sequence ID 187 shows the amino acid sequence of the tau microtubule-binding repeat 4 region (amino acid residues 349-365 of Sequence ID 1).

[0269] Sequence ID 188 shows the amino acid sequence of the tau core motif in MBTR1 linked by 3D6.

[0270] Sequence ID 189 shows the amino acid sequence of the N-terminal tau sequence of the tau core motif in MBTR1 bound by 3D6.

[0271] Sequence ID 190 shows the amino acid sequence of the C-terminal tau sequence of the tau core motif in MBTR1 linked by 3D6.

[0272] Sequence ID 191 shows the amino acid sequence of the 3D6 epitope.

[0273] Sequence ID 192 shows the amino acid sequence of the tau core motif in MBTR2 linked by 3D6.

[0274] Sequence ID 193 shows the amino acid sequence of the tau core motif in MBTR3 linked by 3D6.

[0275] Sequence ID 194 shows the amino acid sequence of the tau core motif in MBTR4 linked by 3D6.

[0276] definition Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that the antibody or other biological entity is typically at least 50% w / w pure with respect to interfering proteins and other contaminants resulting from its manufacture or purification, although this does not exclude the possibility that the monoclonal antibody may be combined with an excess of pharmaceutically acceptable carrier(s) or other vehicle intended to facilitate its use. Sometimes, the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95% or 99% w / w pure with respect to interfering proteins and other contaminants resulting from its manufacture or purification. Isolated monoclonal antibodies or other biological entities are often the major macromolecular species remaining after their purification.

[0277] Specific binding of an antibody to its target antigen means an affinity and / or binding activity of at least 10 6 10 7 10 8 10 9 10 10 10 11 or 10 12 M -1 Specific binding is a binding that is high enough to be detectable in magnitude and is distinguishable from non-specific binding that occurs to at least one irrelevant target. Specific binding can be the result of the formation of a bond between specific functional groups or a specific spatial fit (e.g., lock-and-key type), while non-specific 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.

[0278] 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 (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, primarily involved in antigen recognition. This variable region, linked to a cleavable signal peptide, is the first to be expressed. The variable region that does not contain the signal peptide is often called the mature variable region. Therefore, for example, the mature variable region of a light chain means the light chain variable region that does not contain the light chain signal peptide. The carboxyl-terminus of each chain defines a constant region primarily involved in effector function.

[0279] The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, defining the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 10 or more amino acids. (For a general overview, see Fundamental Immunology, Paul, W., ed., 2nd ed., Raven Press, NY, 1989, Ch. 7 (which is incorporated in its entirety by reference in this application)).

[0280] The immunoglobulin light chain or heavy chain variable region (also referred to herein as the “light chain variable domain” (VL domain) or the “heavy chain variable domain” (“VH domain”), respectively) consists of a “framework” region separated by three “complementarity-determining regions” or “CDRs.” The framework region plays a role in aligning the CDRs for specific binding to the antigen’s epitope. The CDRs contain amino acid residues of the antibody that are primarily involved in antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, 2, and 3 of the VL domain are also referred herein as CDR-L1, CDR-L2, and CDR-L3, respectively; the CDR1, 2, and 3 of the VH domain are also referred herein as CDR-H1, CDR-H2, and CDR-H3, respectively. Where this application discloses a VL sequence having R as the C-terminal residue, this R can instead be considered the N-terminal residue of the light chain constant region. Therefore, it should also be understood that this application discloses VL sequences that do not have a C-terminal R.

[0281] The assignment of each amino acid to the VL and VH domains is consistent with either of the conventional definitions of CDR. These conventional definitions include those by 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 Chothia-Kabat CDR combination, in which case CDR-H1 is the combination of Chothia and Kabat CDR; and Oxford Molecular's antibody modelling. This includes the definition of AbM used by the software, and the definition of contact by Martin et al. (bioinfo.org.uk / abs) (see Table 1). Kabat provides a widely used numbering rule (Kabat numbering) in which the same number is assigned to corresponding residues between different heavy chains or between different light chains. 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 present in the antibody (i.e., Kabat CDRs). This does not preclude the presence of other residues that fall within another conventional CDR definition but are outside that specified definition. For example, an antibody containing a CDR as defined by Kabat includes, among other possibilities, an antibody in which the CDR contains a Kabat CDR residue but does not contain any other CDR residues, and an antibody in which CDR H1 is a Kotia-Kabat combination CDR H1 and other CDRs contain a Kabat CDR residue but do not contain any further CDR residues based on other definitions. [Table 1]

[0282] The term “antibody” includes complete antibodies and their binding fragments. Typically, fragments, including isolated heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dab, nanobodies, and Fv, compete with the complete antibody from which they originate for specific binding to the target. Fragments can be produced by recombinant DNA technology or by enzymatic or chemical separation of complete immunoglobulins. The term “antibody” also includes bispecific antibodies and / or humanized antibodies. Bispecific or bivalent antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). In some bispecific antibodies, the two different heavy / light chain pairs include a humanized 3D6 heavy / light chain pair and a heavy / light chain pair that is more specific to different epitopes on tau than those conjugated by 3D6.

[0283] 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 receptors expressed on the blood-brain barrier, such as insulin receptors, insulin-like growth factor (IGF) receptors, leptin receptors, lipoprotein receptors, or transferrin receptors (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 designing the specific antibody to reduce its affinity for blood-brain barrier receptors. The reduced affinity for the receptor allows for a more widespread distribution within the brain (see, for example, Atwal et al., Sci.Trans.Med.3, 84ra43, 2011; Yu et al., Sci.Trans.Med.3, 84ra44, 2011).

[0284] Representative bispecific antibodies may include the following: (1) a bivariable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains in series via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig®) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) Tandab, which is a fusion of two single-chain diabodies, producing a tetravalent bispecific antibody with two binding sites for each target antigen. (3) Flexibody, which is a combination of scFv and diabody that produces a polyvalent molecule; (4) so-called "dock and lock" molecule, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments bound to different Fab fragments; or (5) so-called scorpion molecule, which, for example, contains two scFv fused to both ends of a human Fc region. Examples of platforms useful for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-modified IgGl (Xencor), or DuoBody (based on Fab arm exchange, Genmab).

[0285] The term "epitope" refers to the site on an antigen to which an antibody binds. Epitopes can be formed from a sequence of amino acids or from discontinuous amino acids juxtaposed by the tertiary folding structure of one or more proteins. Epitopes formed from a sequence of amino acids (also known as linear epitopes) are typically retained even when exposed to denaturing solvents, while epitopes formed by tertiary folding (also known as structural epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and usually at least five or eight to ten, amino acids in a unique spatial higher-order structure. Methods for determining the spatial higher-order structure of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0286] Antibodies that recognize the same or overlapping epitopes can be identified by a simple immunoassay that demonstrates the ability of one antibody to compete for the binding of the other antibody to the target antigen. The epitopes of antibodies can also be determined by identifying contact residues through X-ray crystallography of the antibody bound to that antigen. Alternatively, if all amino acid mutations in the antigen that reduce or disengage the binding of one antibody also reduce or disengage the binding of the other antibody, then the two antibodies have the same epitope. If several amino acid mutations that reduce or disengage the binding of one antibody also reduce or disengage the binding of the other antibody, then the two antibodies have overlapping epitopes.

[0287] Antibody competition is determined by assays in which the test antibody inhibits the specific binding of the reference antibody to a common antigen (e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with the reference antibody if, when measured in a competitive binding assay, an excess (e.g., at least 2x, 5x, 10x, 20x, or 100x) of the test antibody inhibits the binding of the reference antibody by at least 50%. Some test antibodies inhibit the binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes sufficiently proximal to the epitope to which the reference antibody binds due to steric hindrance.

[0288] The term "pharmaceutically acceptable" means that a carrier, diluent, excipient, or auxiliary material is compatible with other formulation components and is not substantially harmful to the recipient.

[0289] The term "patient" includes human and other mammalian subjects receiving either preventive or therapeutic treatment.

[0290] An individual is at high risk of developing a disease if, for example, they have at least one known risk factor (e.g., genetic, biochemical, family history, or situational exposure), and individuals with that risk factor have a statistically significantly higher risk of developing the disease than individuals without that risk factor.

[0291] The term “biological sample” means a sample of biological material in or obtainable from a biological source, such as a human or mammalian subject. Such a sample may be an organ, organelle, tissue, tissue section, body fluid, peripheral blood, plasma, serum, cell, molecule such as proteins and peptides, and any part or combination thereof derived therefrom. The term “biological sample” may also encompass any material obtained by processing the sample. Derived material may include cells or their offspring. Processing of a biological sample may include one or more of the following: filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, etc.

[0292] The term "control sample" means a biological sample that is not known to contain or suspected to contain tau-related disease areas, or at least not known to contain or suspected to contain a given type of disease area. Control samples can be obtained from individuals that do not have tau-related disease. Alternatively, control samples can be obtained from patients that do not have tau-related disease. Such samples can be obtained at the same time as or at a different time as the biological sample is thought to contain tau-related disease. Both the biological sample and the control sample can be obtained from the same tissue. Preferably, the control sample consists basically or entirely of healthy tissue and can be used in comparison to the biological sample that is thought to contain tau-related disease areas. 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 thought to be present in the biological sample are of the same cell type (e.g., neurons or glial cells) as the cell types in the control sample.

[0293] 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 its etiology.

[0294] The term "symptoms" refers to subjective evidence of a disease, such as a change in gait, that the subject notices. "Signs" refers to objective evidence of a disease that a doctor recognizes.

[0295] The term "positive response to treatment" refers to a better or average response in an individual patient within a patient population compared to the average response in a control group that did not receive treatment.

[0296] For the purpose of classifying amino acid substitutions as conserved or non-conserved, 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 affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conserved substitutions include substitutions between amino acids of the same class. Non-conservative substitutions are the exchange of one member of one of these classes with a member of another class.

[0297] Percent sequence identity is determined using antibody sequences aligned to the maximum extent possible according to Kabat numbering rules. After alignment, when comparing the target antibody region (e.g., the entire maturation variable region of the heavy or light chain) with the same region of the reference antibody, the percentage sequence identity between the target antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in both the target antibody region and the reference antibody region by the total number of positions in the two aligned regions (gaps are not counted), and multiplying by 100 to convert it to a percentage.

[0298] A composition or method that “comprising” or “including” one or more descriptive elements may include other elements not specifically described. For example, a composition that “comprises” or “includes” an antibody may include the antibody alone or in combination with other components. Where this disclosure refers to a feature that includes a specified element, this disclosure should be understood to refer instead to a feature that is essentially derived from or consists of the specified element.

[0299] The notation of a range of values ​​includes all integers within or defining that range, as well as all subranges defined by the integers within that range.

[0300] Unless the context makes it clear otherwise, the term "approximately" includes very small variations, such as the standard tolerance for measuring the indicated value (e.g., SEM).

[0301] Statistical significance means p < 0.05.

[0302] The singular articles ("a," "an," and "the") refer to multiple things unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" can refer to multiple compounds, including mixtures of compounds.

[0303] Detailed explanation I. Overview The present invention provides antibodies that bind to tau. Some antibodies specifically bind to one or more epitopes in the microtubule-binding region (MTBR) of human tau, defined by approximately residues 244-372 of SEQ ID NO: 1. Because the MTBR region contains sequence repeats, a single antibody may bind to multiple repeated sites within this region. Some antibodies specifically bind to the epitope KXXSXXNX(K / H)H (SEQ ID NO: 191). Some antibodies bind within residues 199-213 and / or 262-276 of SEQ ID NO: 3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1). Some antibodies bind within residues 259-268 and / or 290-299 and / or 321-330 and / or 353-362 of SEQ ID NO: 1. In some antibodies, binding to the MTBR4 region at residues 353-362 is weaker than binding to MTBR1, 2, and 3. Some antibodies bind to tau regardless of its phosphorylation state. Some antibodies of the present invention help suppress or delay tau-related disease progression and tau-related disease exacerbation. Understanding the mechanism is not necessary for carrying out the present invention, but among several mechanisms, reduced toxicity may result from the induction of tau phagocytosis, inhibition of intermolecular or intramolecular aggregation of tau, or inhibition of binding to other molecules by antibodies, by stabilizing non-toxic higher-order structures, by suppressing intercellular or intracellular transmission of pathogenic tau morphologies, by inhibiting tau phosphorylation, by interfering with tau binding to cells, or by inducing tau protein cleavage. Antibodies or agents of the present invention that induce such antibodies can be used as treatments or effective preventive measures for Alzheimer's disease and other tau-related diseases.

[0304] II.Target molecule Unless otherwise clear from the context, a reference to tau refers to natural human-type tau, including all isoforms, with or without post-translational modifications (e.g., phosphorylation, glycation, or acetylation). Six major isoforms (splice variants) of tau arise in the human brain. The longest of these variants has 441 amino acids, with the first methionine residue cleaved. The residues are numbered according to the 441 isoforms. Therefore, a reference to phosphorylation at position 404, for example, refers to position 404 of the 441 isoform, or the corresponding position of any other isoform when maximally aligned with the 441 isoform. The amino acid sequences and Swiss plot numbers of the isoforms are shown below. P10636-8 (Sequence ID 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(4R0NHitau)(Sequence ID 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 PKSGDRSGYS SPGSPGTPGS 160 170 180 RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 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 (Sequence ID 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 (Accession No. 6) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 [[ID=2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM 340 350 VDSPQLATLA DEVSASLAKQ GL

[0305] References to tau include the approximately 30 known natural variations listed in the Swiss Plot Database and its permutations, as well as variants associated with tau pathologies such as dementia, Pick's disease, and supranuclear palsy (see, e.g., Swiss Plot Database and Poorkaj, et al. Ann Neurol. 43:815-825 (1998)). Some examples of tau mutations numbered by 441 isoforms include a mutation from lysine to threonine at amino acid residue 257 (K257T), a mutation from isoleucine to valine at amino acid position 260 (I260V), a mutation from glycine to valine at amino acid position 272 (G272V), a mutation from asparagine to lysine at amino acid position 279 (N279K), a mutation from asparagine to histidine at amino acid position 296 (N296H), a mutation from proline to serine at amino acid position 301 (P301S), and a mutation from proline to leucine at amino acid position 301 (P301 L) Examples include mutations from glycine to valine at amino acid position 303 (G303V), from serine to asparagine at position 305 (S305N), from glycine to serine at amino acid position 335 (G335S), from valine to methionine at position 337 (V337M), from glutamic acid to valine at position 342 (E342V), from lysine to isoleucine at amino acid position 369 (K369l), from glycine to arginine at amino acid position 389 (G389R), and from arginine to tryptophan at position 406 (R406W).

[0306] Tau may be phosphorylated at one or more amino acid residues, including tyrosine at amino acid positions 18, 29, 97, 310, and 394; serine 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 threonine at amino acid positions 175, 181, 205, 212, 217, 231, and 403. Unless otherwise clear from the context, references to tau or its fragments include native human amino acid sequences, including its isoforms, variants, and allele variants.

[0307] III. Antibodies A. Binding specificity and functional properties The present invention provides antibodies that bind to tau. Some antibodies specifically bind to the epitope in KXXSXXNX(K / H)H (SEQ ID NO: 191). Some antibodies bind to peptides that are essentially composed of amino acid residues or consist of amino acid residues, including amino acid residues 259-268 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to peptides that are essentially composed of amino acid residues or consist of amino acid residues, including amino acid residues 290-299 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to peptides that are essentially composed of amino acid residues or consist of amino acid residues, including amino acid residues 321-330 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to peptides that are essentially composed of amino acid residues or consist of amino acid residues, including amino acid residues 353-362 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies specifically bind to epitopes within residues 199-213 of the 383-amino acid 4R0N human tau protein (SEQ ID NO: 3) (corresponding to residues 257-271 of SEQ ID NO: 1). Some antibodies specifically bind to epitopes within residues 262-276 of the 383-amino acid 4R0N human tau protein (SEQ ID NO: 3) (corresponding to residues 320-334 of SEQ ID NO: 1). Some antibodies of the present invention specifically bind to a peptide consisting of residues 257-271 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies of the present invention specifically bind to a peptide consisting of residues 320-334 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies of the present invention specifically bind to a peptide consisting of residues 259-268 of the 441-amino acid tau protein SEQ ID NO: 1, i.e., KIGSTENLKH (SEQ ID NO: 188). Some antibodies of the present invention specifically bind to a peptide consisting of residues 290-299 of the 441-amino acid tau protein SEQ ID NO: 1, i.e., KCGSKDNIKH (SEQ ID NO: 192). Some antibodies of the present invention specifically bind to a peptide consisting of residues 321-330 of the 441-amino acid tau protein SEQ ID NO: 1, i.e., KCGSLGNIHH (SEQ ID NO: 193).Some antibodies of the present invention specifically bind to a peptide consisting of residues 353-362 of the 441-amino acid tau protein SEQ ID NO: 1, i.e., KIGSLDNITH (SEQ ID NO: 194). Some antibodies of the present invention bind to a peptide consisting of the consensus motif KXXSXXNX(K / H)H (SEQ ID NO: 191). Some antibodies bind to epitopes containing residues 259, 262, 265, 267, 268, 290, 293, 296, 298, 299, 321, 324, 327, 329, 330, or 353, 356, 359, 362 of the 441-amino acid tau protein SEQ ID NO: 1. Some antibodies bind to tau regardless of its phosphorylation state. Some antibodies bind to epitopes that do not contain phosphorylated residues. These antibodies can be obtained by purification from natural sources or by immunization with recombinantly expressed tau polypeptide. Antibodies can be screened for binding to unphosphorylated tau and to tau in a form in which one or more phosphorylated residues are phosphorylated. Such antibodies preferably bind with indistinguishable affinity to unphosphorylated tau, or to phosphorylated tau within a coefficient range of at least 1.5-fold, 2-fold, or 3-fold (i.e., “general-specific”). 3D6 is an example of a general-specific monoclonal antibody. The present invention also provides antibodies that bind to the same epitopes as any of the above antibodies, such as the 3D6 epitope. It also includes antibodies that compete with any of the above antibodies for binding to tau, such as those that compete with 3D6.

[0308] The antibodies described above can be produced de novo by immunizing with peptides that are essentially composed of or consist of residues containing residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to residues 257-271 or 320-334 of SEQ ID NO: 1, respectively), or by immunizing with peptides that are essentially composed of or consist of residues containing residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, or by immunizing with full-length tau polypeptides or fragments thereof containing such residues, and by screening for specific binding to peptides containing such residues. Such peptides are preferably conjugated to heterologous conjugate molecules that help induce an antibody response to the peptide. Binding may be direct or mediated via spacer peptides or amino acids. Cysteine ​​is used as a spacer amino acid because its free SH group facilitates the binding of carrier molecules. Polyglycine linkers (e.g., 2-6 glycines) with or without cysteine ​​residues between glycine and peptide may also be used. Carrier molecules help provide T cell epitopes that support the induction of an antibody response to the peptide. Multiple carriers, particularly hemocyanin (KLH) derived from water oysters, ovalbumin, and bovine serum albumin (BSA), are commonly used. Peptide spacers may be added to peptide immunogens as part of solid-phase peptide synthesis. Carriers are typically added by chemical crosslinking.Some examples of chemical crosslinking agents that can be used include cross-N-maleimide-6-aminocaproyl ester or m-maleimidebenzoyl-N-hydroxysuccinimide ester (MBS) (e.g., Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1988; Sinigaglia et al., Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47 (1993); Hammer et al., Cell 74:197-203 (1993); Falk K. et al., Immunogenetics, 39:230-242 (1994); International Publication No. 98 / 23635; and Southwood et al. See al. J. Immunology, 160:3363-3373 (1998). Carriers and spacers, if present, can bind to either end of the immunogen.

