Antibodies recognizing tau

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 TO 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 applications is incorporated by reference in its entirety into this application.

[0002] Array list reference This application contains an electronic sequence listing with filename 536323WO_ST25.TXT, which was created on February 24, 2020, and contains 168,875 bytes. This sequence listing is incorporated by reference in its entirety into this application. [Background technology]

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

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

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

[0006] In one aspect, the invention provides an isolated antibody that specifically binds human tau, comprising a mature heavy chain variable region that comprises 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, wherein the mature heavy chain variable region is at least 90% identical to SEQ ID NO:18, and a mature light chain variable region that comprises CDR-L1 comprising SEQ ID NO:12, 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, wherein the mature light chain variable region 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 comprises SEQ ID NO: 150, 151, 163, 167, 168, or 169. In some antibodies, the heavy chain variable region comprises SEQ ID NO: 18 and the light chain variable region comprises SEQ ID NO: 110, 121, 122, or 123.

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

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

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

[0012] In another aspect, the invention provides a mature heavy chain variable region comprising CDRs H1, H2 and H3 comprising SEQ ID NOs: 8, 9 and 10, respectively, except that position H28 may be occupied by N or T, H54 may be occupied by N or D, H56 may be occupied by D or E, position H58 may be occupied by V or I, and position H60 may be designated by D or E; and CDRs H1, H2 and H3 comprising SEQ ID NOs: 12, 13 and 14, respectively, except that position L24 may be occupied by K or R, position L50 may be occupied by L, E, D, G, or V, position L52 may be occupied by S or G, and position L54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S. and a mature light chain variable region comprising L1, L2, and L3, wherein at least one of the following positions is occupied by the specified amino 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, and L10 is occupied by T. L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L83 is occupied by L, L86 is occupied by H, L100 is occupied by Q, and L106 is occupied by L.

[0013] In another aspect, the invention provides an isolated monoclonal antibody that binds human tau comprising the 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. 3D6 is a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:7 and a light chain variable region having an amino acid sequence comprising SEQ ID NO:11, except that position L54 may be occupied by L, D, G, N, E, Q, K, R, T, V, or S, and at least one of the following positions is occupied by the specified amino acid: L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L100 is occupied by Q, H60 is occupied by E, and H82 is occupied by V.

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

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

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

[0017] Some antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 18 and 146-148, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any 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 the 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 is occupied by V, H11 is occupied by V, H20 is occupied by I, H23 is occupied by K, H38 is occupied by R, H42 is occupied by G, H43 is occupied by K, H66 is occupied by R, H75 is occupied by T, H76 is occupied by D, H81 is occupied by E, H108 is occupied by L, and H109 is occupied by V. In some antibodies, positions H1, H5, H11, H20, H23, H38, H42, H43, H66, H75, H76, H81, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, L, and V, respectively.

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

[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 the specified amino acid: H28 is occupied by T and H67 is occupied by V. In some antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.

[0023] In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H54 is occupied by D and H56 is occupied by E. In some antibodies, positions H54 and H56 in the VH region are occupied by D and E, respectively.

[0024] In some antibodies, at least one of the following positions in the VH region is occupied by a designated amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H17 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, H54 is occupied by N or D, H56 is occupied by D or E, and H5 8 is occupied by V or I, H60 is occupied by D or E, H66 is occupied by K or R, H67 is occupied by A or V, H75 is occupied by S or T, H76 is occupied by N or D, H80 is occupied by L or M, H81 is occupied by Q or E, H82c is occupied by L or V, H83 is occupied by T or R, H91 is occupied by F or Y, H93 is occupied by S, H94 is occupied by T, H108 is occupied by T or L, and H109 is occupied by L or V.

[0025] In some antibodies, at least one of the following positions in the VH region is occupied by a designated amino acid: H10 is occupied by E or D, H12 is occupied by K or V, H13 is occupied by K or R, H17 is occupied by T, L or S, H24 is occupied by V or A, H27 is occupied by F or Y, H28 is occupied by N or T, H29 is occupied by I or F, H30 is occupied by K or T, H38 is occupied by Q or R, H40 is occupied by A or R, H42 is occupied by G or E, H43 is occupied by K or Q, H48 is occupied by M or I, and H51 is occupied by V. or I, H54 is occupied by N or D, H60 is occupied by D, A or E, H61 is occupied by P or E, H66 is occupied by R or K, H67 is occupied by V or A, H76 is occupied by D or N, H80 is occupied by M or L, H81 is occupied by E or Q, H82 is occupied by S or G, H82c is occupied by L or V, H83 is occupied by T or R, H91 is occupied by Y or F, H93 is occupied by A or S, H102 is occupied by F or Y, H108 is occupied by T or L, and H109 is occupied 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 is occupied by S, L15 is occupied by L, L83 is occupied by V, L86 is occupied by Y, and L106 is occupied by I. In some antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.