[0309] Peptides with any spacer and carrier may be used to immunize experimental animals or B cells as described in more detail below. Hybridoma supernatant may be tested for its ability to bind to one or more peptides that are essentially composed of residues, or consist of residues, including residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to residues 257-271 or 320-334, respectively, of SEQ ID NO: 1), or one or more peptides that are essentially composed of residues, or consist of residues, including residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, and / or phosphorylated or unphosphorylated forms of tau, such as the full-length isoform of tau having position 404 in the phosphorylated form. Peptides may be bound to carriers or other tags to facilitate screening assays. In this example, the carrier or tag is selectively different from the spacer-carrier molecule combination used in immunization to exclude antibodies specific to the spacer or carrier rather than the tau peptide. Any of the tau isoforms may be used.

[0310] This invention provides a monoclonal antibody that binds to an epitope in tau. The antibody designated 3D6 is one such representative mouse antibody. Unless otherwise clear from the context, a reference to 3D6 should be understood as referring to one of the mouse, chimeric, veneer, or humanized forms of this antibody. This antibody is deposited as [accession number]. This antibody specifically binds to the epitope KXXSXXNX(K / H)H (SEQ ID NO: 191). This antibody binds within amino acid residues 199-213 and / or 262-276 of the 383-amino acid 4R0N human tau protein (SEQ ID NO: 3) (corresponding to amino acid residues 257-271 and / or 320-334, respectively, of SEQ ID NO: 1). The antibody specifically binds to amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1, and any combination of two, three, or all four of them. This antibody is further characterized by its ability to bind to both phosphorylated and unphosphorylated tau, both non-pathological and pathological forms as well as tau in higher-order structures, and tau in misfolded / aggregated forms. The antibody designated 6A10 is one such representative mouse antibody. Unless otherwise clear from the context, a reference to 6A10 should be understood as referring to any of the mouse, chimeric, veneer, and humanized forms of this antibody. The Kabat / Cotia combination CDRs of the heavy chain of 6A10 are shown in SEQ ID NOs: 67, 68, and 69, respectively, and the Kabat CDRs of the light chain of 6A10 are shown in SEQ ID NOs: 12, 13, and 14, respectively. Mouse 6A10 shares 82.1% VH sequence identity and 100% VL sequence identity with the VH and VL strands of mouse 3D6, respectively.

[0311] Some antibodies of the present invention bind to the same or overlapping epitopes as the antibody designated 3D6. The sequences of the heavy chain and light chain maturation variable regions of this antibody are shown in SEQ ID NOs: 7 and 11, respectively. Other antibodies with such binding specificity may be produced by immunizing mice with tau or a portion thereof that is essentially made up of or composed of an epitope containing a desired epitope (e.g., 199-213 and / or 262-276 of SEQ ID NO: 3, corresponding to residues 257-271 and / or 320-334 of SEQ ID NO: 1, or residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1, or any combination of two, three or all four of these), and optionally by screening the resulting antibodies for binding to tau in competition with antibodies having the variable region of mouse 3D6 (IgG1 kappa). A tau fragment containing a desired epitope may be ligated to a carrier that assists in inducing an antibody response to the fragment, and / or may be mixed with an adjuvant that assists in inducing such a response. Such antibodies may be screened for differential binding to tau or its fragment compared to variants of specified residues. Screening for such variants allows for a more precise definition of binding specificity, enabling the identification of antibodies whose binding is inhibited by the mutagenicity of a particular residue, and antibodies that may share the functional properties of other exemplified antibodies. The mutations may involve systematically replacing one residue at a time, or a wider spatial distance, with alanine (or serine if alanine is already present) across the entire target or a segment thereof where the epitope is known to exist. If the same set of mutations significantly reduces the binding of two antibodies, then those two antibodies bind to the same epitope.

[0312] Antibodies with binding specificity to a selected mouse antibody (e.g., 3D6) can also be generated using a variant of the phage display method. See Winter's International Publication No. 92 / 20791. This method is particularly suitable for generating human antibodies. In this method, either the heavy chain or light chain variable region of a selected mouse antibody is used as a starting material. For example, if the light chain variable region is selected as the starting material, a phage display is constructed in which members present the same light chain variable region (i.e., the mouse starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Strong specific binding to tau or its fragments (e.g., at least 10) 8 Preferably at least 10 9 M -1 A phage exhibiting the ) is selected. The heavy chain variable region from this phage then serves as a starting material for constructing a further phage library. In this library, each phage presents the same heavy chain variable region (i.e., the region identified from the initial display library) and a different light chain variable region. The light chain variable region is obtained, for example, from a library of rearranged human variable light chain regions. Again, a phage exhibiting strong specific binding to tau or its fragments is selected. The resulting antibody typically has the same or similar epitope specificity as the mouse starting material.

[0313] The Kabat / Cotia combination CDRs for the heavy chain of 3D6 are shown in SEQ ID NOs: 8, 9, and 10, respectively, and the Kabat CDRs for the light chain of 3D6 are shown in SEQ ID NOs: 12, 13, and 14, respectively.

[0314] Table 2 shows the 3D6 CDRs as defined by Kabat, Cotia, combinations of Cotia and Kabat (which are also referred to herein as “Kabat / Cotia combinations”), AbM, and Contact. [Table 2]

[0315] Other antibodies can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of representative antibodies such as 3D6. The present invention also includes monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 3D6 in the amino acid sequence of the mature heavy and / or light chain variable region, and that retain its functional properties, and / or monoclonal antibodies that differ from each other by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. This also includes monoclonal antibodies having at least one or all six CDRs, preferably according to Kabat's conventional definition, that are 90%, 95%, 99%, or 100% identical to the corresponding CDR of 3D6, as defined by Kabat.

[0316] The present invention also provides antibodies having several or all (e.g., 3, 4, 5, and 6) CDRs derived entirely or substantially from 3D6. Such antibodies may include a heavy chain variable region having at least two, and usually all three, CDRs derived entirely or substantially from the heavy chain variable region of 3D6, and / or a light chain variable region having at least two, and usually all three, CDRs derived entirely or substantially from the light chain variable region of 3D6. Antibodies may include both heavy and light chains. A CDR is substantially derived from the corresponding 3D6 CDR, except that if it contains 4, 3, 2, or 1 or fewer substitutions, insertions, or deletions, CDR-H2 (as defined by Kabat) may have 6, 5, 4, 3, 2, or 1 or fewer substitutions, insertions, or deletions. Such antibodies may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with 3D6 in the amino acid sequence of the mature heavy chain and / or light chain variable region, retain its functional properties, and / or may differ from antibodies of 3D6 by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0317] Some antibodies identified by such assays can bind to monomers, misfolded, aggregated, phosphorylated, or non-phosphorylated forms of tau, etc. Similarly, some antibodies are immunoreactive against non-pathological and pathological morphologies and higher-order structures of tau.

[0318] B. Non-human antibodies The production of other non-human antibodies against tau or its fragments (e.g., amino acid residues 199-213 or 262-276 of SEQ ID NO: 3, corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO: 1, or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1), such as antibodies in mice, guinea pigs, primates, rabbits, or rats, can be achieved, for example, by immunizing animals with tau or its fragments. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference). Such immunogens can be obtained from natural sources by peptide synthesis or recombinant expression. In some cases, the immunogen may be administered fused with a carrier protein or otherwise complexed. In some cases, the immunogen may be administered with an adjuvant. Several types of adjuvants may be used as described below. An incomplete adjuvant following a complete Freund's adjuvant is preferred for immunization of experimental animals. Rabbits or guinea pigs are typically used to produce polyclonal antibodies. Mice are typically used to produce monoclonal antibodies. Antibodies are screened for specific binding to tau or epitopes within tau (e.g., epitopes containing one or more amino acid residues 199-213 or 262-276 of SEQ ID NO: 3, corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO: 1, respectively, or epitopes containing one or more amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1).Such screening can be accomplished by determining the binding of antibodies to a collection of tau variants, such as tau variants containing amino acid residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO: 1, respectively), or tau variants containing amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, or mutations within these residues, and determining which tau variants bind to the antibody. Binding can be evaluated, for example, by Western blotting, FACS, or ELISA.

[0319] C. Humanized antibodies Humanized antibodies are genetically modified antibodies in which CDRs derived from non-human "donor" antibodies are grafted onto human "receptor" antibody sequences (see, for example, Queen's U.S. Patents 5,530,101 and 5,585,089; Winter's U.S. Patent 5,225,539; Carter's U.S. Patent 6,407,213; Adair's U.S. Patent 5,859,205; and Foote's U.S. Patent 6,881,557). The receptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of a human antibody sequence, or a germline region sequence. Thus, a humanized antibody is an antibody that has at least three, four, five, or all of the CDRs, as well as a variable region framework sequence and, if present, a constant region, which is entirely or substantially derived from the human antibody sequence. Similarly, the humanized heavy chain has at least one, two, usually three CDRs, entirely or substantially derived from the donor antibody heavy chain, and, if present, a heavy chain variable region framework sequence and a heavy chain constant region substantially derived from the human heavy chain variable region framework and constant region sequence. Similarly, the humanized light chain has at least one, two, usually three CDRs, entirely or substantially derived from the donor antibody light chain, and, if present, a light chain variable region framework sequence and a light chain constant region substantially derived from the human light chain variable region framework and constant region sequence. In addition to nanobodies and dAbs, the humanized antibody includes a humanized heavy chain and a humanized light chain. The CDRs in the humanized antibody are substantially derived from the corresponding CDRs in the non-human antibody if at least 85%, 90%, 95%, or 100% of the corresponding residues (defined by either conventional method, but preferably by Kabat) are identical between the respective CDRs. The variable region framework sequence or the constant region of the 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.According to the 2014 World Health Organization (WHO) definition of the International Generic Name (INN) for humanized antibodies, an antibody must be classified as humanized if it is at least 85% identical to a human germline antibody sequence (i.e., before somatic hypermutation). A mixed antibody is one in which one antibody chain (e.g., heavy chain) meets the threshold, while the other chain (e.g., light chain) does not. If neither chain meets the threshold, the antibody is classified as a chimera, even if it is substantially human with some reverse mutations in the variable framework regions of both chains. See Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1,1-9, DOI:10.1080 / 19420862.2015.1114320. It is also available from “WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review)” (Internet) 2014 (http: / / www.who.int / medicines / services / inn / BioRev2014.pdf). This document is incorporated herein by reference. To avoid misunderstanding, the term “humanized” as used herein is not intended to be limited to the 2014 WHO INN definition of humanized antibodies. Some humanized antibodies provided herein have at least 85% sequence identity to human germline sequences, while 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 sequence identity of approximately 60% to 100% with respect to human germline sequences, for example, in the range of approximately 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%.Some heavy chains fall below the 2014 WHO INN definition, for example, having sequence identity of approximately 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% to human germline sequences, while other heavy chains conform to the 2014 WHO INN definition and have sequence identity of approximately 85%, 86%, 87%, 88%, 89%, or more to human germline sequences. Some light chains of the humanized antibodies provided herein have sequence identity of approximately 60% to 100% to human germline sequences, for example, in the range of 80% to 84%, or 85% to 89%. Some light chains fall below the 2014 WHO INN definition, for example, having approximately 81%, 82%, 83%, or 84% sequence identity to the human germline sequence, while other light chains meet the 2014 WHO INN definition, having approximately 85%, 86%, 87%, 88%, 89%, or more sequence identity to the human germline sequence. Some humanized antibodies provided herein that are “chimeric” according to the 2014 WHO INN definition have a heavy chain with less than 85% identity to the human germline sequence paired with a light chain with less than 85% identity to the human germline sequence. For example, some humanized antibodies provided herein that have a heavy chain with at least 85% identity to the human germline sequence paired with a light chain with less than 85% identity to the human germline sequence are “mixed” according to the 2014 WHO INN definition, and vice versa. Some of the humanized antibodies provided herein conform to the 2014 WHO INN definition of “humanized” and have a heavy chain having at least 85% identity to a human germline sequence paired with a light chain having at least 85% identity to a human germline sequence. Further humanized antibodies of the present invention conform to the 2014 WHO INN definition of “mixed.”

[0320] Humanized antibodies often include all six CDRs derived from mouse antibodies (defined by any conventional method, preferably as defined by Kabat), but can also be produced with fewer than all of the mouse antibody-derived CDRs (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).

[0321] In some antibodies, only a portion of the CDR, i.e., a subset of the CDR residues required for binding, called the SDR, is necessary to maintain binding in the humanized antibody. CDR residues that do not come into contact with the antigen and are not present in the SDR can be identified based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), by molecular modeling and / or empirically from the region of the Kabat CDR outside the Chothia high-frequency variable loop (Chothia, J. Mol. Biol. 196:901, 1987), or as described in Gonzales et al., Mol. Immunol. 41:863, 2004. In such humanized antibodies, at positions where one or more donor CDR residues are absent or the entire donor CDR is removed, the amino acid occupying that position may be the amino acid occupying the corresponding position (by Kabat numbering) in the receptor antibody sequence. The number of such receptor substitutions for donor amino acids included in the CDR reflects a balance of competing considerations. Such substitutions are potentially beneficial in that they reduce the number of mouse amino acids in the humanized antibody, thereby reducing potential immunogenicity, and / or in order to conform to the WHO INN definition of "humanization." However, substitutions can also result in changes in affinity, and it is preferable to avoid significant decreases in affinity. The location of the substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0322] Human receptor antibody sequences can be selected from a large number of known human antibody sequences as needed to provide a high degree of sequence identity (e.g., 65-85% identity) between the variable region framework of the human receptor sequence and the corresponding variable region framework of the donor antibody chain.

[0323] Some humanized and chimeric antibodies have the same (within experimental error) or improved functional properties as the derived mouse antibodies, e.g., binding affinity to human tau as described in the examples, and inhibition of tau internalization in neurons. For example, some humanized and chimeric antibodies have binding affinity that is 3 times, 2 times, or 1 times higher than the mouse antibodies derived from them, or affinity that is indistinguishable within experimental error. Some humanized and chimeric antibodies inhibit tau internalization in neurons as described in the examples by 3 times, 2 times, or 1 time higher than the mouse antibodies derived from them, or they inhibit tau internalization similarly to the mouse antibodies derived from them within experimental error. Some humanized antibodies exhibited reduced immunogenicity, increased affinity, increased thermal stability, and / or improved expression compared to previously described humanized forms of 3D6 antibodies (see International Publication No. 2017 / 191560). Hu3D6VHv1bA11 / L2-DIM4 showed improved affinity compared to the parent hu3D6VHv1bA11 / hu3D6VLv2, as indicated by the on-rate, off-rate, and Kd number. Hu3D6VHv1bA11 / L2-DIM4 also showed higher thermal stability and titer than the parent hu3D6VHv1bA11 / hu3D6VLv2.

[0324] An example of a heavy chain receptor sequence is the human mature heavy chain variable region of humanized 48G7Fab, PDB accession code 2RCS-VH_huFrwk (SEQ ID NO: 75). The variable domains of 3D6 and 48G7Fab also share the same length for the CDR-H1, H2 loops. Another example of a heavy chain receptor sequence is IMGT number IGHV1-69-2. * This is the human mature heavy chain variable region of 01 (SEQ ID NO: 25). IMGT number IGHV1-69-2* 01 (SEQ ID NO: 25) shares 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 the human antibody ARX71335VL (SEQ ID NO: 82), PDB accession code. The variable light chain domains of 3D6 and ARX71335VL antibodies also share identical lengths for the CDR-L1, L2, and L3 loops. An example of a light chain receptor sequence is IMGT number IGKV2-30. * This is the human mature light chain variable region of 02 (SEQ ID NO: 27). IMGT number IGKV2-30 * 02 (Sequence ID 27) has the same canonical CDR-L1, CDR-L2, and L3 classes as mouse 3D6. IMGT number IGKV2-30 * 02 (sequence number 27) belongs to the hinotkappa subgroup 2.

[0325] When two or more human receptor antibody sequences are selected, a complex or hybrid of these receptors can be used, and the amino acids used at different positions in the humanized light chain and heavy chain variable regions can be selected from any of the human receptor antibody sequences used. For example, IMGT number IGHV1-69-2 * The human mature heavy chain variable region 01 (SEQ ID NO: 25) and PDB accession code number 2RCS-VH_huFrwk (SEQ ID NO: 75) were used as receptor sequences for the humanization of the 3D6 mature heavy chain variable region. An example of these two receptors being at different positions is position H17 (T or S). The humanized version of the 3D6 heavy chain variable region can include either amino acid at this position. For example, the human mature light chain variable region IMGT number IGKV2-30. * 02 (SEQ ID NO: 27) and PDB code number ARX71335VL_huFrwk (SEQ ID NO: 82) were used as receptor sequences for humanization of the 3D6 mature light chain variable region. An example of these two receptors being at different positions is position L100 (Q or A). The humanized form of the 3D6 light chain variable region may contain either amino acid at this position.

[0326] Certain amino acids derived from human variable region framework residues can be selected for substitution based on their possible effects on the CDR higher-order structure and / or binding to antigens. Studies of such possible effects can be conducted through modeling, characterization of amino acids at specific locations, or empirical observation of the effects of substitution or mutagenesis of specific amino acids.

[0327] For example, if the amino acids differ between the mouse variable region framework residues and the selected human variable region framework residues, the human framework amino acids can be substituted with equivalent framework amino acids derived from the mouse antibody, in which case the amino acids are... (1) Directly binds non-covalently to the antigen, (2) Located adjacent to the CDR area or within the CDR as defined by Kotia rather than Kabat, (3) Otherwise, interacting with the CDR region (e.g., within approximately 6 Å of the CDR region), (e.g., identified by modeling the light or heavy chain on the analyzed structure of homologous known immunoglobulin chains), or (4) Residues involved in the VL-VH interface, It is reasonable to assume that this will happen.

[0328] In one embodiment, the humanized sequence is generated using a two-stage 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].

[0329] As defined by Queen's U.S. Patent No. 5,530,101, framework residues of classes (1) to (3) are sometimes alternately called canonical and vernier residues. Framework residues that help determine the higher-order structure of the CDR loop are sometimes called 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 higher-order structure of the antigen-binding loop and play a role in fine-tuning the fit of the antibody to the antigen are sometimes called vernier residues (Foote & Winter, J Mol.Bio 224:487-499 (1992)).

[0330] Other framework residues that are candidates for substitution create potential glycosylation sites. Further other substitution candidates are receptor human framework amino acids that are not typically found at their positions in human immunoglobulins. These amino acids can be substituted with amino acids at the equivalent positions of mouse donor antibodies or amino acids derived from the more typical equivalent positions of human immunoglobulins.

[0331] Another framework residue that is a candidate for substitution is the N-terminal glutamine residue (Q), which may be substituted with glutamate (E) to minimize the possibility of pyroglutamate conversion [Y. Diana Liu, et al., 2011, J. Biol. Chem., 286:11211-11217]. The conversion of glutamate (E) to pyroglutamate (pE) occurs more slowly than from glutamine (Q). Due to the decrease in the primary amine in the conversion of glutamine to pE, the antibody becomes more acidic. Incomplete conversion leads to antibody heterogeneity, which can be observed as multiple peaks by charge-based analytical methods. Differences in heterogeneity may indicate insufficient process control.

[0332] A representative humanized antibody is the humanized form of mouse 3D6, known as Hu3D6.

[0333] The mouse antibody 3D6 includes a mature heavy chain and a light chain variable region having amino acid sequences including SEQ ID NOs. 7 and 11, respectively. The present invention includes 10 exemplified humanized mature heavy chain variable regions (hu3D6VHvb1 (SEQ ID NOs. 76), hu3D6VHvb2 (SEQ ID NOs. 77), hu3D6VHvb3 (SEQ ID NOs. 78), hu3D6VHvb4 (SEQ ID NOs. 79), hu3D6VHvb5 (SEQ ID NOs. 80), hu3D6VHvb6 (SEQ ID NOs. 90), hu3D6VHvb7 (SEQ ID NOs. 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NOs. 146), hu3D6VHv1bA11 L82cV (SEQ ID NOs. 147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R(h3D6VHvb9, SEQ ID NO: 148)) and 56 examples of human mature light chain variable regions (hu3D6VLvb1(SEQ ID NO: 83), hu3D6VLvb2(SEQ ID NO: 84), hu3D6VLvb3(SEQ ID NO: 85), hu3D6VLv2 L54D(SEQ ID NO: 93), hu3D6VLv2 L54G(SEQ ID NO: 94), hu3D6VLv2 L54N(SEQ ID NO: 95), hu3D6VLv2 L54E(SEQ ID NO: 96), hu3D6VLv2 L50E(SEQ ID NO: 97), hu3D6VLv2 L54Q(SEQ ID NO: 98), hu3D6VLv2 L50D(SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (Sequence ID 134), hu3D6VLv2 L37Q_L50E_L54R (Sequence ID 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (Sequence ID 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (Sequence ID 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (Sequence ID 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (Sequence ID 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 This invention provides humanized forms of mouse 3D6 antibodies, including L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).

[0334] Figures 2 and 3 show sequence comparisons of the heavy chain variable region and light chain variable region of mouse 3D6 and various humanized antibodies, respectively. Figures 9A and 9B show sequence comparisons of the heavy chain variable region of mouse 3D6 and the heavy chain variable region of various humanized antibodies. Figures 10A, 10B, 10C, and 10D show sequence comparisons of the light chain variable region of hu3D6VLv2 and the light chain variable region of various humanized antibodies.

[0335] For reasons including the potential impact on CDR higher-order structure and / or antigen binding, mediation of heavy-light chain interactions, interaction with constant regions, being a site for desirable or undesirable post-translational modifications, potential immunogenicity due to being an unusual residue for its position in the human variable region, and the acquisition of aggregation potential, and for other reasons, as further illustrated by examples, the following 35 variable region framework locations were considered as substitution candidates in 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions: L7(T7S), L10(T10S), L15(I15L), L17(Q17E), L37(L37Q), L45(K45R), L47(L47G, L47N, L47D, L47E, L47P, L47T, L47S, or L47A). L48 (I48G or I48D), L49 (Y49E), L83 (L83V), L86 (H86Y), L100 (A100Q), L106 (L106I), H1 (Q1E), H5 (Q5V), H11 (L11V), H17 (S17T), H20 (L20I), H23 (T23K), H38 (K38R), H42 (E42G), H43 (Q43K), H66 (K66R), H67 (A67V), H75 (S75T), H76 (N76D), H80 (L80M), H81 (Q81E), H82c (L82cV), H83 (T83R), H91 (Y91F), H93 (A93S), H94 (S94T), H108 (T108L), and H109 (L109V). The following nine variable region CDR locations were considered as substitution candidates in 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions, as further illustrated by examples: L24(K24R), L50(L50E, L50D, L50G, or L50V), L52(S52G), L54(L54D, L54G, L54N, L54E, L54Q, L54K, L54R, L54T, L54V, or L54S), H28(N28T), H54(N54D), H56(D56E), H58(V58I), and H60(D60E). In some humanized 3D6 antibodies, Kabat CDR-H2 has the amino acid sequence containing SEQ ID NO: 87.In some humanized 3D6 antibodies, Kabat-CDR-H2 has an amino acid sequence containing SEQ ID NO: 149. In some humanized 3D6 antibodies, Kabat-Cotia combination CDR-H1 has an amino acid sequence containing SEQ ID NO: 86, and Kabat-CDR-H2 has an amino acid sequence containing SEQ ID NO: 87. In some humanized 3D6 antibodies, Kabat-Cotia combination CDR-H1 has an amino acid sequence containing SEQ ID NO: 86, and Kabat-CDR-H2 has an amino acid sequence containing SEQ ID NO: 88. In some humanized 3D6 antibodies, Kabat-Cotia combination CDR-H1 has an amino acid sequence containing SEQ ID NO: 86, and Kabat-CDR-H2 has an amino acid sequence containing SEQ ID NO: 92. In some humanized 3D6 antibodies, Kabat-CDR-L1 has an amino acid sequence containing SEQ ID NO: 89. In some humanized 3D6 antibodies, Kabat-CDR-L2 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.

[0336] Here, as in other sections, the first residue mentioned is the residue of the humanized antibody formed by grafting the cotia-kabat combination CDR (in the case of Kabat CDR or CDR-H1) onto the human receptor framework, and the second residue mentioned is the residue being considered for substitution. Thus, the first residue mentioned within the variable region framework is human, and the first residue mentioned within the CDR is mouse.

[0337] The exemplified antibodies include any permutations or combinations of the following exemplified mature heavy chain and light chain variable regions: VHvb1 / VLvb1, VHvb1 / VLvb2, VHvb1 / VLvb3, VHvb2 / VLvb1, VHvb2 / VLvb2, VHvb2 / VLvb3, VHvb3 / VLvb1, VHvb3 / VLvb2, VHvb3 / VLvb3, VHvb4 / VLvb1, VHvb4 / VLvb2, VHvb4 / VLvb3, VHvb5 / VLvb1, VHvb5 / VLvb2, VHvb5 / VLv b3, VHvb6 / VLvb1, VHvb6 / VLvb2, VHvb6 / VLvb3, VHvb7 / VLvb1, VHvb7 / VLvb2, VHvb7 / VLvb3. The exemplified antibodies include the exemplified mature heavy chain variable region hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) and humanized 3D6VL light chain variable regions hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), hu3D6VLvb3 (SEQ ID NO: 85), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), hu3D6VLv2 L50D (SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (Sequence ID 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (Sequence ID 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (Sequence ID 137), hu3D6VLv2This includes any permutations or combinations with any of the following: L37Q_S52G_L54R_G100Q (sequence number 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (sequence number 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (sequence number 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (sequence number 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (sequence number 142), hu3D6VLv2 L37Q (sequence number 143), and hu3D6VLv2 G100Q (sequence number 144).

[0338] The exemplified antibodies include the exemplified mature heavy chain variable region hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), and hu3D6VHv1bA11 This includes any permutation or combination of D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) and any of the humanized 3D6VL light chain variable regions hu3D6VLv1 (SEQ ID NO: 20), hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv3 (SEQ ID NO: 22), and hu3D6VLv4 (SEQ ID NO: 22). The exemplified antibodies are the exemplified mature light chain variable regions hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), hu3D6VLvb3 (SEQ ID NO: 85), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), hu3D6VLv2 L50D (SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), and hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54t (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (Sequence ID 132), hu3D6VLv2 L37Q_L50D_L54R (Sequence ID 133), hu3D6VLv2 L37Q_L50E_L54G (Sequence ID 134), hu3D6VLv2 L37Q_L50E_L54R (Sequence ID 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (Sequence ID 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (Sequence ID 137), hu3D6VLv2 L37Q_s52G_L54R_G100Q (Sequence ID 138), hu3D6VLv2 L37Q_s52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2G100Q (sequence number 144) and humanized 3D6 heavy chain variable regions hu3D6VHv1 (sequence number 15); hu3D6VHv2 (sequence number 16); hu3D6VHv1b (sequence number 17); hu3D6VHv1bA11 (sequence number 18); hu3D6VHv5 (sequence number 19); hu3D6VHv1bA11b6G2 (sequence number 46); hu3D6VHv1bA11b6H3 (sequence number 47); hu3D6VHv1c (sequence number 48); This includes any permutations or combinations with any of the following: 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).

[0339] The present invention provides an antibody obtained by combining the humanized heavy chain variable region hu3D6VHv1bA11, also known as h3D6Hu5 (SEQ ID NO: 18), with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54R (L2-DIM4, SEQ ID NO: 122). The present invention provides an antibody obtained by combining the humanized heavy chain variable region hu3D6VHv1bA11, also known as h3D6Hu5 (SEQ ID NO: 18), with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54T (L2-DIM5, SEQ ID NO: 123). The present invention provides an antibody obtained by combining the humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146), with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54R (L2-DIM4, SEQ ID NO: 122). The present invention provides an antibody obtained by combining the humanized heavy chain variable region hu3D6VHv1bA11, also known as h3D6Hu5 (SEQ ID NO: 18), with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54G (L2-DIM3, SEQ ID NO: 121). The present invention provides an antibody obtained by combining the humanized heavy chain variable region hu3D6VHv1bA11, also known as h3D6Hu5 (SEQ ID NO: 18), with the humanized light chain variable region hu3D6VLv2 S52G (L2-DIM9, SEQ ID NO: 110). The present invention provides an antibody obtained by combining the humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) with the humanized light chain variable region hu3D6VLv2 L54G (L2-DIM7, SEQ ID NO: 94). The present invention provides an antibody obtained by combining the humanized heavy chain variable region hu3D6VHv1bA11, also known as h3D6Hu5 (SEQ ID NO: 18), with the humanized light chain variable region hu3D6VLv2 L50G (L2-DIM22, SEQ ID NO: 103).

[0340] The present invention provides antibodies in which any one of the exemplified humanized heavy chain variable regions is combined with a human heavy chain constant region. A representative human heavy chain constant region is provided as SEQ ID NO: 176 (IgG1: allotype G1m17,1). For example, SEQ ID NO: 178 shows the amino acid sequence of the mature heavy chain of a 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype). For example, SEQ ID NO: 180 shows the amino acid sequence of the heavy chain of a 3D6 humanized variant having a bovine alpha-lactalbumin signal peptide at the N-terminus (hu3D6VHv1bA11 IgG1 G1m17 allotype). The present invention also provides antibodies in which any one of the exemplified humanized light chain variable regions is combined with a light chain constant region. A representative light chain constant region is provided as SEQ ID NO: 177 (Kappa). For example, Sequence ID No. 179 shows the amino acid sequence of the mature light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa). For example, Sequence ID No. 181 shows the amino acid sequence of the light chain of a 3D6 humanized variant having a bovine alpha-lactalbumin signal peptide at its N-terminus (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa).

[0341] The present invention provides variants of 3D6 humanized antibodies in which the humanized mature heavy chain variable region is hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6VHvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), or hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity, and the humanized maturation light chain variable region is hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), hu3D6VLvb3 (SEQ ID NO: 85), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2 L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), hu3D6VLv2 L50D (SEQ ID NO: 99), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2 L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (Sequence ID 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (Sequence ID 142), hu3D6VLv2 L37Q (Sequence ID 143), or hu3D6VLv2We provide variants of 3D6 humanized antibodies that exhibit at least 90%, 95%, 96%, 97%, 98%, or 99% identity with G100Q (SEQ ID NO: 144). 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, 36, 37, 38, 39, 40, 41, 42, 43, or all 44 reverse mutations or other mutations from SEQ ID NOs: 76-80, SEQ ID NOs: 90-91, SEQ ID NOs: 146-148, SEQ ID NOs: 83-85, and SEQ ID NOs: 93-145.

[0342] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H93 is occupied by S, and H94 is occupied by T. In some humanized 3D6 antibodies, positions H93 and H94 are occupied by S and T, respectively.

[0343] In some humanized 3D6 antibodies, position H91 in the VH region is occupied by F.

[0344] 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 by V, H11 by V, H20 by I, H23 by K, H38 by R, H42 by G, H43 by K, H66 by R, H75 by T, H76 by D, H81 by E, H108 by L, and H109 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.

[0345] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H17 is occupied by T, H80 by M, and H83 by R. In some humanized 3D6 antibodies, positions H17, H80, and H83 in the VH region are occupied by T, M, and R, respectively.

[0346] In some humanized 3D6 antibodies, position H58 in the VH region is occupied by I.

[0347] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by a 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.

[0348] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H54 is occupied by D, and H56 is occupied by E.

[0349] 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 Q or E, H5 by Q or V, H11 by L or V, H17 by S or T, H20 by L or I, H23 by T or K, H28 by N or T, H38 by K or R, H42 by E or G, H43 by Q or K, and H54 by N or D. Furthermore, H56 is occupied by D or E, H58 by V or I, H66 by K or R, H67 by A or V, H75 by S or T, H76 by N or D, H80 by L or M, H81 by Q or E, H83 by T or R, H91 by F or Y, H93 by S, H94 by T, H108 by T or L, and H109 by L or V.

[0350] In some humanized 3D6 antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively, similar to 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, similar to 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, similar to 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, similar to huVHvb7.

[0351] In some humanized 3D6, position H60 is occupied by E, as in hu3D6VHv1bA11 D60E(h3D6VHvb8). In some humanized 3D6, position H82C is occupied by V, as in hu3D6VHv1bA11 L82cV. In some humanized 3D6, positions H60, H80, H81, H82c, and H83 are occupied by E, M, E, V, and R, as in hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R(h3D6VHvb9).

[0352] The heavy chain variable region of any of the previously referenced antibodies may be modified to further reduce immunogenicity. For example, in some humanized antibodies, position H80 is occupied by M and / or position H82c is occupied by V.

[0353] 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 S, L10 by S, L15 by L, L83 by V, L86 by Y, and L106 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.

[0354] 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 by T or S, L15 by I or L, L17 by Q or E, L24 by K or R, L37 by L or Q, L45 by K or R, L83 by L or V, L86 by H or Y, L100 by A or Q, and L106 by L or I.

[0355] 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.

[0356] The light chain variable region of any of the previously referenced antibodies may be modified to further reduce immunogenicity. For example, in some humanized antibodies, position L47 is occupied by G, N, D, E, P, T, S, or A; position L48 is occupied by G or D; position L49 is occupied by E; position L50 is occupied by E, D, G, or V; position L52 is occupied by G; and / or position L54 is occupied by D, G, N, E, Q, K, R, T, V, or S. The heavy chain variable region of any of the previously referenced antibodies may be modified to further reduce immunogenicity. For example, in some humanized antibodies, position H80 is occupied by M, and / or H82c is occupied by V.

[0357] In some humanized 3D6 antibodies, position L54 is occupied by D, similar to hu3D6VLv2 L54D. In some humanized 3D6 antibodies, position L54 is occupied by G, similar to hu3D6VLv2 L54G. In some humanized 3D6 antibodies, position L54 is occupied by N, similar to hu3D6VLv2 L54N. In some humanized 3D6 antibodies, position L54 is occupied by E, similar to hu3D6VLv2 L54E. In some humanized 3D6 antibodies, position L50 is occupied by E, similar to hu3D6VLv2 L50E. In some humanized 3D6 antibodies, position L54 is occupied by Q, similar to hu3D6VLv2 L54Q. In some humanized 3D6 antibodies, position L50 is occupied by D, similar to hu3D6VLv2 L50D. In some humanized 3D6 antibodies, position L54 is occupied by K, similar to hu3D6VLv2 L54K. In some humanized 3D6 antibodies, position L54 is occupied by R, similar to hu3D6VLv2 L54R. In some humanized 3D6 antibodies, position L54 is occupied by T, similar to hu3D6VLv2 L54T. In some humanized 3D6 antibodies, position L50 is occupied by G, similar to hu3D6VLv2 L50G. In some humanized 3D6 antibodies, position L48 is occupied by G, similar to hu3D6VLv2 I48G. In some humanized 3D6 antibodies, position L48 is occupied by D, similar to hu3D6VLv2 I48D. In some humanized 3D6 antibodies, position L47 is occupied by G, similar to hu3D6VLv2 L47G. In some humanized 3D6 antibodies, position L49 is occupied by E, similar to hu3D6VLv2 Y49E. In some humanized 3D6 antibodies, position L54 is occupied by V, similar to hu3D6VLv2 L54V. In some humanized 3D6 antibodies, position L54 is occupied by S, similar to hu3D6VLv2 L54S. In some humanized 3D6 antibodies, position L52 is occupied by G, similar to hu3D6VLv2 S52G. In some humanized 3D6 antibodies, position L47 is occupied by N, similar to hu3D6VLv2 L47N.In some humanized 3D6 antibodies, position L47 is occupied by D, similar to hu3D6VLv2 L47D. In some humanized 3D6 antibodies, position L47 is occupied by E, similar to hu3D6VLv2 L47E. In some humanized 3D6 antibodies, position L47 is occupied by P, similar to hu3D6VLv2 L47P. In some humanized 3D6 antibodies, position L47 is occupied by T, similar to hu3D6VLv2 L47T. In some humanized 3D6 antibodies, position L47 is occupied by S, similar to hu3D6VLv2 L47S. In some humanized 3D6 antibodies, position L47 is occupied by A, similar to hu3D6VLv2 L47A. In some humanized 3D6 antibodies, position L50 is occupied by V, similar to hu3D6VLv2 L50V.

[0358] In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and R, respectively, similar to hu3D6VLv2 L37Q_L50G_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and G, respectively, similar to hu3D6VLv2 L37Q_L50G_L54G. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and G, respectively, similar to hu3D6VLv2 L37Q_L50G_L54G. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and R, respectively, similar to hu3D6VLv2 L37Q_S52G_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and T, respectively, as in hu3D6VLv2 L37Q_S52G_L54T. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively, as in hu3D6VLv2 L37Q_S52G_L54D.

[0359] In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and R, respectively, similar to hu3D6VLv2 L37Q_L54R. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and G, respectively, similar to hu3D6VLv2 L37Q_L54G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and D, respectively, similar to hu3D6VLv2 L37Q_L54D. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and G, respectively, similar to hu3D6VLv2 L37Q_L50G. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and D, respectively, similar to hu3D6VLv2 L37Q_L50D. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and T, respectively, similar to hu3D6VLv2 L37Q_L54T. In some humanized 3D6 antibodies, positions L37 and L52 are occupied by Q and G, respectively, similar to hu3D6VLv2 L37Q_S52G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and E, respectively, similar to hu3D6VLv2 L37Q_L54E.

[0360] In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, D, and G, respectively, similar to hu3D6VLv2 L37Q_L50G_L54G. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, D, and R, respectively, similar to hu3D6VLv2 L37Q_L50D_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, E, and G, respectively, similar to hu3D6VLv2 L37Q_L50E_L54G. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, E, and R, respectively, similar to hu3D6VLv2 L37Q_L50E_L54R.