[0031] In some antibodies, at least one of the following positions in the VL region is occupied by a designated amino acid: L7 is occupied by T or S, L10 is occupied by T or S, L15 is occupied by I or L, L17 is occupied by Q or E, L24 is occupied by K or R, L37 is occupied by L or Q, L45 is occupied by K or R, and L47 is occupied 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 is occupied by S or T, L12 is occupied by P or S, L15 is occupied by L or I, L36 is occupied by L, L37 is occupied by L or Q, L45 is occupied by R or K, L47 is occupied 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, L60 is occupied by D or S, and L100 is occupied 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 the amino acid sequence of any one of SEQ ID NOs: 76 to 80 and 90 to 91, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 83 to 85. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 18 and 146 to 148, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 93 to 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 veneered antibody, or a humanized antibody.

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

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

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

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

[0059] In some nucleic acids, the heavy chain is encoded by a sequence comprising SEQ ID NO:182 and the light chain is encoded by a sequence comprising SEQ ID NO:183.

[0060] Also provided are methods for producing such humanized, chimeric, or veneered antibodies, such as humanized, chimeric, or veneered versions of 3D6. In such methods, cells transformed with nucleic acids encoding the antibody heavy and light chains are cultured to secrete the antibody, which can then be purified from the cell culture medium.

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

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

[0063] In yet another aspect, the invention provides a method of expressing an antibody in a mammalian cell, comprising incorporating a nucleic acid disclosed herein into the genome of a transgenic animal in which the antibody is expressed.

[0064] In yet another aspect, 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, each operably linked to one or more regulatory sequences for effecting expression of any of the antibodies disclosed herein, and a host cell containing 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, respectively. In some first and second vectors, the heavy chain constant region has the sequence of SEQ ID NO: 176, with or without a C-terminal lysine, and the light chain constant region has the sequence of SEQ ID NO: 177.

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

[0066] Also provided are methods for producing antibodies, such as humanized, chimeric, or veneered antibodies. 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 antibody. The antibody can then be purified from the cell culture medium.

[0067] A cell line producing any of the antibodies disclosed herein can be generated by introducing into cells a vector encoding the heavy and light chains of the antibody of claim 1 and a selectable marker, growing the cells under conditions that select for cells with increased copy number of the vector, isolating single cells from the selected cells, and banking the cloned cells from the selected single cells based on antibody yield.

[0068] Some cells were grown under selective conditions and spontaneously expressed at least 100 mg / L / 10 6 Cell lines secreting 3D6 / 24 hours can be screened. Single cells can be isolated from the selected cells. Cells cloned from the single cells can then be stored. Single cells can be selected based on desirable properties, such as antibody yield. Exemplary cell lines are those expressing 3D6 or a humanized version of 3D6.

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

[0070] Also provided are methods for treating or preventing a tau-related disease in a subject, comprising administering an effective dosing regimen of an antibody disclosed herein, thereby treating or preventing the disease. Examples of such diseases include Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some methods, the tau-associated disease is Alzheimer's disease. In some methods, the patient is an ApoE4 carrier.

[0071] Also provided are methods for reducing the aberrant propagation of tau, the methods comprising administering an effective dosage regimen of an antibody disclosed herein, thereby reducing the propagation of tau.

[0072] Also provided is a method of inducing phagocytosis of tau, the method comprising administering an effective dosage regimen of an antibody disclosed herein, thereby inducing phagocytosis of tau.

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

[0074] Also provided are methods for inhibiting the formation of tau tangles, the methods comprising administering an effective dosage regimen of the antibodies disclosed herein.

[0075] The present invention also provides a method for detecting tau protein deposits in a subject suffering from or at risk for a disease associated with tau aggregation or deposition, comprising administering an antibody disclosed herein to the subject and detecting antibody binding to tau in the subject. Examples of such diseases include Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some embodiments, the antibody is administered to the subject's body by intravenous injection. In some embodiments, the antibody is administered directly to the subject's brain by intracranial injection or by drilling a hole through the subject's skull. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

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

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

[0078] In another aspect, the invention provides a method for generating antibodies that specifically bind to human tau at an epitope within a motif of the 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 antibodies and screening for one of the generated antibodies that specifically binds within the motif. In another aspect, the invention provides a method for generating antibodies that specifically bind 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 antibodies and screening for one of the generated antibodies that specifically binds to the peptide. In another aspect, the invention provides a method for generating antibodies that specifically bind to an epitope comprising 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 the epitope.