[0361] In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, G, G, and Q, respectively, as in hu3D6VLv2 L37Q_L50G_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L520, L54, and L100 are occupied by Q, G, R, and Q, respectively, as in hu3D6VLv2 L37Q_S52G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54, and L100 are occupied by Q, G, D, and Q, respectively, as in hu3D6VLv2 L37Q_S52G_L54D_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, G, and Q, respectively, as in hu3D6VLv2 L37Q_L50D_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, D, R, and Q, respectively, as in hu3D6VLv2 L37Q_L50D_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54, and L100 are occupied by Q, V, D, and Q, respectively, as in hu3D6VLv2 L37Q_L50V_L54D_G100Q.

[0362] In some humanized 3D6 antibodies, position L37 is occupied by Q, similar to hu3D6VLv2 L37Q. In some humanized 3D6 antibodies, position L100 is occupied by Q, similar to hu3D6VLv2 G100Q.

[0363] Some humanized 3D6 antibodies include a mature heavy chain variable region containing H1, H2, and H3, respectively, with the exception that position H28 may be occupied by N or T, H54 by N or D, H56 by D or E, position H58 by V or I, and position H60 by D or E, as indicated by SEQ ID NOs. 8, 9, and 10; and a mature heavy chain variable region containing H1, H2, and H3, respectively, with the exception that position L24 may be occupied by K or R, position L50 by L, E, D, G, or V, position L52 by S or G, and position L54 by L, D, G, N, E, Q, K, R, T, V, or S, as indicated by SEQ ID NOs. 12, 13, and 14. It includes a mature light chain variable region including L1, L2 and L3, and at least one of the following positions is occupied by a specified amino acid: H1 is occupied by Q, H5 is occupied by Q, H11 is occupied by L, H20 is occupied by L, H23 is occupied by T, H38 is occupied by K, H75 is occupied by S, H56 is occupied by E, H58 is occupied by I, H60 is occupied by E, H82 is occupied by V, L10 is occupied by T, and L17 is occupied by E. L24 is occupied by R, L37 by Q, L47 by G, N, D, E, P, T, S, or A, L48 by G or D, L49 by E, L50 by E, D, G, or V, L52 by G, L54 by D, G, N, E, Q, K, R, T, V, or S, L83 by L, L86 by H, L100 by Q, and L106 by L.

[0364] Some humanized 3D6 antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody 3D6, where position H27 may be occupied by F or Y, position H28 by N or T, position H29 by I or F, position H30 by K or T, position H51 by I or V, position H54 by N or D, position H60 by D, A or E, position H61 by P or E, position H102 by F or Y, position L50 by L, E, D, G or V, position L52 by S or G, position L A mouse antibody characterized by a heavy chain variable region having an amino acid sequence including SEQ ID NO: 7, and a light chain variable region having an amino acid sequence including SEQ ID NO: 11, except that 54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by a specified amino acid: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L100 is occupied by Q, H60 is occupied by E, and H82c is occupied by V.

[0365] In some humanized 3D6 antibodies, the heavy chain variable region has ≥85% identity with the human sequence. In some humanized 3D6 antibodies, the light chain variable region has ≥85% identity with the human sequence. In some humanized 3D6 antibodies, both the heavy chain variable region and the light chain variable region have ≥85% identity with the human germline sequence. In some humanized 3D6 antibodies, the three heavy chain CDRs are as defined by the Kabat / Cotia combination (SEQ ID NOs: 8, 9, and 10), and the three light chain CDRs are as defined by the Kabat / Cotia combination (SEQ ID NOs: 12, 13, and 14), provided that position H28 is occupied by N or T, position H54 by N or D, position H56 by D or E, position H58 by V or I, position H60 by D or E, position L24 by K or R, position L50 by L, E, D, G, or V, position L52 by S or G, and position L54 by L, D, G, N, E, Q, K, R, T, V, or S. In some humanized 3D6 antibodies, the Kabat / Cotia combination CDR-H1 has the amino acid sequence including SEQ ID NO: 86. In some humanized 3D6 antibodies, Kabat-CDR-H2 has an amino acid sequence containing SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 92, or SEQ ID NO: 149. In some humanized 3D6 antibodies, Kabat-CDR-L1 has an amino acid sequence containing SEQ ID NO: 89. In some humanized 3D6 antibodies, Kabat-CDR-L2 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.

[0366] The CDR region of such humanized antibodies may be identical or substantially identical to the CDR region of 3D6. The CDR region can be defined by any conventional definition (e.g., cotia, or a combination of cotia and kavat), but is preferably defined by kavat.

[0367] Unless otherwise specified, variable region framework locations correspond to Kabat numbering. Other such variants typically differ from the exemplary Hu3D6 heavy and light chain sequences by only a few substitutions, deletions, or insertions (e.g., typically 1, 2, 3, 5, 10, or 15). Such differences are usually found within the framework but can also occur within the CDR.

[0368] One possibility for further diversity in humanized 3D6 variants is further reverse mutations within the variable region framework. Many framework residues in humanized mAbs that do not contact the CDR can adapt to amino acid substitutions at the corresponding positions in donor mouse mAbs or other mouse or human antibodies, and many more potential CDR contact residues are also susceptible to substitution. Even amino acids within the CDR can be modified, for example, by residues found at the corresponding positions in the human receptor sequence used to provide the variable region framework. Furthermore, different human receptor sequences can be used, for example, for the heavy and / or light chains. If different receptor sequences are used, one or more of the reverse mutations recommended above may not need to be performed, as the corresponding donor and receptor residues already do not contain reverse mutations.

[0369] It is preferable that substitutions or reverse mutations in the humanized 3D6 variant (whether conservative or not) do not substantially affect the binding affinity or ability of the humanized mAb, i.e., its ability to bind to tau.

[0370] Humanized 3D6 antibodies are further characterized by their ability to bind to both phosphorylated and unphosphorylated tau, as well as to misfolded / aggregated forms of tau.

[0371] D. Chimeric and Benya Antibodies The present invention further provides non-human antibodies, particularly chimeric and veneer forms of the 3D6 antibody of the examples.

[0372] A chimeric antibody is an antibody in which the maturation variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are combined with the constant regions of the human light and heavy chains. Such antibodies exhibit, or entirely retain, the binding specificity of the mouse antibody and are approximately two-thirds the size of the human sequence. In one embodiment, a chimeric 3D6 antibody has the heavy chain amino acid sequence of SEQ ID NO: 72 and the light chain amino acid sequence of SEQ ID NO: 73.

[0373] A veneer antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some of the non-human variable region framework residues of a non-human antibody, but replaces other variable region framework residues that may contribute to B or T cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions of the human antibody sequence. The result is an antibody in which the CDRs are derived entirely or substantially from a non-human antibody, and the variable region framework of the non-human antibody has been made more human-like by substitution. A veneer-type 3D6 antibody is included in this invention.

[0374] E. Human antibodies Human antibodies against tau or its fragments (for example, amino acid residues 199-213 and / or 262-276 of SEQ ID NO: 3, corresponding to amino acid residues 257-271 and / or 320-334 of SEQ ID NO: 1, or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1, or any combination of two, three, or all four thereof) are provided by various techniques described below. Several human antibodies are selected by competitive binding experiments, by Winter's phage display method described above, or by other methods to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. Human antibodies can also be screened for specific epitope specificity by using only tau fragments, such as tau fragments containing only amino acid residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1), or only amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, and / or by screening antibodies against collections of tau variants, such as tau variants containing various mutations within amino acid residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1), or within amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, as target antigens, and / or by screening antibodies against collections of tau variants, such as tau variants containing various mutations within amino acid residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1).

[0375] Methods for generating human antibodies include the trioma method described in Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666, and the use of genetically modified mice containing human immunoglobulin genes (e.g., Lonberg et al. See also Dower et al., WO International Publication No. 93 / 12227 (1993); US Patent Nos. 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996); and Kucherlapati, International Publication No. 91 / 10741 (1991), phage display method (e.g., Dower et al., International Publication No. 91 / 17271; McCafferty et al.) Examples include methods described in al., International Publication No. 92 / 01047; U.S. Patent Nos. 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and 5,565,332); and International Publication No. 2008 / 081008 (e.g., immortalizing memory B cells isolated from humans, for example with EBV, screening them for desired properties, and cloning and expressing recombinant forms).

[0376] F. Selection of the steady-state region The variable regions of the heavy and light chains of chimeric antibodies, benija antibodies, or humanized antibodies can be linked to at least a portion of the human constant region. The selection of the constant region depends, to some extent, on whether antibody-dependent cytotoxicity, antibody-dependent phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 possess complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce more potent cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa. Numbering rules 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, ibid.).

[0377] One or more amino acids in the light chain, such as the C-terminal lysine of the heavy chain, and / or in the amino-terminus or carboxy-terminus of the heavy chain, may be partially or entirely missing or derivatized. Substitutions may be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Typical substitutions for increasing the half-life of an antibody include Gln at position 250 and / or Leu at position 428 (EU numbering of the constant region is used in this paragraph). Substitutions of 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. Several antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. If necessary, positions 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 positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 of human IgG1 in EU numbering. In some antibodies, EU numbering uses mutations at one or more positions of human IgG1 at positions 318, 320, and 322. 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.

[0378] 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 maturation variable domains of the heavy and light chains are linked via spacers.

[0379] The human constant region exhibits allotypic and isoallotypic variations between different individuals. That is, the constant region can differ in one or more polymorphic positions in different individuals. Isoallotypes differ from allotypes in that the serum recognizing the isoallotype binds to a non-polymorphic region of one or more other isotypes. For example, another heavy chain constant region is IgG1 G1m3 with or without C-terminal lysine. References to the human constant region include any native allotype or any permutation of residues occupying positions in a native allotype. A representative heavy chain constant region is SEQ ID NO: 176 with or without C-terminal lysine, and a representative light chain constant region is SEQ ID NO: 177.

[0380] G. Expression of recombinant antibody Several methods are known for generating chimeric and humanized antibodies using antibody-expressing cell lines (e.g., hybridomas). For example, the immunoglobulin variable region of an antibody can be cloned and sequenced using well-known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are used with a VH region leader peptide that includes the translation initiation as a 5' primer and the g2b constant region as a specific 3' primer. Representative primers are described in U.S. Patent Application Publication No. 2005 / 0009150 by Schenk et al. (hereinafter referred to as "Schenk"). By comparing sequences from multiple independently induced clones, it is possible to ensure that no changes are introduced during amplification. The VH region sequence can also be determined or confirmed by sequencing the VH fragment obtained by 5'RACE RT-PCR and 3' g2b-specific primers.

[0381] The variable light chain (VL) region can be cloned using similar methods. In one method, a consensus primer set is designed for amplification of the VL region using 5' primers that are designed to hybridize with 3' primers specific to the VL region containing the translation start codon and the Ck region downstream of the VJ binding region. In a second method, the cDNA encoding the VL is cloned using 5'RACE RT-PCR. Representative primers are described in Schenk (see above). The cloned sequence is then bound to a sequence encoding a human (or other non-human species) constant region.

[0382] In one method, the heavy and light chain variable regions are re-modified 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 cassette. After sequence validation, the heavy and light chain expression vectors can be simultaneously transduced into CHO cells to generate chimeric antibodies. The culture supernatant is collected 48 hours after gene transfer, and antibody production is analyzed by Western blotting or antigen binding by ELISA. The chimeric antibodies are then humanized as described above.

[0383] Chimeric, veneer, humanized antibodies, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably ligated to an antibody chain coding sequence, which in turn includes a naturally related or heterologous expression control sequence, e.g., a promoter. The expression control sequence may be a promoter system in a vector that can transform or transfect eukaryotic host cells. After the vector is incorporated into a suitable host, the host is maintained under conditions favorable for high-level expression of the nucleotide sequence, as well as collection and purification of cross-reactive antibodies.

[0384] These expression vectors are typically replicable in host organisms, either as episomes or as integrated portions of host chromosomal DNA. Generally, expression vectors contain selective markers, such as ampicillin resistance or hygromycin resistance, enabling the detection of cells transformed with the desired DNA sequence.

[0385] Escherichia coli is a useful prokaryotic host for expressing antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces species are yeast hosts that have suitable vectors containing, as needed, expression regulatory sequences, origins of replication, and stop sequences. Typical promoters include 3-phosphoglycerate kinase and other glycosphagocyte enzymes. Inducible yeast promoters include, in particular, promoters derived from alcohol dehydrogenases, isocytochrome C, and enzymes involved in maltose and galactose utilization.

[0386] 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 myeloma cells including SP2 / 0 and NS0. The cells may be non-human. Expression vectors for these cells may include expression regulatory sequences such as origins of replication, promoters, and 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 regulatory sequences may include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, and bovine papillomavirus, etc. See Co et al., J.Immunol. 148:1149 (1992).

[0387] Alternatively, antibody coding sequences can be incorporated into the transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, for example, U.S. Patents 5,741,957, 5,304,489, and 5,849,992). Suitable transgenes include light and / or heavy chain coding sequences operably linked to a mammary gland-specific gene, such as a promoter and enhancer derived from casein or beta-lactoglobulin.

[0388] Vectors containing the target DNA segment can be introduced into host cells using methods appropriate to the host cell type. For example, calcium chloride gene transfer is typically used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene guns, or viral gene transfer can be used for other cell hosts. Other methods used to transform mammalian cells include the use of polyblens, plasmofusion, liposomes, electroporation, and microinjection. For the production of genetically modified animals, the transgene can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs), and the cell nuclei of such cells can be introduced into enucleated oocytes.

[0389] By introducing a vector (one or more) encoding the heavy and light chains of an antibody into a cell culture, the cell pool can be screened in serum-free medium for growth productivity and product quality. The highest-producing cell pool can then be subjected to FACS-based single-cell cloning to generate monoclonal strains. Specific productivity exceeding 50 pg or 100 pg per cell per day can be used, corresponding to the product titer of cultures with over 7.5 g / L. The antibodies produced by the 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. The selected clones can then be placed in multiple vials and cryopreserved for later use.

[0390] Once expressed, the antibody can be purified using standard procedures in the art, including protein A capture, HPLC purification, column chromatography, and gel electrophoresis (see Scopes, Protein Purification (Springer-Verlag, NY, 1982) for an overview).

[0391] Methodologies for the commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell preservation, use of selection markers for copy number amplification, CHO terminators, or improvement of protein titer (see, for example, U.S. Patents 5,786,464, 6,114,148, 6,063,598, 7,569,339, International Publications 2004 / 050884, 2008 / 012142, 2008 / 012142, 2005 / 019442, 2008 / 107388, and 2009 / 027471, as well as U.S. Patent 5,888,809).

[0392] IV. Active immunogens The drugs used in active immunization help induce the same type of antibody in the patient as those described above in relation to passive immunization. The drugs used for active immunization may be of the same type as the immunogen used to produce monoclonal antibodies in experimental animals, for example, peptides of 3 to 15, 3 to 12, 5 to 12, or 5 to 8 adjacent amino acids from the tau region corresponding to residues 199 to 213 or 262 to 276 of SEQ ID NO: 3 (corresponding to residues 257 to 271 or 320 to 334 of SEQ ID NO: 1, for example, peptides containing residues 199 to 213 or 262 to 276 of SEQ ID NO: 3 (corresponding to residues 257 to 271 or 320 to 334 of SEQ ID NO: 1), or peptides containing the tau region corresponding to residues 259 to 268, 290 to 299, 321 to 330, or 353 to 362 of SEQ ID NO: 1, for example, peptides containing residues 259 to 268, 290 to 299, 321 to 330, or 353 to 362 of SEQ ID NO: 1. To induce antibodies that bind to the same or overlapping epitopes as 3D6, the epitope specificity of these antibodies can be mapped (e.g., by examining their binding to a series of overlapping peptides across tau). Fragments of tau consisting of, containing, or overlapping epitopes can then be used as immunogens. Such fragments can typically be used in their non-phosphorylated form.

[0393] If used, heterologous carriers and adjuvants may be the same as those used to produce monoclonal antibodies, but may be selected for better pharmaceutical suitability for human use. Suitable carriers include serum albumin, hemocyanin from water oysters, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin, or toxins or attenuated derivatives from other pathogenic bacteria such as diphtheria (e.g., CRM197), Escherichia coli, cholera, or H. pylori. T cell epitopes are also suitable carrier molecules. Some conjugates may be formed by linking the agents of the present invention to immunostimulatory polymer molecules (e.g., tripalmitoyl-S-glycerol cysteine ​​(Pam3Cyc), mannan (mannose polymer), or glucan (αβ1→2 polymer)), cytokines (e.g., IL-1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1-α and β, and RANTES). The immunogen may be linked to a carrier with or without the use of a spacer amino acid (e.g., gly-gly). Further carriers include virus-like particles. Virus-like particles (VLPs), also called pseudovirions or virus-inducing particles, represent a subunit structure composed of multiple copies of a viral capsid and / or envelope protein that can self-assemble in vivo into a spherically symmetric VLP (Powilleit, et al., (2007) PLoS ONE 2(5):e415). Alternatively, peptide immunogens can be linked to at least one artificial T-cell epitope capable of binding a large portion of MHC class II molecules, such as the panDR epitope ("PADRE"). PADRE is described in U.S. Patent No. 5,736,142, International Publication No. 95 / 07707, and Alexander J et al, Immunity, 1:751-761 (1994). Active immunogens may be provided in a multimeric form in which multiple copies of the immunogen and / or its carrier are provided as a single covalent molecule.

[0394] The fragment is often administered with a pharmaceutically acceptable adjuvant. The adjuvant increases the titer and / or binding affinity of the induced antibody compared to when the peptide is used alone. Various adjuvants can be used in combination with the immunogenic fragment of tau to induce an immune response. Preferred adjuvants enhance the intrinsic response to the immunogen without causing conformational changes to the immunogen that would affect the nature and morphology of the response. Preferred adjuvants include aluminum salts such as aluminum hydroxide and aluminum phosphate, and 3-O-deacylated monophosphoryl lipid A (MPL®) (UK Patent No. 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa)). Stimulon® QS-21 is a triterpene glycoside or saponin isolated from the bark of Quillaja saponaria molina, found in South America (see Kensil et al., Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540), (Aquila BioPharmaceuticals, Framingham, Massachusetts (now Antigenics, Inc., New York, New York). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), which are sometimes combined with immunostimulants such as monophosphoryl lipid A (see Stoute et al., N.Engl. J.Med. 336, 86-91 (1997)), Pluronic polymers, and dead mycobacteria. The Ribi adjuvant is an oil-in-water emulsion. Ribi contains a metabolizable oil (squalene) emulsified in saline containing Tween 80. Ribi also contains a purified mycobacterial product and bacterial monophosphoryl lipid A, which act as immunostimulants. Another adjuvant is CpG (International Publication No. 98 / 40100).The adjuvant can be administered as a component of a therapeutic composition containing an activator, or separately before, simultaneously with, or after the administration of the therapeutic agent.

[0395] Analogues of the natural fragment of tau that induce antibodies against tau can also be used. For example, one, more, or all L-amino acids may be substituted with D-amino acids in such peptides. The order of amino acids may also be reversed (retropeptides). In some cases, the peptide may contain all of the D-amino acids in reverse order (retroinversopeptides). Peptides and other compounds that do not necessarily have a significant amino acid sequence similarity to tau peptides can still serve as mimics of tau peptides and induce similar immune responses. Anti-idiotype antibodies against the monoclonal antibodies against tau described above can also be used. Such anti-Id antibodies mimic the antigen and produce an immune response to it (see Essential Immunology, Roit ed., Blackwell Scientific Publications, Palo Alto, CA 6th ed., p.181).

[0396] Peptides (and optionally carriers fused to peptides) can also be administered in the form of nucleic acids encoding the peptide and expressed in situ in the patient. The nucleic acid segment encoding the immunogen is typically ligated to regulatory elements such as promoters and enhancers, which enable the expression of the DNA segment in the patient's intended target cells. For expression in blood cells, promoter and enhancer elements from light-chain and heavy-chain immunoglobulin genes, or the CMV major pre-early promoter and enhancer, are suitable for directing expression because they are desirable for inducing an immune response. The ligated regulatory elements and coding sequences are often cloned into a vector. 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 ligated as single-chain antibodies. Antibodies for passive administration can also be prepared, for example, by affinity chromatography from the serum of a patient treated with a peptide immunogen.

[0397] DNA can be delivered in its naked form (i.e., colloidal or without encapsulation material). Alternatively, it can be delivered using retroviruses (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenovirus vectors, including retrovirus-derived vectors such as MMLV, HIV-1, and ALV (see, e.g., Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)); lentiviral vectors, such as those based on HIV or FIVgag sequences; pox family viral vectors, including vaccinia virus and avian poxvirus; and alphavirus viral vectors, such as those derived from Sindbis and Semlik Forest Virus (see, e.g., Dubensky et al.). Several viral vector systems can be used, including rhabdoviruses such as al., J. Virol. 70, 508-519 (1996), Venezuelan encephalitis virus (see U.S. Patent Nos. 5,643,576) and vesicular stomatitis virus (see International Publication No. 96 / 34625), as well as papillomavirus (see Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al, International Publication No. 94 / 12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).

[0398] DNA encoding an immunogen, or DNA encoding antibody heavy and / or light chains, or vectors containing them, can be packaged in liposomes. Suitable lipids and related analogues are described in U.S. Patents 5,208,036, 5,264,618, 5,279,833, and 5,283,185. Vectors and DNA encoding immunogens or antibody heavy and / or light chains can also be adsorbed or bound to microparticle carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides) (see, e.g., McGee et al., J. Micro Encap. 1996).

[0399] Vectors or segments from which antibody heavy and / or light chains are encoded ex vivo into cells, for example, cells explanted from individual patients (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or generic donor hematopoietic stem cells, and then, after selecting cells that have typically incorporated transgenes, the cells can be re-transplanted into the patient (see, for example, International Publication No. 2017 / 091512). Typical patient-derived cells include patient-derived pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal).

[0400] Vectors or segments thereof encoding antibody heavy and / or light chains can be introduced ex vivo into any region of interest in a cell, such as the albumin gene or other safe Haber genes. Cells incorporating the vector can be transplanted with or without prior differentiation. Cells can be transplanted into specific tissues, such as secretory tissue or sites of lesions, or systemically, such as by intravenous infusion into the bloodstream. For example, cells can be transplanted into the patient's secretory tissue, such as the liver, and may be pre-differentiated into cells present in that tissue, such as hepatocytes in the case of the liver. Antibody expression in the liver leads to the secretion of antibodies into the bloodstream.

[0401] H. Antibody Screening Assay As described above, antibodies can first be screened for their desired binding specificity. Active immunogens can similarly be screened for their ability to induce antibodies with such binding specificity. In this case, experimental animals are immunized with the active immunogen, and the resulting serum is tested for appropriate binding specificity.

[0402] Subsequently, antibodies with the desired binding specificity can be tested in cell and animal models. Neuronal cells should be prioritized as the cells used for such screening. Cell models of tau pathology have been reported in which neuroblastoma cells are genetically modified with the 4-repeat domain of tau, along with mutations associated with tau pathology as needed (see, e.g., Delta K280, Khlistunova, Current Alzheimer Research 4, 544-546 (2007)). In another model, tau is induced in neuroblastoma N2a cell lines by adding doxycycline. This cell model allows for the study of the toxicity of tau to cells in soluble or aggregated states, the appearance of tau aggregates after switching on tau gene expression, the lysis of tau aggregates after switching off gene expression again, and the efficiency of antibodies in suppressing the formation of tau aggregates or deaggregating them.

[0403] Antibodies or active immunogens can also be used to screen for tau-related diseases in transgenic animal models. Such transgenic animals may contain a tau transgene (e.g., any human isoform) and, if necessary, human APP transgenes such as tau, ApoE, presenilin, or alpha-synuclein phosphorylating kinases, among others. Such transgenic animals are treated to develop at least one sign or symptom of a tau-related disease.

[0404] A representative gene transgenic animal is the K3 mouse strain (Itner et al., Proc. Natl. Acad. Sci. USA 105(41):15997-6002 (2008)). These mice possess a human tau transgene with the K369I mutation (associated with Pick's disease) and the Thy1.2 promoter. This model exhibits a rapid course of neurodegeneration, motor impairment, and degeneration of afferent fibers and cerebellar granule cells. Another representative animal is the JNPL3 mouse strain. These mice possess a human tau transgene with the P301L mutation (associated with frontotemporal dementia) and the Thy1.2 promoter (Taconic, Germantown, NY, Lewis, et al., Nat Genet. 25:402-405 (2000)). These mice exhibit a slower course of neurodegeneration. These mice develop neurofibrillary hives in several brain regions and spinal cord, which are incorporated herein by reference in their entirety. This is an excellent model for studying the consequences of hives development and for screening for therapies that can suppress the formation of these aggregates. Another advantage of these animals is the relatively early onset of the lesions. In homozygous lines, tau pathology and associated behavioral abnormalities can be observed at least as early as 3 months, but these animals remain relatively healthy until at least 8 months of age. That is, at 8 months, these animals are able to walk around, feed on their own, 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 produced titers of approximately 1,000, with fewer neurofibrillary hives, reduced pSer422, and reduced weight loss compared to untreated control ice.

[0405] The activity of antibodies or activators can be assessed by a variety of criteria, including reductions in total tau or phosphorylated tau levels, reductions in other pathological features such as α-β amyloid deposits, and inhibition, delay, or behavioral defects. Active immunogens can also be tested for the induction of antibodies in serum. Both passive and active immunogens can be tested for the passage of antibodies across the blood-brain barrier into the brain of genetically modified animals. Antibodies or antibody-inducing fragments can also be tested in non-human primates that develop symptoms of tau-characterized diseases, either naturally or through induction. Testing of antibodies or activators is usually performed concurrently with control experiments, which are carried out in parallel except that the antibody or activator is absent (e.g., replaced by a vehicle). Subsequently, the reduction, delay, or suppression of signs or symptoms of the disease attributable to the antibody or activator during testing can be evaluated compared to the control.

[0406] V. Patients who are eligible for treatment The presence of neurofibrillary folds has been observed in several diseases, including Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glial tauopathy (GGT), and progressive supranuclear palsy (PSP). This treatment regimen can also be used to treat or prevent any of these diseases. Due to the extensive relationship between neurological disorders and disease conditions and tau, this dosing regimen can be used to treat or prevent any subject showing elevated levels of tau or phosphorylated tau (e.g., in CSF) compared to the mean levels of individuals without neurological disorders. This regimen can also be used to treat or prevent neurological disorders in individuals with tau mutations associated with neurological disorders. This method is particularly suitable for the treatment or prevention of Alzheimer's disease, and especially in patients.

[0407] Patients eligible for treatment include individuals at risk of the disease but without symptoms, as well as patients currently exhibiting symptoms. Patients at risk of the disease include those with a known genetic risk for the disease. Such individuals include those with relatives experiencing the disease, and those 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 disorders. For example, heterozygous and homozygous ApoE4 alleles are also associated with the risk of Alzheimer's disease. Other markers of Alzheimer's disease risk include mutations in the APP gene, particularly at position 717 and at positions 670 and 671, known as the Hardy and Swedish mutations, PS1 and PS2 mutations in the presenilin gene, and a family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be identified by PET imaging due to characteristic dementia and the presence of the aforementioned risk factors. Furthermore, several diagnostic tests are available to identify individuals with AD. These include measuring CSF tau or phosphorylated tau and αβ42 levels. Elevated levels of tau or phosphorylated tau and decreased αβ42 levels indicate the presence of AD. Several mutations are associated with Parkinson's disease. Mutations in Ala30Pro or Ala53, or other genes such as PARK8 of leucine-rich repeat kinase, are associated with Parkinson's disease. Individuals may also be diagnosed with any of the aforementioned neurological disorders according to the DSM IV TR criteria.

[0408] In asymptomatic patients, treatment can be initiated at any age (e.g., 10, 20, 30). However, it is usually not necessary to start treatment until the patient reaches 40, 50, 60, or 70 years of age. Treatment typically requires multiple doses over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response declines, additional immunization is required. In patients with potential Down syndrome, treatment can be initiated prenatally by administering the medication to the mother, or immediately after birth.

[0409] I. Nucleic acids The present invention further provides nucleic acids encoding either the heavy chain or the light chain (e.g., SEQ ID NOs: 7, SEQ ID NOs: 11, SEQ ID NOs: 76-80, SEQ ID NOs: 90-91, SEQ ID NOs: 146-148, SEQ ID NOs: 83-85, SEQ ID NOs: 93-145, and SEQ ID NOs: 178-181). A representative nucleic acid encoding the heavy chain of the present invention is SEQ ID NOs: 182, and a representative nucleic acid encoding the light chain of the present invention is SEQ ID NOs: 183. Optionally, such nucleic acids may further encode a signal peptide and express a signal peptide linked to a variable region. The coding sequence of the nucleic acid can be operably linked to a regulatory sequence to ensure the expression of coding sequences such as promoters, enhancers, ribosome binding sites, and transcription termination signals. The regulatory sequence may include a promoter, e.g., a prokaryotic promoter or a eukaryotic promoter. Nucleic acids encoding the heavy chain or light chain can be codon-optimized for expression in host cells. Nucleic acids encoding the heavy chain and light chain can encode selectable genes. Nucleic acids encoding the heavy chain and light chain can be generated in isolation or inserted into one or more vectors. Nucleic acids can be synthesized, for example, by solid-phase synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding the heavy and light chains can be linked together, for example, as a single continuous nucleic acid within an expression vector, or they can be inserted into separate expression vectors, for example, each in its own expression vector.

[0410] J. Conjugate Antibody Conjugate antibodies that specifically bind to antigens such as tau are used for: detection of tau presence; monitoring and evaluation of therapeutic agents used to treat patients diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glial tauopathy (GGT), or progressive supranuclear palsy (PSP); and tau aggregation. It is useful for the suppression or reduction of tau fibrillation; the suppression or reduction of tau fibrillation; the reduction or removal of tau deposits; the stabilization of the non-toxic higher-order structure of tau; or for the treatment or prevention of Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glial tauopathy (GGT), or progressive supranuclear palsy (PSP). For example, such antibodies can be conjugated with parts of other therapeutic agents, other proteins, other antibodies, and / or detectable labels. See International Publication No. 03 / 057838; U.S. Patent No. 8,455,622.Such therapeutic components may be any medications that can be used to treat, combat, alleviate, prevent, or improve undesirable conditions or diseases in patients, such as Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroidal glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroidal glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0411] The conjugated therapeutic portion may contain any of the following bioactive substances: cytotoxic agents, cell division inhibitors, neurodifferentiation inducers, neuroprotective agents, radiotherapeutic agents, immunomodulators, or substances that promote or enhance the activity of an antibody. Cytotoxic agents may be any agents that are toxic to cells. Cytotoxic agents may be any agents that inhibit cell proliferation. Neurodifferentiation inducers may be any agents containing chemical or protein substances that promote neuronal maintenance, proliferation, or differentiation. Neuroprotective agents may be agents containing chemical or protein substances that protect neurons from acute invasion or degenerative processes. Immunomodulators may be any agents that stimulate or suppress the development or maintenance of an immune response. Radiotherapeutic agents may be any molecules or compounds that emit radiation. When such a therapeutic portion is conjugated to a tau-specific antibody, such as the antibodies described herein, the conjugated therapeutic portion will have a specific affinity for tau-related disease-affected cells that is superior to that of normal cells. Therefore, administration of the conjugated antibody directly targets cancer cells with minimal damage to surrounding healthy tissue. This may be particularly useful when the therapeutic portions are too toxic to be administered alone. Furthermore, a smaller amount of the therapeutic agent can be used.

[0412] Some such antibodies can be modified to act as immunotoxins. See, for example, U.S. Patent No. 5,194,594. For example, lysine, a cytotoxin derived from plants, can be conjugated to antibodies using the bifunctional reagent S-acetylmercaptosuccinate anhydride and succinimidyl 3-(2-pyridyldithio)propionate for lysine. See Pietersz et al., Cancer Res. 48(16):4469-4476 (1998). Conjugation reduces the B-chain binding activity of lysine, but neither the toxic potential of the lysine A-chain nor the antibody activity is impaired. Similarly, saporins, inhibitors of ribosome assembly, can also be conjugated to antibodies via disulfide bonds between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

[0413] Some such antibodies can be bound to radioactive isotopes. Examples of radioactive isotopes include, for example, yttrium. 90 (90Y) Indium 111 (111In) 131 I, 99 mTc, radioactive silver-111, radioactive silver-199, and bismuth 213 These include: Linking of radioisotopes to antibodies can be carried out using conventional difunctional chelates. Sulfur-based linkers can be used for linking radioactive silver-111 and radioactive silver-199. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linking of silver radioisotopes may require reduction of immunoglobulins with ascorbic acid. Ibritumomabutiuxetane can be used for radioisotopes such as 111In and 90Y, reacting with these isotopes to form 111In-ibritumomabutiuxetane and 90Y-ibritumomabutiuxetane, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0414] Some such antibodies can be conjugated to other therapeutic moieties. These therapeutic moieties may be, for example, cytotoxic, cell division-arresting, neurotrophic, or neuroprotective. For instance, antibodies can be conjugated to tubulin inhibitors such as maytansine, geldanamycin, and tubulin conjugates (e.g., auristatin), or to toxic chemotherapeutic agents such as small groove conjugates like calitiamycin. Other representative therapeutic agents include drugs known to be useful in treating, managing, or improving patients with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroid glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroid glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0415] Antibodies may also be bound to other proteins. For example, antibodies may be bound to finomers. Finomers are small binding proteins (e.g., 7 kDa) derived from the human oncogene Fyn SH3 domain. They can be stable and soluble and may lack cysteine ​​residues and disulfide bonds. Finomers can be genetically modified to bind to 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 an antibody to generate bi- or tri-specific FynomAb with different structures. Finomers can be selected from a finomer library through screening techniques using FACS, Biacore, and cell-based assays, which enable efficient selection of finomers with optimal properties. Examples of finomers are disclosed in Grabulovski et al., J. Biol. Chem. 282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner et al., Acta. Crystallogr. D. Biol. Crystallogr. 69 (Pt6):1124-1137 (2013); and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

[0416] The antibodies disclosed herein can also be conjugated to or attached to one or more other antibodies (e.g., to form an antibody heteroconjugate). Such other antibodies can bind to different epitopes within tau or to different target antigens.

[0417] Antibodies may also be conjugated with detectable labels. Such antibodies can be used, for example, to assess the efficacy of treatment for Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroidal glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroidal glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Such antibodies are particularly useful in making such determinations in subjects with or susceptible to Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glial tauopathy (GGT), or progressive supranuclear palsy (PSP), or in appropriate biological samples obtained from such subjects. Representative detectable labels that can bind to or link to antibodies 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, dansilchloride, or phycoerythrin; luminescent materials such as luminol; bioluminescent materials such as luciferase, luciferin, and aequorin; and radioactive silver-111, radioactive silver-199, and bismuth. 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 Enjoy, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117 Examples include radioactive materials such as tin; positron-emitting metals obtained using various positron emission tomography techniques; non-radioactive paramagnetic metal ions; and molecules that are radiolabeled or conjugated with specific radioisotopes.

[0418] The linkage of radioisotopes to antibodies can be carried out using conventional difunctional chelates. Sulfur-based linkers can be used for the linkage of radioactive silver-111 and radioactive silver-199. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes may require reduction of immunoglobulins with ascorbic acid. Ibritumomabutiuxetane can be used for radioisotopes such as 111In and 90Y, reacting with these isotopes to form 111In-ibritumomabutiuxetane and 90Y-ibritumomabutiuxetane, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0419] A portion of the therapeutic agent, other proteins, other antibodies, and / or a detectable label may be directly or indirectly bound to or conjugated to the antibody of the present invention via an intermediary (e.g., a linker). For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); 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 al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers can be used that release the bound therapeutic moiety, protein, antibody, and / or detectable label upon exposure to a specific protease under acidic or reducing conditions, or under other predetermined conditions.

[0420] VI. Pharmaceutical Compositions and Methods of Use For preventative use, antibodies, or agents for inducing antibodies, or pharmaceutical compositions thereof, are administered to patients susceptible to or at risk of the disease (e.g., Alzheimer's disease) in a dosage regimen (dosage, frequency, and route of administration) effective in reducing the risk of the disease, reducing the severity of the disease, or delaying the onset of at least one sign or symptom of the disease. In particular, the dosage regimen is preferably effective in inhibiting or delaying tau or phosphorylated tau and the filament pairs formed therefrom in the brain, and / or inhibiting or delaying their toxic effects, and / or inhibiting or delaying the onset of behavioral defects. For therapeutic use, antibodies, or agents for inducing antibodies, are administered to patients suspected of having the disease (e.g., Alzheimer's disease) or already suffering from the disease in a dosage regimen (dosage, frequency, and route of administration) effective in improving or at least preventing further deterioration of at least one sign or symptom of the disease. In particular, this dosage regimen is preferably effective in reducing or at least suppressing further increases in the levels of tau, phosphorylated tau, or filament pairs formed therefrom, and associated toxicity and / or behavioral disorders.

[0421] The dosing regimen is considered therapeutically or prophylactically effective if individual treated patients achieve better treatment outcomes than the average treatment outcomes in a control population of equivalent patients not treated by the method of the present invention, or if a comparative clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial) demonstrates better treatment outcomes for treated patients compared to control patients at a level of p<0.05 or 0.01, or even 0.001.

[0422] The effective dose varies depending on many different factors, such as the method of administration, the target site, the patient's physiological state, whether the patient is an ApoE carrier, whether the patient is human or animal, whether other drugs are administered, and whether the treatment is prophylactic or therapeutic.

[0423] Typical antibody dose ranges are approximately 0.01–60 mg / kg per kg of patient body weight, or approximately 0.1–3 mg / kg, 0.15–2 mg / kg, or 0.15–1.5 mg / kg. Antibodies can be administered daily, every other day, weekly, bi-weekly, monthly, quarterly, or according to any other schedule determined by empirical analysis. Typical treatment requires administration over a long period, for example, at least 6 months, in multiple doses. Further typical dosing regimens require administration every two weeks, monthly, or every 3–6 months.