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

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

[0081] [Figure 1] 1 shows the results of experiments designed to map the epitope(s) bound by the murine 3D6 monoclonal antibody. [Figure 2]1 shows the heavy chain variable region of the murine 3D6 antibody (SEQ ID NO: 7) and the heavy chain variable regions of the humanized versions of the 3D6 antibody (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7), as well as a sequence comparison of 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 SEQ ID NO: 76, hu3D6VHvb2 is SEQ ID NO: 77, hu3D6VHvb3 is SEQ ID NO: 78, hu3D6VHvb4 is SEQ ID NO: 79, hu3D6VHvb5 is SEQ ID NO: 80, hu3D6VHvb6 is SEQ ID NO: 90, and hu3D6VHvb7 is SEQ ID NO: 91. CDRs defined by the Kabat / Chothia combination are shown in bold. [Figure 3] 1 shows a sequence comparison of the light chain variable region of the murine 3D6 antibody (SEQ ID NO: 11) and the light chain variable regions of 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. The CDRs as defined by Kabat are shown in bold. [Figure 4A] 1 shows the results of an ELISA screen for selected mouse monoclonal anti-tau antibodies. [Figure 4B] 1 shows the results of an ELISA screen for selected mouse monoclonal anti-tau antibodies. [Figure 4C] 1 shows the results of an ELISA screen for selected mouse monoclonal anti-tau antibodies. [Figure 5] Figure 1 shows the binding kinetics of selected mouse monoclonal anti-tau antibodies to recombinant human tau. [Figure 6]1 shows the results of a functional blocking assay for selected mouse monoclonal anti-tau antibodies. [Figure 7] 1 shows the results of a disaggregation assay for selected mouse monoclonal anti-tau antibodies. [Figure 8] 1 shows the results of experiments demonstrating that 3D6 and 5G8 immunocapture tau from human Alzheimer's disease tissue. [Figure 9A] 1 shows a sequence comparison of the heavy chain variable region of murine 3D6 (SEQ ID NO: 7) and the heavy chain variable regions of humanized versions of the 3D6 antibody (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHv1bA11, h3D6VHvb8, and h3D6VHvb9) with 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 SEQ ID NO: 76, hu3D6VHvb2 is SEQ ID NO: 77, hu3D6VHvb3 is SEQ ID NO: 78, hu3D6VHvb4 is SEQ ID NO: 79, hu3D6VHvb5 is SEQ ID NO: 80, hu3D6VHvb6 is SEQ ID NO: 90, hu3D6VHvb7 is SEQ ID NO: 91, hu3D6VHv1bA11 is SEQ ID NO: 18, h3D6VHvb8 is SEQ ID NO: 146, and h3D6VHvb9 is SEQ ID NO: 148. Residues identical to those in the heavy chain variable region of murine 3D6 (SEQ ID NO: 7) are annotated with a "." CDRs defined by the Kabat / Chothia combination are shown in bold. [Figure 9B]1 shows a sequence comparison of the heavy chain variable region of murine 3D6 (SEQ ID NO: 7) and the heavy chain variable regions of humanized versions of the 3D6 antibody (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHv1bA11, h3D6VHvb8, and h3D6VHvb9) with 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 SEQ ID NO: 76, hu3D6VHvb2 is SEQ ID NO: 77, hu3D6VHvb3 is SEQ ID NO: 78, hu3D6VHvb4 is SEQ ID NO: 79, hu3D6VHvb5 is SEQ ID NO: 80, hu3D6VHvb6 is SEQ ID NO: 90, hu3D6VHvb7 is SEQ ID NO: 91, hu3D6VHv1bA11 is SEQ ID NO: 18, h3D6VHvb8 is SEQ ID NO: 146, and h3D6VHvb9 is SEQ ID NO: 148. Residues identical to those in the heavy chain variable region of murine 3D6 (SEQ ID NO: 7) are annotated with a "." CDRs defined by the Kabat / Chothia combination are shown in bold. [Figure 10A]3D6 antibody: 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 (SEQ ID NO: 132), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 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 hu3D6VLv2

[0023] Figure 1 shows a sequence comparison of the humanized light chain variable region of 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 annotated with a ".". The CDRs as defined by Kabat are shown in bold. [Figure 10B]3D6 antibody: 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 (SEQ ID NO: 132), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 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 hu3D6VLv2

[0023] Figure 1 shows a sequence comparison of the humanized light chain variable region of 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 annotated with a ".". The CDRs as defined by Kabat are shown in bold. [Figure 10C]3D6 antibody: 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 (SEQ ID NO: 132), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 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 hu3D6VLv2

[0023] Figure 1 shows a sequence comparison of the humanized light chain variable region of 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 annotated with a ".". The CDRs as defined by Kabat are shown in bold. [Figure 10D]3D6 antibody: 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 (SEQ ID NO: 132), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 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 hu3D6VLv2