[0424] The amount of drug for active administration varies in humans from 0.1 to 500 μg / patient, more commonly from 1 to 100 or 1 to 10 μg / injection. The timing of injections can vary greatly, from once daily to once a year or once every 10 years. A typical regimen consists of immunization at time intervals such as every 6 weeks or every 2 months, followed by booster injections. Another regimen consists of immunization and booster injections at 1, 2, and 12 months. Yet another regimen requires injections every 2 months for life. Alternatively, booster injections may be based on irregular criteria, as indicated by monitoring of the immune response.

[0425] Antibodies, or agents for inducing antibodies, are preferably administered via peripheral routes (i.e., routes through which the administered or induced antibody crosses the blood-brain barrier and reaches the intended site in the brain). Routes of administration include local, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, or intramuscular administration. Preferred routes of administration for antibodies are intravenous and subcutaneous. Preferred routes for active immunization are subcutaneous and intramuscular. These types of injections are most typically performed in the muscles of the arm or leg. In some methods, the agent is injected directly into specific tissues where deposits have accumulated, e.g., intracranial injection.

[0426] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be supplied in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated with one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solution, preferably with a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulation agents such as suspensions, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form to prepare a suitable vehicle before use, e.g., sterile, pyrogen-free water.

[0427] This treatment regimen may be administered in combination with another drug effective in treating or preventing the disease being treated. For example, in the case of Alzheimer's disease, this regimen may be combined with immunotherapy against Aβ (International Publication No. 2000 / 072880), cholinesterase inhibitors, or memantine, or in the case of immunotherapy for Parkinson's disease, it may be combined with immunotherapy against α-synuclein (International Publication No. 2008 / 103472), levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, amantadine, or anticholinergics.

[0428] Antibodies are administered in an effective dosing regimen, which means a regimen including a dose, route of administration, and frequency of administration that delays the onset, reduces the severity, suppresses further exacerbation, and / or improves at least one sign or symptom of the disorder being treated. If the patient already has the disorder, the regimen may be referred to as a therapeutically effective regimen. If the patient is at high risk of the disorder compared to the population but is not yet asymptomatic, the regimen may be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in individual patients compared to historical controls or past experience of the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated in the treated patient population preclinically or during clinical trials compared to a control population of untreated patients.

[0429] Typical antibody doses 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 a fixed dose of 10–4000 mg or 10–1500 mg. The dose depends particularly on the patient's condition and, if any, their response to prior treatment, whether the treatment was prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0430] Administration may be parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular. Some antibodies can be administered intravenously or subcutaneously into the systemic circulation. Intravenous administration may be, for example, an infusion over 30 to 90 minutes.

[0431] The frequency of administration depends particularly on the half-life of the circulating antibody, the patient's condition, and the route of administration. Depending on the patient's condition or the progression of the disorder being treated, the frequency can be daily, weekly, monthly, every three months, or at irregular intervals. Typical frequencies for intravenous administration range from weekly to every three months, depending on the cause of treatment, but more frequent or less frequent administration is also possible. For subcutaneous administration, typical frequencies range from daily to monthly, but more frequent or less frequent administration is also possible.

[0432] The number of doses depends on whether the disorder is acute or chronic and the disorder's response to treatment. For acute disorders or acute exacerbations of chronic disorders, a dose of 1 to 10 times is often sufficient. Sometimes, for acute disorders or acute exacerbations of chronic disorders, a single rapid dose, divided as needed, is sufficient. Treatment may be repeated for recurrences of acute disorders or exacerbations. For chronic disorders, antibodies may be administered at regular intervals, e.g., weekly, bi-weekly, monthly, every three months, every six months, for at least one, five, or ten years, or for the patient's lifetime.

[0433] 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 condensates and tau inclusions) in a patient. The method works by administering a reagent such as an antibody that binds to tau (e.g., a mouse, humanized, chimeric, or veneerated 3D6 antibody) to the patient, and then detecting the drug after it has bound. Antibodies that bind to tau epitopes in amino acid residues 199-213 or 262-276 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO: 1), or in amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, are preferred. In some methods, the antibody binds to epitopes within amino acid residues 199-213 of SEQ ID NO: 3 (corresponding to amino acid residues 257-271 of SEQ ID NO: 1) or within amino acid residues 262-276 of SEQ ID NO: 3 (corresponding to amino acid residues 320-334 of SEQ ID NO: 1). In some methods, the antibody binds to epitopes within amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1. The removal reaction of administered antibodies can be avoided or reduced by using antibody fragments that lack a full-length constant region, such as Fab. In some methods, the same antibody can function as both a treatment and a diagnostic reagent.

[0434] Diagnostic reagents can be administered intravenously into the patient's body, intracranially, or directly to the brain by drilling a hole in the skull. The dosage of the reagent must be within the same range as that used for treatment methods. Typically, the reagents are labeled, although in some methods, the primary reagent, which has an affinity for tau, is left unlabeled and a secondary label is used to bind it to the primary reagent. The choice of labeling depends on the means of detection. For example, fluorescent labeling is suitable for optical detection. The use of paramagnetic labeling is suitable for tomographic detection without surgical intervention. Radioactive labeling can also be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0435] In vivo imaging of tau protein deposits is useful for diagnosing or confirming tauopathy, such as Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, and Pick's disease, or susceptibility to such diseases. For example, this method can be used in patients exhibiting symptoms of dementia. If a patient has abnormal neurofibrillary folds, that patient may have Alzheimer's disease. Alternatively, if a patient has abnormal tau inclusions, depending on the location of the inclusions, that patient may have frontotemporal dementia. This method can also be used in asymptomatic patients. The presence of abnormal tau protein deposits indicates a future susceptibility to symptomatic disease. This method is also useful for monitoring disease progression and / or response to treatment in patients previously diagnosed with tau-related disorders.

[0436] Diagnosis may be made by comparing the number, size, and / or intensity of labeled loci to corresponding baseline values. Baseline values ​​may represent the mean level in a non-disease population. Baseline values ​​may also represent previous levels determined in the same patient. For example, a patient's baseline value can be measured before initiating tau immunotherapy treatment, and then the measured value can be compared to the baseline value. A decrease in value relative to the baseline signal indicates a positive response to treatment.

[0437] In some patients, the diagnosis of tauopathy may be aided by performing a PET scan. The PET scan can be performed, for example, using conventional PET imaging devices and auxiliary equipment. The scan typically includes one or more areas of the brain commonly known to be associated with tau protein deposits, and one or more areas where deposits, if any, are usually present and serve as controls.

[0438] Signals detected by PET scans can be represented as multidimensional images. These images may represent a cross-section of the brain in two dimensions, a three-dimensional brain in three dimensions, or changes in the three-dimensional brain over time in four dimensions. Color scales can be used, indicating different labeling levels and the estimated tau protein deposits detected. Scan results can also be presented numerically, in terms of the amount of labeling detected and the resulting number of tau protein deposits. Labeling present in brain regions known to be associated with deposition in specific tauopathies (e.g., Alzheimer's disease) can be compared to labeling present in regions not known to be associated with deposition to provide a ratio representing the degree of deposition in the former regions. Such ratios for the same radiolabeled ligand provide a comparable measure of tau protein deposition and its variation between different patients.

[0439] In some cases, PET scans are performed simultaneously with or during the same patient visit as MRI or CAT scans. MRI or CAT scans provide more anatomical detail of the brain than PET scans. However, images from PET scans can be superimposed onto MRI or CAT scan images, which more accurately show the location of PET ligands and putative tau deposits relative to anatomical structures within the brain. Some devices can perform both a patient's PET scan and MRI or CAT scan without changing the patient's position between scans, facilitating image superposition.

[0440] Suitable PET ligands include the radiolabeled antibodies of the present invention (e.g., mouse, humanized, chimeric, or veneer 3D6 antibodies). The radioisotopes used are, for example, C 11 , N 13 , O 15 F 18 , or I 123 This is possible. The interval between PET ligand administration and scan execution may depend on the PET ligand, particularly its uptake and removal rates in the brain, as well as the half-life of its radiolabeled form.

[0441] PET scans can also be performed prophylactically in asymptomatic patients or those with mild cognitive impairment who have not been diagnosed with tauopathy but are still at high risk of developing it. In asymptomatic patients, scans are particularly useful for individuals considered to be at high risk of tauopathy due to family history, genetic or biochemical risk factors, or age. Prophylactic scans can be initiated, for example, in patients aged 45–75 years. In some patients, the first scan is performed at age 50.

[0442] Prophylactic scans may be performed, for example, at intervals of 6 months to 10 years, preferably 1 to 5 years. In some patients, prophylactic scans are performed annually. If a PET scan performed as a prophylactic measure shows abnormally high levels of tau protein deposits, immunotherapy may be initiated, and thereafter PET scans may be initiated in the same way as in patients diagnosed with tauopathy. If a PET scan performed as a prophylactic measure shows levels of tau protein deposits within the normal range, 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 normal levels of tau protein deposition are detected as described above, combining prophylactic scanning with tau-targeted immunotherapy can reduce the level of tau protein deposition to or near normal levels, or at least prevent it from increasing further, allowing the patient to remain tauopathy-free for a longer period (e.g., at least 5, 10, 15, or 20 years, or for the rest of the patient's life) than if they had not received prophylactic scanning and tau-targeted immunotherapy.

[0443] Normal levels of tau protein deposition can be determined by the amount of neurofibrillary condensates or tau inclusions in the brain of a representative sample of an individual from a population that has not been diagnosed with a specific tauopathy (e.g., Alzheimer's disease) and is 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 obtained by the method of the present invention in a brain region known to produce tau protein deposition does not differ (within the precision of the measurement) from the signal from a brain region known to normally produce such deposition. Elevated levels in an individual can be recognized by comparison with normal levels (e.g., levels outside the mean + standard deviation variance) or simply by an elevated signal in a brain region associated with tau protein deposition compared to a region not known to be associated with deposition, exceeding the experimental error. For the purpose of comparing tau protein deposition levels in individuals and populations, tau protein deposition should preferably be determined in the same region(s) of the brain, which includes at least one region known to form tau protein deposition associated with a specific tauopathy (e.g., Alzheimer's disease). Patients with elevated tau protein deposition levels are candidates for initiating immunotherapy.

[0444] A decrease in tau protein deposition levels after initiating immunotherapy can initially be seen as an indication that the treatment is having the desired effect. The observed decrease may be, for example, within the range of 1–100%, 1–50%, or 1–25% of the baseline value. Such an effect can be measured in one or more brain regions known to form deposits, or from the mean value of such regions. The overall effect of the treatment can be estimated by adding the percentage decrease relative to baseline to the increase in tau protein deposition that would occur in the average untreated patient without treatment.

[0445] Maintaining a nearly constant level of tau protein deposition or even a slight increase in tau protein deposition, while suboptimal, can serve as an indicator of the response to treatment. Such responses can be compared over time to the levels of tau protein deposition in untreated patients with certain tauopathy (e.g., Alzheimer's disease) to determine whether immunotherapy has the effect of suppressing further increases in tau protein deposition.

[0446] Monitoring changes in tau protein deposits allows for adjustments to immunotherapy or other treatment plans in response to the treatment. PET monitoring provides an indicator of the nature and extent of the response to the treatment. This allows for a determination of whether or not to adjust the treatment, and if necessary, the treatment can be adjusted in accordance with the PET monitoring. Therefore, PET monitoring allows for adjustments to the administration plan of tau-targeted immunotherapy or other treatments before other biomarkers, MRI, or cognitive measurements show a detectable response. Significant changes mean that by comparing the post-treatment parameter values ​​to the baseline, evidence can be obtained that the treatment has produced or not produced a beneficial effect. In some cases, changes in parameter values ​​in the patient themselves provide evidence that the treatment has produced or not produced a beneficial effect. In other cases, if there are changes in values ​​in a patient, these are compared to changes in values ​​in a representative control population of patients not receiving immunotherapy, if such changes exist. The difference between a particular patient's response and the normal response of control patients (e.g., mean plus standard deviation variance) can also provide evidence that the immunotherapy administration plan is achieving or not producing a beneficial effect in the patient.

[0447] In some patients, monitoring shows a detectable decrease in tau protein deposits, but the level of tau protein deposits remains above normal. In such patients, the treatment plan can be continued at the same frequency and / or dose, or increased if not at the maximum recommended dose, provided there are no unacceptable side effects.

[0448] If monitoring indicates that the patient's tau protein deposition levels have decreased to normal or near-normal, the immunotherapy regimen may be adjusted from an induction regimen (i.e., a regimen to reduce tau protein deposition levels) to a maintenance regimen (i.e., a regimen to maintain tau protein deposition levels at a near-constant level). Such a regimen may be achieved by reducing the dose and / or frequency of immunotherapy.

[0449] In other patients, monitoring may indicate that immunotherapy has some beneficial effects but is suboptimal. Optimal effect can be defined as the percentage reduction in the upper half or quartile levels of tau protein deposits (measured or calculated across the entire brain or representative regions (one or more) where tau protein deposits are known to form) of a representative sample of tauopathy patients receiving immunotherapy at a given time after the start of treatment. Patients with a slight decrease in tau protein deposits, or whose tau protein deposits remain constant, or who experience an increase but to a level lower than expected in the absence of immunotherapy (e.g., when inferred from a control group of patients not receiving immunotherapy), may be classified as experiencing a positive but suboptimal response. Such patients may be given adjustments to their dosing regimen, increasing the dose and / or frequency of administration of the drug, as needed.

[0450] In some patients, tau protein deposition may increase to a level similar to or exceeding that of patients not receiving immunotherapy. If such an increase persists over a period such as 18 months or 2 years, immunotherapy may be discontinued, if necessary, to prioritize other treatments, even after the frequency or dose of the drug has been increased.

[0451] The preceding descriptions regarding the diagnosis, monitoring, and treatment of tauopathy have primarily focused on the use of PET scans. However, any other techniques for the visualization and / or measurement of tau protein deposits susceptible to the use of the tau antibodies of the present invention (e.g., mouse, humanized, chimeric, or veneer 3D6 antibodies) can be used instead of PET scans to perform such methods.

[0452] A method for detecting the immune response to tau in patients with or susceptible to tau-related diseases is also provided. Using this method, the course of therapeutic and prophylactic treatment with the active ingredients provided herein can be monitored. The antibody profile after passive immunization typically shows an immediate peak in antibody concentration followed by 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 porous material. For example, the half-life of some human antibodies is approximately 20 days.

[0453] In some methods, a baseline measurement of antibodies against tau in the target group is performed before administration, a second measurement is performed immediately thereafter to measure the peak level of the antibody, and one or more further measurements are performed 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 the peak minus baseline, an additional dose of antibody is administered. In some methods, the subsequent measured level minus the peak or background is compared to a predetermined reference level to create a beneficial prophylactic or therapeutic treatment administration plan for other subjects. If the measured antibody level is significantly lower than the reference level (e.g., less than the mean minus 1 standard deviation, preferably 2 standard deviations, of the reference value in the target population that would benefit from the treatment), an additional antibody dose is required.

[0454] For example, methods are provided for detecting tau in a subject by measuring tau in a sample derived from the subject, or by in vivo imaging of the subject's tau. Such methods are useful for diagnosing or confirming tau-related diseases or susceptibility to them. These methods can also be used in asymptomatic subjects. The presence of tau indicates a future susceptibility to symptomatic diseases. This method is also useful for monitoring disease progression and / or response to treatment in subjects who have previously been diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroidal glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroidal glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0455] Biological samples obtained from subjects who have, are suspected of having, or are at risk of having Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroidal glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroidal glial tauopathy (GGT), or progressive supranuclear palsy (PSP) can be contacted with the antibodies 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 tau levels of such subjects receiving treatment for a disease can be compared to the tau levels of subjects who have never received treatment for Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroid glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroid glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Some such tests require biopsy material from tissues obtained from such subjects. ELISA assays can also be a useful method for evaluating tau in fluid samples, for example.

[0456] VII. Kit The present invention further provides a kit (e.g., a container) comprising the antibody and related products disclosed herein, e.g., instructions for use (e.g., a package insert). The instructions for use may include, for example, instructions for the administration of the antibody and, if necessary, one or more additional drugs. The antibody container may be in the form of a unit dose, a bulk package (e.g., a multi-dose package) or a divided unit dose.

[0457] The package insert refers to the instruction manual that is customarily included with the commercial packaging of a therapeutic drug, containing information about indications, usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic drugs.

[0458] The kit also includes a second container containing pharmaceutically acceptable buffers 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, which include other buffers, diluents, filters, needles, and syringes.

[0459] VIII. Other Uses In clinical diagnosis, treatment, or research, antibodies can be used to detect tau or fragments thereof. For example, antibodies can be used to detect the presence of tau in a biological sample as an indicator that the biological sample contains tau deposits. The binding of antibodies to a biological sample can be compared to the binding of antibodies to a control sample. Control samples and biological samples may contain cells of the same tissue origin. Control samples and biological samples may be obtained from the same or different individuals, at the same or different times. If necessary, multiple biological samples and multiple control samples are evaluated at multiple times to protect against random variability unrelated to differences between samples. Subsequently, a direct comparison is made between the biological sample (single or multiple) and the control sample (single or multiple) to determine whether antibody binding to the biological sample (single or multiple) (i.e., the presence of tau) is increased, decreased, or the same as antibody binding to the control sample (single or multiple). An increase in antibody binding to the biological sample (single or multiple) compared to the control sample (single or multiple) indicates the presence of tau in the biological sample (single or multiple). In some cases, the increase in antibody binding is statistically significant. In some instances, antibody binding to biological samples is at least 1.5, 2, 3, 4, 5, 10, 20, or 100 times greater than antibody binding to control samples.

[0460] Furthermore, antibodies can be used to detect the presence of tau in biological samples, allowing for the monitoring and evaluation of the efficacy of therapeutic agents used to treat patients diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spherical glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spherical glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Biological samples from patients diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glial tauopathy (GGT), or progressive supranuclear palsy (PSP) are evaluated to establish a baseline of antibody binding to the sample (i.e., a baseline for the presence of tau in the sample) before initiating treatment with the therapeutic agent. In some cases, multiple patient-derived biological samples are evaluated at multiple time points to establish both baseline and criteria for random variability unrelated to the treatment. The therapeutic agent is then administered according to a dosing plan. The dosing plan may include multiple doses of the drug 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 criteria for random variability and to 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 comparison is made 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 changes over time starting before treatment with the drug and as changes over time progressing throughout the treatment process. If antibody binding to a biological sample (i.e., the presence of tau) is reduced in a patient, it can be concluded that the therapeutic agent is effective in treating the patient's Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), glial tauopathy (GGT), or progressive supranuclear palsy (PSP). The reduction in antibody binding is statistically significant. Depending on the circumstances, the binding may decrease by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The evaluation of antagonistic binding can be performed in conjunction with the assessment of signs and symptoms of Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis parkinsonism, post-traumatic dementia or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granulopathy, spheroidal glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), Lewy body dementia, Lewy body Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), spheroidal glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0461] Antibodies can also be used as research reagents for the detection of tau or its fragments. For such applications, antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be supplied in kit form along with all the reagents necessary to perform the detection assay. Furthermore, antibodies can be used to purify tau, or its binding partners, for example, by affinity chromatography.

[0462] All patent applications, websites, other publications, acceptance numbers, etc., cited above or below are incorporated in whole by reference to the same extent as individual items are specifically and individually incorporated by reference. Where different versions of an arrangement are associated with acceptance numbers of different times, the version associated with the acceptance number of the effective filing date of this application is meant. The effective filing date means a date earlier than the actual filing date or, where applicable, the filing date of the priority application relating to the acceptance number. Similarly, where different versions of publications, websites, etc., are published at different times, unless otherwise indicated, the most recently published version on the effective filing date of this application is meant. Any feature, process, element, embodiment, or aspect of the present invention may be used in combination with any other unless otherwise indicated. Although the present invention has been described in some detail by examples and embodiments for the purpose of clarity and understanding, it will be apparent that certain changes and modifications may be implemented within the scope of the appended claims.

[0463] Examples Example 1. Identification of tau monoclonal antibodies Monoclonal antibodies against tau were generated as follows: Immunization was performed using recombinant 383a.a. human tau (4R0N) [immunogen A] containing the P301S mutation and with an N-terminal His tag, or recombinant 383a.a. human tau (4R0N) [immunogen B] containing the P301S mutation but lacking an N-terminal His tag. The immunogens were emulsified in RIBI adjuvant.

[0464] Five-week-old female Balb / c mice were intraperitoneally immunized with 25 μg of immunogen A on day 0, and then intraperitoneally immunized with 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. Blood samples were taken from the mice on days 43 and 98, and their titers to immunogen A were measured; on day 101, the animals with the highest titer were given a terminal immunization with 50 μg of immunogen B. This additional immunization was delivered 1 / 2 intraperitoneally and 1 / 2 intravenously. Fusion hybridomas were screened against both immunogens by ELISA, and those with the highest signal were epitope-mapped (see Example 2). 3D6 reacted with peptides corresponding to amino acid residues 199-213 and 262-276 of SEQ ID NO: 3 (according to the numbering of the longest CNS isoform of tau, this corresponds to amino acid residues 257-271 and 320-334 of SEQ ID NO: 1).

[0465] Example 2. Epitope mapping of antibody 3D6 A series of overlapping biotinylated peptides across the entire 383aa 4R0N human tau protein were used for mapping with a mouse 3D6 antibody. Another peptide was used to model potential post-translational modifications at the C-terminus and N-terminus of the protein.

[0466] Biotinylated peptides were conjugated to separate wells of a streptavidin-coated ELISA plate. The plate was blocked and treated with mouse 3D6, followed by incubation with horseradish peroxidase-labeled anti-mouse antibody. After thorough washing, OPD was applied to the plate for color development. The absorbance of the plate was read at 450 nm. Background removal was performed for absorbance values ​​from wells without primary antibody, and the positive binding threshold was set to 0.2 absorbance units. Positive binding was detected for peptides spanning amino acid residues 199-213 (SEQ ID NO: 3) and amino acid residues 262-276 (SEQ ID NO: 3). Using the numbering of the full-length 4R2N human tau protein (441 amino acids), these peptides correspond to amino acid residues 257-271 (SEQ ID NO: 1) and 320-334 (SEQ ID NO: 1). (Figure 1)

[0467] Example 3. Design of Humanized 3D6 The starting point or donor antibody for humanization was mouse 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 kavat / cotia combination CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs: 8-10, respectively. The light chain kavat CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs: 12-14, respectively. Kabat numbering is used throughout.

[0468] 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]. Cotia CDR-L1, consisting of 16 residues, belongs to canonical class 4 of Vκ, Cotia CDR-L2, consisting of 7 residues, belongs to class 1, and Cotia CDR-L3, consisting of 9 residues, also belongs to class 1 [Martin AC, and Thornton JM (1996) J.Mol.Biol.263:800-15. [Martin & Thornton, 1996]. Cotia CDR-H1, consisting of 10 residues, belongs to class 1, and Cotia CDR-H2, consisting of 17 residues, belongs to class 2 [Martin & Thornton, 1996]. CDR-H3 does not have a canonical class. To find a structure that gives a rough structural model of the 3D6 antibody, a search was performed against protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33: D233-7]. To construct the Fv model of 3D6, the structure of mouse anti-pyroglutamate A beta 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] was used. This retained the same canonical structure as the 3D6 loop.

[0469] The framework of 3D6 VH shares a high degree of sequence similarity with the corresponding region of the 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 identical lengths for the CDR-H1 and H2 loops. Similarly, the framework of 3D6 VL shares a high degree of sequence similarity with the corresponding region of the 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 identical lengths for the CDR-L1, L2, and L3 loops. Therefore, the framework regions of 48G7 VH (2RCS-VH) and ARX71335 VL were selected as receptor sequences for the CDR of 3D6. Models of 3D6 CDR grafted onto the VH and VL human frameworks were constructed and used as guidance for further reverse mutations.

[0470] 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, and the degree of humanization of the heavy and light chains was evaluated as outlined in the WHO INN Committee Guidelines. (WHO-INN: International General Names for Biological and Biotechnological Substances (INN) (Overview) (Internet) 2014. Available from http: / / www.who.int / medicines / services / inn / BioRev2014.pdf) Where possible, residues were modified to correspond to the corresponding human germline sequences to increase the degree of humanization and decrease potential immunogenicity. In the humanized VLvb2 and VLvb3 variants, the sequences were aligned to the human germline gene IGKV2-30. * To make it more similar to 02 (SEQ ID NO: 27), a mutation was introduced. In humanized VHvb2, VHvb3, VHvb4, VHvb5, VHvb6, and the VHvb6 variant, the sequence was changed to that of the human germline gene IGHV1-69-2.* A mutation was introduced to make it more similar to 01 (sequence number 25).

[0471] The hu3D6-VH and hu3D6-VL types were designed to allow evaluation of various framework residues for their contributions to antigen binding, thermal stability, and immunogenicity, as well as for the optimization of glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface-exposed charged patches, deamination, and proteinase sensitivity. The locations examined for mutations included: - The position that defines the canonical CDR three-dimensional structure (summarized in Martin, ACR (2010) Protein sequence and structure analysis of antibody variable domains, in Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.), - Location 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.) - Localized at the VH / VL domain interface (summarized in Leger 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 that are susceptible to post-translational modifications such as glycosylation or pyroglutamine oxidation, - According to the model of 3D6 CDR grafted onto the VH and VL frameworks, the positions occupied by residues predicted to collide with the CDR, or - A position occupied by a rare residue in the sequenced human antibody, where either the parent mouse 3D6 residue or several other residues are found far more frequently in the human antibody repertoire. These are some examples.

[0472] Sequence comparisons of mouse 3D6 and various humanized antibodies are shown for the light chain variable region (Table 4 and Figure 3) and the heavy chain variable region (Table 3 and Figure 2).

[0473] Seven humanized heavy chain variable region variants and three humanized light chain variable region variants were constructed, including different permutation permutations: hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, or hu3D6VHvb7 (sequence codes 76-80 and 90-91, respectively), and hu3D6VLvb1, hu3D6VLvb2, or hu3D6VLvb3 (sequence codes 83-85, respectively) (Tables 3 and 4). Representative humanized Vk and Vh designs with reverse mutations and other mutations based on the Selected Human Framework are shown in Tables 3 and 4, respectively. Regions in bold in Tables 3 and 4 indicate CDRs defined by Kabat / Cotia combinations. Sequence codes 76-80 and 90-91 include reverse mutations and other mutations, as shown in Table 5. Table 6 shows the amino acids at the positions in hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7. Table 7 shows the amino acids at the positions in hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3. [Table 3] TIFF0007911799000004.tif239161TIFF0007911799000005.tif239161TIFF00079117990 00006.tif238160TIFF0007911799000007.tif239159TIFF0007911799000008.tif130161 [Table 4] TIFF0007911799000010.tif239162TIFF0007911799000011.tif238160TIFF0007911799000012.tif238162TIFF0007911799000013.tif209162 [Table 5] [Table 6] TIFF0007911799000016.tif128162 [Table 7]

[0474] The positions where canonical, vernier, or interface residues differ between mouse and human receptor sequences are candidates for substitution. Examples of canonical / CDR interaction 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.

[0475] The principle for selecting replacement candidates for the positions shown in Table 3 within the heavy chain variable region is as follows:

[0476] Heavy chain variable region hu3D6VHvb1 - Consists of CDR-H1, H2, and H3 loops of 3D6-VH grafted onto a framework of 48G7-VH(RCS-VH) with reverse mutations at positions H91(Y91F), H93(A93S), and H94(S94T).

[0477] hu3D6VHvb2 - Restores all framework substitutions at locations crucial to defining the canonical class of Kotia, locations that are part of the vernier zone, locations localized at the VH / VL domain interface, or locations that contribute to structural stability. 3D6-VH_vb2 incorporates reverse mutations or substitutions Q1E, Q5V, L11V, L20I, T23K, K38R, E42G, Q43K, K66R, S75T, N76D, Q81E, Y91F, A93S, S94T, T108L, and L109V to enable evaluation of the contributions of these locations to antigen-binding affinity and immunogenicity.

[0478] hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 This consists of further substitutions that increase antibody stability and / or optimize glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface-exposed charge patch, deamination, and proteinase sensitivity.

[0479] Q1E is a mutation that enhances stability by reducing pyroglutamate formation ability (Liu, op. cit.).

[0480] Q5V is a frequency-based mutation and a germline line-aligning mutation. Val is the most abundant gene at this position in the human sequence. Val is located at the human germline gene IMGT number IGHV1-69-2. * It is located in 01 (sequence number 25).

[0481] L11V is a germline alignment mutation. Val is the human germline gene IMGT number IGHV1-69-2 at this position. * It is located in 01 (sequence number 25).

[0482] S17T is a germline alignment mutation. Thr is the human germline gene IMGT number IGHV1-69-2 at this position. * It is located in 01 (sequence number 25).

[0483] L20I is a germline alignment mutation. Ile is the human germline gene IMGT number IGHV1-69-2 at this position. * It is located in 01 (sequence number 25).

[0484] T23K is a frequency-based germline alignment variant. Lys is most frequently found at this position. Lys is found at this position in the human germline gene IMGT number IGHV1-69-2. * It is located in 01 (sequence number 25).

[0485] N28T: This is a CDR-H1 residue substitution to Thr.

[0486] K38R ​​is a frequency-based reverse mutation. Arg is most abundant at this position. Arg at this position is predicted to generate one H bond plus two H bonds with Glu46, and H bonds with Asp86 and Tyr90 respectively in the heavy chain, and therefore Arg may enhance stability compared to Lys at this position.

[0487] E42G is a frequency-based and germline alignment variant. Gly is the human germline gene IMGT number IGHV1-69-2 at this position. * It is located in 01 (sequence number 25). Gly is most abundant at this position. Gly substitution is not expected to affect stability.

[0488] Q43K: The Lys side chain at this position is predicted to form H-bonds with the G42 side chain in addition to the main chain and with Gln39 and Arg40, such that the Lys substitution may enhance stability over Q at this position.

[0489] N54D and D56E are substitutions of CDR residues and are predicted to be at non-antigen contact positions by the homology model. The N54D and D56E substitutions are predicted to stabilize the antibody structure.

[0490] V58I is a substitution of a CDR-H2 residue. Germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25) has Ile at this position. This residue is predicted not to contact the antigen.

[0491] K66R: The Arg at this position is predicted to form H-bonds and a salt bridge with Asp86 and to form H-bonds with Ser82a and Thr83.

[0492] A67V is a substitution of a framework residue. Germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25) has Val at this position.

[0493] S75T: The Ser at this position is predicted to form H-bonds with Asp72 and Tyr76. The Thr at this position is also predicted to form these contacts, but Thr, being a surface-exposed residue, may enhance antibody stability.

[0494] N76D: Asp is a germline alignment variant. Asp is present at this position in the human germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25).

[0495] L80M: Met is a germline alignment variant. Met is present at this position in the human germline gene IMGT number IGHV1-69-2 *It is located in 01 (sequence number 25).

[0496] Q81E:Glu is predicted to form an H-bond + salt crosslink with K19, and therefore Glu at this position enhances antibody stability.

[0497] T83R enhances thermal stability and increases humanization. Arg is a germline alignment mutation. Arg is a human germline gene IMGT number IGHV1-69-2 at this position. * It is located in 01 (sequence number 25). Arg is most abundant at this position.

[0498] F91Y is an interfacial residue mutation and is a frequency-based inverse mutation. Tyr is typically located at this position and may enhance antibody stability.

[0499] A93S is a reverse mutation in the vernier zone and interface zone residues.

[0500] S94T is a reverse mutation of the canonical and vernier residues as defined by Kotia.

[0501] T108L:Leu is a germline alignment mutation. Leu is associated with the human germline gene IMGT number IGHV1-69-2 at this position. * It is located in 01 (SEQ ID NO: 25). Leu at this position is expected to reduce the immunogenicity of the antibody and have no effect on antibody stability.

[0502] L109V is a frequency-based mutation. Val is most frequently found at this location.

[0503] The principle for selecting replacement candidates for the positions shown in Table 4 within the light chain variable region is as follows:

[0504] Kappa Light Chain Variable Region

[0505] hu3D6VLvb1 - It consists of the CDR-L1, L2, and L3 loops of 3D6-VL grafted onto the framework of ARX71335VL.

[0506] hu3D6VLvb2 and hu3D6VLvb3 - Restore all framework substitutions at positions important for defining the kappa canonical class, positions that are part of the Verner zone, or positions localized at the VH / VL domain interface. Hu3D6-VLvb2 and hu3D6-VLvb3 also contain substitutions that contribute to structural stability. Hu3D6-VL-vb2 incorporates the reverse mutations T7S, I15L, L83V, H86Y, and L106I to enable evaluation of the contribution of these positions to antigen-binding affinity and immunogenicity at these positions. All substitutions of Hu3D6-VL-vb3 are described for vb2 with additional changes at Q17E, K24R, L37Q, K45R, and L106I.

[0507] T7S is a germline alignment mutation. Ser is present at this position in the human germline gene IGKV2-30 * 02 (SEQ ID NO: 27).

[0508] T10S is a frequency-based and germline alignment mutation. Ser is present at this position in many cases. Ser is present at this position in the human germline gene IGKV2-30 * 02 (SEQ ID NO: 27).

[0509] I15L is a germline alignment mutation. Leu is present at this position in the human germline gene IGKV2-30 * 02 (SEQ ID NO: 27).

[0510] Q17E: Glu at this position is predicted to form an H-bond with T14 and a salt bridge with Lys107, both of which are light chain residues, and is predicted to enhance antibody stability.

[0511] K24R is a mutation in the CDR residue. Both Lys and Arg are predicted to form H bonds and salt crosslinks with Asp70 in the light chain. Arg is predicted to fit better in three dimensions. Arg is also a germline alignment mutation. Arg is a mutation in the human germline gene IGKV2-30 at this position. * It is located in 02 (sequence number 27).

[0512] L37Q: This is predicted to be a deeply embedded residue, and Leu is not predicted to interact with surrounding residues, but Gln is predicted to generate H bonds with Q38 and Asp82 in the light chain. Gln is also a germline alignment mutation. Gln is present in the human germline gene IGKV2-30 at this position. * It is located in 02 (sequence number 27).

[0513] K45R:Lys is predicted to generate H bonds with S56 and Gly57; it is also predicted to form a salt bridge with D55, an H bond with Arg46, and a double H bond with S56; therefore, the predicted interactions of Arg with adjacent residues are much more extensive. Arg is also a germline alignment mutation. Arg is present in the human germline gene IGKV2-30 at this position. * It is located in 02 (sequence number 27).

[0514] L83V: This is a mutation based on the frequency of residues that are predicted to be surface-exposed. Val is also a germline alignment mutation. Val is a mutation of the human germline gene IGKV2-30 at this position. * It is located in 02 (sequence number 27).

[0515] H86Y: Mouse 3D6 VL has Tyr at this position. Tyr is also the most abundant residue at this position.

[0516] 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 at this position and is also a germline alignment mutation. Gln is present at this position in the human germline gene IGKV2-30. * It is located in 02 (sequence number 27). Gln is predicted to stabilize the chain by forming an H bond with Ser7.

[0517] L106I is a frequency-based and germline alignment variant. Ile is most frequently found at this position. Ile is found at this position in the human germline gene IGKV2-30. * It is located in 02 (sequence number 27).

[0518] The following design is based on these human frameworks:

[0519] Heavy chain variable region >hu3D6VHvb1(sequence number 76) QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0520] >hu3D6VHvb2(sequence number 77) EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLELSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0521] >hu3D6VHvb3(sequence number 78) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRATITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0522] hu3D6VHvb4(Sequence ID 79) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0523] >hu3D6VHvb5(sequence number 80) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0524] >hu3D6VHvb6(sequence number 90) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYFCSTLDFWGQGTLVTVSS

[0525] >hu3D6VHvb7(sequence number 91) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0526] Kappa Light Chain Variable Region hu3D6VLvb1(Sequence ID 83) DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCWQGTHFPYTFGAGTKLELK

[0527] >hu3D6VLvb2(sequence number 84) DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGAGTKLEIK

[0528] >hu3D6VLvb3(sequence number 85) DVVMTQSPLSLSVTLGEPASISCRSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0529] Humanized sequences are generated using a two-stage 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].

[0530] Example 4. Mouse monoclonal antibody binds to tau in ELISA assay. Method: Indirect ELISA: 96-well polystyrene plates were coated with capture antibody anti-6xHis (Figure 4A) or polyclonal anti-tau (Dako#A20024, Figure 4B) suspended in 1xPBS at room temperature for 2 hours or at 4°C for 16 hours. After removing the coating solution, the plates were blocked with 1% BSA in 1xPBS for 1 hour, and then incubated with human recombinant tau, with and without polyhistidine tags attached to the N-terminus of the protein (Figure 5A and Figure 4B). After washing, the plates were incubated with the antibodies shown and then incubated with HRP-labeled goat anti-mouse secondary antibody. The plates were stained with TMB, and A 450 The measurement was taken using a plate reader.

[0531] Sandwich ELISA: 96-well polystyrene plates were coated with anti-mouse antibody in 1x PBS at room temperature for 2 hours or at 4°C for 16 hours. The coating was removed and the plates were blocked with 1% BSA in 1x PBS for 1 hour. The plates were then incubated with the same concentrations of antibody and diluted with 0.1% BSA in 1x PBS. The plates were sequentially treated with human tau, polyclonal rabbit anti-tau (Dako#A0024), and HRP-labeled goat anti-rabbit antibody. All were diluted with 0.1% BSA in PBS and washed between each step. Streptavidin-HRP was added, and the plates were stained with TMB. 450 The values ​​were measured using a plate reader. See Figure 4C.

[0532] Results: Several different ELISA formats were used to assay the binding of a range of hybridoma-generating antibodies to tau. Tau detection was confirmed using an indirect format with tau proteins immobilized by a polyhistidine tag fused to the N-terminus (Figure 4A). Undenatured, untagged proteins were similarly confirmed (Figure 4B). To evaluate the nutrient solution affinity of various antibodies, a sandwich ELISA format was used, with the tested hybridoma antibodies as capture reagents (Figure 4C).

[0533] Example 5. Affinity of mouse monoclonal antibody against 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 antibody (GE Life Sciences) was immobilized on the sensor tip CM5 via amine coupling, and the antibody was captured at a level that ensured maximum binding of 50 RU. Recombinant tau at various concentrations ranging from 10 to 0.14 nM was 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 seconds of binding and 900 seconds of dissociation. Data were used as a dual standard against both an unrelated sensor without antibody-ligand and an analyte concentration of 0 nM to capture ligand dissociation from the captured portion. Subsequently, the data were analyzed using a global 1:1 fit.

[0534] Results: Multiple mouse antibodies were selected based on the results of a series of ELISA assays, and their binding affinity was evaluated by SPR. Several sets of antibodies were tested simultaneously, and their binding and dissociation rates were compared to select the antibody with the highest binding affinity to recombinant human tau. The highest binding affinity was observed with antibody clone 3D6. Binding affinities are shown in Figure 5.

[0535] Example 6. Mouse monoclonal antibody interferes with the binding of human tau to the surface of immortalized nerve cells. Method: Inhibition of tau binding to B103 neuroblastoma cells by anti-tau monoclonal antibody. 1. B103 cells 5x10 5 Resuspend the cells at a concentration of cells / mL. Seed 50 μL of cell suspension per well into an MSD high-binding plate. This yields 25k cells / well. Cover the plate and allow the cells to adhere for 2 hours at 37°C under 5% CO2. 2. After cell attachment, remove PBS from the wells by inverting the plate and gently tap to remove excess buffer. Add 3% MSD Blocker A buffer or other preferred blocking buffer in 50 μl of PBS to each well and incubate the plate at room temperature for 1 hour without shaking. 3. During the plate blocking step, co-incubate the tau and anti-tau antibodies as follows: a. Starting with 2 mg / mL anti-tau antibody, seven further dilutions are performed using a 1:2 serial dilution method with PBS. b. Dilute tau to 20 nM in PBS. The tau concentration will be constant in each well. c. Mix tau and anti-tau antibody in a 1:1 ratio to achieve a final tau concentration of 10 nM and an initial anti-tau concentration of 1 mg / mL. d. Incubate the mixture at room temperature for approximately 1 hour while shaking (600 rpm). 4. After plate blocking in Step 2, invert the plate to remove the blocking buffer from the wells, gently tap, and then wash the plate twice with PBS using a multichannel pipette to ensure complete removal of excess buffer. Tau: Cool the seeded cells to 4°C before adding the anti-tau complex. 5. Add 50 μL of the cooled complex from Step 3 to the seeded cells and incubate on ice for 30 minutes. 6. As previously mentioned, wash the plate twice with chilled PBS. 7. To detect cell surface-bound tau, add 50 μL of 16B5.SULFO-TAG per well. Incubate on ice for 30 minutes. 8. As previously mentioned, wash the plate twice again with chilled PBS. 9. Add 150 μL of 1X surfactant-free reed buffer T (diluted with H2O) per well and immediately read the reading using an MSD SECTOR® 600 meter. Avoid introducing air bubbles when adding the reed buffer. 10. Report the MSD signal versus tau concentration.

[0536] The antibodies tested were anti-tau antibodies 3D6, 16G7, 3H9, 4C5, and 5G8, as well as an isotype control.

[0537] Results: The decrease in sulfotagled anti-tau signaling, accompanied by an increase in the tested antibody, indicates functional blocking of tau binding to neurons. No blocking was observed with the isotype control, 16G7, or 3H9. Increased levels of functional blocking activity were observed with 4C5, 5G8, and 3D6. 3D6 showed the strongest blocking activity among the antibodies tested. See Figure 6.

[0538] Example 7. De-aggregation activity Methods: Purified recombinant tau with a recombinant tau-N-terminal 6xHis tag was mixed with an equimolar amount of low molecular weight heparin in 1x PBS (pH 7.4) and incubated at 37°C for 96 hours using a swirling apparatus. Agglomeration of the sample was confirmed by binding to thioflavin T. Antibody incubation – Antibodies were incubated with agglutinated recombinant tau at 37°C for 96 hours without the use of a rotating or swirling apparatus. At the end of the experiment, the samples were incubated with 25 mM thioflavin T, and agglutination was measured by measuring the emitted fluorescence (450 / 482 ex / em). Background was subtracted from the signal to the buffer sample.

[0539] Results: As shown in Figure 7, 3D6 selectively degrades complete tau fibrils. Various molar ratios of 3D6 (triangle), isotype control (circle), and 16G7 (square) were incubated with amyloid-containing tau fibrils for 96 hours. At the end of this period, the degree of aggregation was evaluated by binding to thioflavin T. 3D6 selectively reduces the thioflavin T signal present in the sample compared to the isotype control antibody and 16G7, an anti-tau antibody that binds to tau in different regions.

[0540] Examples 8.3D6 and 5G8 immunocapture tau derived from human disease tissue. Methods: High-salt soluble protein fractions were prepared to 1 mg / ml. 200 μg of sample was used for each immunoprecipitation. 10 μg 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 a further 1 hour to capture the antibody / antigen complex. The samples were thoroughly washed with 1x PBS, and the beads were boiled in reducing / denaturing sample buffer to release the captured proteins. The resulting samples were separated by SDS-PAGE, and Western blotting was performed using polyclonal anti-tau antibody (Dako, #A0024).

[0541] Results: As shown in Figure 8, 3D6 and 5G8 were immunoprecipitated with tau derived from Alzheimer's disease tissue. Immunoprecipitation was performed with an antibody showing a high salt soluble fraction, and the region of the tau molecule separated from the binding site of 3D6 and tau antibody A was detected with a polyclonal anti-tau antibody. 3D6 reliably captured tau from this fraction. The input (high salt soluble sample) is shown on the right.

[0542] Immunohistochemical analysis of immunoreactivity in Example 9.3D6 Frontotemporal cortices were obtained from patients without neurodegenerative disease or those with Alzheimer's disease, confirmed at postmortem evaluation. Immunohistochemistry was performed on 10 μm frozen sections mounted on slides and lightly fixed with acetone. All staining steps were performed using a Leica BOND Rx automated immunostaining system and Leica consumables. Mouse or human-type 3D6 was incubated with the tissue sections, followed by the addition of species-appropriate secondary antibodies conjugated to HRP polymer. To prevent nonspecific binding of endogenous immunoglobulins when humanized antibodies are used against human tissue, the antibodies were in vitro non-covalently labeled with biotin-conjugated anti-human monovalent Fab fragments before tissue incubation. Tissues labeled with the primary antibody-biotin Fab fragment complex were further amplified using an avidin-biotin amplification system (Vector Laboratories, Burlingame, CA). Staining was visualized using DAB chromogens, which produced brown deposits. Negative controls were prepared by performing a total immunohistochemical procedure on adjacent sections using an IgG isotype control antibody.

[0543] The antibodies tested were mouse CD6, chimeric 3D6 (which included VH and VL derived from mouse antibodies with a human constant region, heavy chain SEQ ID NO: 72 and light chain SEQ ID NO: 73), and the humanized variant hu3D6VHv5 / hu3D6VLv2.

[0544] Staining of mouse, chimeric, and humanized 3d6 was qualitatively compared and evaluated based on staining intensity, brightness, and localization of immunoreactivity. Staining brightness was similar in 3D6 chimeric and humanized forms compared to mouse antibodies, and similar localization patterns were observed. Tau was detected in neurofibrillary condensates, fibrils, filamentous structures, and degenerated axons. Significant cell body staining was also detected.

[0545] Example 10. Affinity of humanized variants for tau Method; Indirect ELISA: Coat a 96-well polystyrene plate with human recombinant tau suspended in 1x PBS at room temperature for 2 hours or at 4°C for 16 hours. Remove the coating solution and block the plate with 1% BSA in 1x PBS for 1 hour. Add 1 μg / mL of humanized variant antibody in 0.1% BSA in 1x PBS to the plate, retain for 1 hour, then wash and add HRP-labeled goat anti-human antibody. Chromomorphize the plate with TMB and A 450 Measure it with a plate reader.

[0546] Sandwich ELISA: Coat a 96-well polystyrene plate with anti-human antibody in 1x PBS at room temperature for 2 hours or at 4°C for 16 hours. Remove the coating solution and block the plate with 1% BSA in 1x PBS for 1 hour. Add humanized variant antibodies of various concentrations diluted with 0.1% BSA in 1x PBS to the plate, hold for 1 hour, then wash, and add biotinylated recombinant human tau diluted with 0.1% BSA in 1x PBS. After washing, add streptavidin-HRP and color the plate with TMB. 450 Measure it with a plate reader.

[0547] SPR analysis is performed using a Biacore T200 to measure the binding kinetics of h3D6-VHv5-L2 to recombinant human tau. To prepare the sensor surface, an anti-human antibody (GE Life Sciences) is immobilized on the sensor chip CM5 via amine coupling, and a humanized variant antibody is captured at a level ensuring maximum binding of 50 RU. Recombinant tau at various concentrations ranging from 10 to 0.14 nM is 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) with binding for 180 seconds and dissociation for 900 seconds. The data is double-referenced to both an unrelated sensor without antibody-ligand and to an analyte concentration of 0 nM to account for ligand dissociation from the capture portion. The data is then analyzed using a 1:1 global fit.

[0548] Example 11. Immunogenicity of the variable region of the hu3D6VLv2 light chain. The amino acid sequence of the hu3D6VLv2 light chain variable region (SEQ ID NO: 21) was analyzed using the iedb.org Deimmunization Tool (Dhanda et al, Immunology. 2018 Jan;153(1):118-132). Table 9 shows the peptides that can be selected for deimmunization of hu3D6VLv2, i.e., the areas where further substitutions may be made to reduce potential immunogenicity. [Table 8]

[0549] Based on the analysis results shown in Table 9, variants of the hu3D6VLv2 light chain variable region were designed, targeting the amino acid residues shown in bold in Table 10. Each variant incorporates one of the following amino acids, as shown in Table 11. [Table 9] [Table 10] TIFF0007911799000021.tif28162

[0550] Further variants of the hu3D6VLv2 light chain variable region, incorporating substitutions in one or two of the amino acids highlighted in Table 10, were designed as shown in Table 12. Some of the variants in Table 12 also incorporate substitution L37Q (sequences 119-135 and 145), or both substitution L37Q and substitution G100Q (sequences 136-142).

[0551] The principle for selecting positions L37Q and G100Q in the light chain variable region, as shown in Tables 12 and 13, as substitution candidates is as follows:

[0552] L37Q is a mutation that increases the degree of humanization of the sequence. Gln is a germline alignment mutation. Gln is a mutation that affects the human germline gene IGKV2-30 at this position. * It is located in 02 (sequence number 27).

[0553] G100Q is a mutation that increases the degree of humanization of the sequence. Gln is a germline alignment mutation. Gln is a mutation that affects the human germline gene IGKV2-30 at this position. * It is located in 02 (sequence number 27). [Table 11] TIFF0007911799000023.tif107161

[0554] Further variants of the hu3D6VLv2 light chain variable region were designed to incorporate L37Q (SEQ ID NO: 143) or G100Q (SEQ ID NO: 144), as shown in Table 13. [Table 12]

[0555] Figures 10A, 10B, 10C, and 10D show an alignment comparison of the light chain variable regions of humanized 3D6 antibodies: hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv2 L37Q (SEQ ID NO: 143), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2 L54D (SEQ ID NO: 93), hu3D6VLv2 L54K (SEQ ID NO: 100), hu3D6VLv2 L54R (SEQ ID NO: 101), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54T (SEQ ID NO: 130), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_L50D_L54R (Sequence ID 133), hu3D6VLv2 L37Q_L50D_L54G (Sequence ID 132), hu3D6VLv2 L37Q_S52G_L54D (Sequence ID 124), hu3D6VLv2 L37Q_S52G_L54G (Sequence ID 121), hu3D6VLv2 L37Q_S52G_L54T (Sequence ID 123), hu3D6VLv2 L37Q_S52G_L54R (Sequence ID 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (Sequence ID 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (Sequence ID 141), hu3D6VLv2 L37Q_L50G_L54R_G100Q (Sequence ID 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (Sequence ID 137), hu3D6VLv2 L37Q_L50V_L54D_G100Q (Sequence ID 142), hu3D6VLv2L37Q_S52G_L54D_G100Q (Sequence ID 139), and hu3D6VLv2 L37Q_S52G_L54R_G100Q (Sequence ID 138).

[0556] Example 12: Immunogenicity of hu3D6VHv1bA11 (heavy chain variable region) Further heavy-chain variable region variants of hu3D6VHv1bA11 (also known as h3D6Hu5) (Sequence ID 18) were designed as shown in Table 14.

[0557] Figures 9A and 9B show a comparison of the alignment of the mouse 3D6 heavy cha...

Claims

1. An antibody or antigen-binding antibody fragment that specifically binds to human tau, A mature heavy chain variable region comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 8, CDR-H2 containing the amino acid sequence of SEQ ID NO: 9, and CDR-H3 containing the amino acid sequence LDF, and A mature light chain variable region comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 12, CDR-L2 containing the amino acid sequence of SEQ ID NO: 168, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

14. Including; The mature heavy chain variable region includes a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18; The mature light chain variable region includes a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 138; Antibodies or antigen-binding antibody fragments.

2. The antibody or antigen-binding antibody fragment according to claim 1, wherein the mature heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

18.

3. Kabat position H12 is V; Kabat position H13 is K; Kabat position H17 is T; Kabat position H24 is A; Kabat position H40 is R; Kabat position H43 is Q; Kabat position H48 is I; Kabat position H66 is R; Kabat position H67 is A; Kabat position H76 is D; Kabat position H80 is L; Kabat position H81 is Q; and / or Kabat position H91 is F; The antibody or antigen-binding antibody fragment according to claim 1.

4. The antibody or antigen-binding antibody fragment according to any one of claims 1 to 3, wherein the mature light chain variable region comprises the amino acid sequence of SEQ ID NO:

138.

5. Kabat position L2 is V; Kabat position L12 is P; Kabat position L15 is L; Kabat position L37 is Q; Kabat position L39 is R; Kabat position L45 is R; Kabat position L60 is D; and / or The Kabat position L100 is Q; The antibody or antigen-binding antibody fragment according to any one of claims 1 to 3.

6. The antibody or antigen-binding antibody fragment according to any one of claims 1 to 5, which is a chimeric antibody, a chimeric antigen-binding antibody fragment, a benia antibody, a benia antigen-binding antibody fragment, a humanized antibody, or a humanized antigen-binding antibody fragment.

7. The antibody according to any one of claims 1 to 6.

8. The antigen-binding antibody fragment according to any one of claims 1 to 6.

9. The antigen-binding antibody fragment is a single-chain antibody, a Fab fragment, or Fab' ２ A fragment, which is an antigen-binding antibody fragment according to claim 8.

10. The antibody according to claim 7, wherein the antibody has the isotype of human IgG1, human IgG2, or human IgG4.

11. An antibody or antigen-binding antibody fragment according to any one of claims 1 to 6, comprising a light chain including the mature light chain variable region fused to the light chain constant region, and a heavy chain including the mature heavy chain variable region fused to the heavy chain constant region.

12. The antibody or antigen-binding antibody fragment according to claim 11, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 176 with or without a C-terminal lysine.

13. The antibody or antigen-binding antibody fragment according to claim 11, wherein the mature heavy chain variable region fused to the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 178, with or without a C-terminal lysine.

14. The antibody or antigen-binding antibody fragment according to claim 11, further comprising a bovine alpha-lactalbumin signal peptide fused to the mature heavy chain variable region and / or the mature light chain variable region.

15. The antibody or antigen-binding antibody fragment according to claim 14, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 180 with or without a C-terminal lysine.

16. The antibody or antigen-binding antibody fragment according to claim 11, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

177.

17. The antibody or antigen-binding antibody fragment according to claim 11, comprising at least one mutation in the heavy chain constant region.

18. The antibody or antigen-binding antibody fragment according to claim 17, wherein the at least one mutation reduces complement binding or activation by the heavy chain constant region, or reduces binding to the Fcγ receptor, compared to the natural human heavy chain constant region.

19. The antibody or antigen-binding antibody fragment according to claim 18, wherein the at least one mutation is located at one or more of EU numbering positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331, or is alanine at EU numbering positions 318, 320, and 322.

20. An antibody or antigen-binding antibody fragment according to any one of claims 1 to 19, conjugated to a therapeutic agent, cytotoxic agent, cell division arrester, neurotrophic agent, or neuroprotective agent.

21. A pharmaceutical composition comprising an antibody or antigen-binding antibody fragment according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. A nucleic acid encoding the heavy chain and light chain of an antibody according to any one of claims 1 to 7 and 10 to 19.

23. The nucleic acid according to claim 22, comprising the nucleotide sequence of sequence number 182 which codes for the heavy chain.

24. A method for producing antibodies, (a) culturing cells transformed with the nucleic acid according to claim 22 or 23 so that the cells secrete the antibodies; and (b) Purify the antibody from the cell culture medium. Methods that include...

25. A method for producing antibody-producing cell lines, (a) Introducing a vector comprising the nucleic acid and a selection marker according to claim 22 or 23 into cells; (b) Growing the cells under conditions for selecting cells having an increased copy number of the vector; (c) Isolating single cells from selected cells; and (d) Banking cells cloned from single cells selected based on antibody yield, Methods that include...

26. A method for producing antibodies, (a) Culturing cells containing nucleic acids encoding the heavy and light chains of the antibody according to any one of claims 1 to 7 and 10 to 19 so that the cells secrete the antibody; and (b) Purify the antibody from the cell culture medium. Methods that include...

27. A method for producing antibody-producing cell lines, (a) Introducing into cells a vector encoding the heavy and light chains of an antibody and a selection marker as described in any one of claims 1 to 7 and 10 to 19; (b) Growing the cells under conditions for selecting cells having an increased copy number of the vector; (c) Isolating single cells from selected cells; and (d) Banking cells cloned from single cells selected based on antibody yield, Methods that include...

28. The pharmaceutical composition according to claim 21, for use in a method for inhibiting or reducing tau aggregation in subjects having tau-mediated amyloidosis or subjects at risk of developing tau-mediated amyloidosis.

29. The pharmaceutical composition according to claim 28, wherein the antibody or antigen-binding antibody fragment comprises a mature heavy chain variable region containing the amino acid sequence of SEQ ID NO: 18 and a mature light chain variable region containing the amino acid sequence of SEQ ID NO:

138.

30. The pharmaceutical composition according to claim 21 for use in a method of treating or preventing tau-related diseases in a subject.

31. The pharmaceutical composition according to claim 21 for use in a method for reducing abnormal propagation of tau, inducing phagocytosis of tau, inhibiting deposition or aggregation of tau, or inhibiting the formation of tau concentrates in a subject.

32. The pharmaceutical composition according to claim 21 for use in a method for measuring the efficacy of a treatment in a subject treated for a disease related to the aggregation or deposition of tau.

33. A pharmaceutical composition according to any one of claims 28 to 32, formulated for intravenous injection or infusion.

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