[0023] Figure 1 shows a sequence comparison of the humanized light chain variable region of 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 annotated with a ".". The CDRs as defined by Kabat are shown in bold. [Figure 11] 1 shows the results of a tau internalization assay for selected humanized 3D6 variants. [Figure 12A]

[0013] Figure 12 shows the results of substitution mapping microarray experiments. Figure 12A shows a plot of substitution effects, and Figure 12B shows a sequence comparison of a portion of the tau microtubule-binding repeat, highlighting the critical residues for binding to 3D6. aa255-271 is SEQ ID NO: 184, aa286-302 is SEQ ID NO: 185, aa317-333 is SEQ ID NO: 186, and aa349-365 is SEQ ID NO: 187. [Figure 12B]

[0013] Figure 12 shows the results of substitution mapping microarray experiments. Figure 12A shows a plot of substitution effects, and Figure 12B shows a sequence comparison of a portion of the tau microtubule-binding repeat, highlighting the critical residues for binding to 3D6. aa255-271 is SEQ ID NO: 184, aa286-302 is SEQ ID NO: 185, aa317-333 is SEQ ID NO: 186, and aa349-365 is SEQ ID NO: 187. [Figure 13] 1 shows the results of immunohistochemistry experiments demonstrating that 3D6 binds tau in normal tissue (top panel) and Alzheimer's disease tissue (middle and bottom panels). [Figure 14] 1 shows the results of mass spectrometry experiments assessing the binding stoichiometry of 3D6. [Figure 15] 1 shows that 3D6 disrupts tau seeding in an in vivo disease model of Alzheimer's disease. [Figure 16] 1 shows that the 3D6 humanized variants hu3D6VHv1bA11 / hu3D6VLv2 and hu3D6VHv1bA11 / L2-DIM4 block the interaction of tau with heparin. [Figure 17]1 shows that the 3D6 humanized variants hu3D6VHv1bA11 / hu3D6VLv2 and hu3D6VHv1bA11 / L2-DIM4 bind the fibrillar form of tau. DETAILED DESCRIPTION OF THE INVENTION

[0082] A brief description of arrays SEQ ID NO: 1 shows the amino acid sequence of a human tau isoform (Swissprot P10636-8).

[0083] SEQ ID NO: 2 shows the amino acid sequence of a human tau isoform (Swissprot P10636-7).

[0084] SEQ ID NO: 3 shows the amino acid sequence of an isoform of human tau (Swissprot P10636-6), (4R0N human tau).

[0085] SEQ ID NO: 4 shows the amino acid sequence of a human tau isoform (Swissprot P10636-5).

[0086] SEQ ID NO: 5 shows the amino acid sequence of a human tau isoform (Swissprot P10636-4).

[0087] SEQ ID NO: 6 shows the amino acid sequence of a human tau isoform (Swissprot P10636-2).

[0088] SEQ ID NO: 7 shows the amino acid sequence of the heavy chain variable region of the mouse 3D6 antibody.

[0089] SEQ ID NO: 8 shows the amino acid sequence of the Kabat / Chothia combination CDR-H1 of the murine 3D6 antibody.

[0090] SEQ ID NO: 9 shows the amino acid sequence of Kabat CDR-H2 of the murine 3D6 antibody.

[0091] SEQ ID NO: 10 shows the amino acid sequence of the Kabat CDR-H3 of the murine 3D6 antibody.

[0092] SEQ ID NO: 11 shows the amino acid sequences of the light chain variable regions of the murine 3D6 antibody and the murine 6A10 antibody.

[0093] SEQ ID NO: 12 shows the amino acid sequence of Kabat CDR-L1 of the murine 3D6 antibody and the murine 6A10 antibody.

[0094] SEQ ID NO: 13 shows the amino acid sequence of Kabat CDR-L2 of the murine 3D6 antibody and the murine 6A10 antibody.

[0095] SEQ ID NO: 14 shows the amino acid sequence of the Kabat CDR-L3 of the murine 3D6 antibody and the murine 6A10 antibody.

[0096] SEQ ID NO: 15 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1.

[0097] SEQ ID NO: 16 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv2.

[0098] SEQ ID NO: 17 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1b.

[0099] SEQ ID NO: 18 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1bA11.

[0100] SEQ ID NO: 19 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv5.

[0101] SEQ ID NO: 20 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv1.

[0102] SEQ ID NO: 21 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv2.

[0103] SEQ ID NO: 22 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv3.

[0104] SEQ ID NO: 23 shows the amino acid sequence of the light chain variable region of humanized 3D6 antibody hu3D6VLv4.

[0105] SEQ ID NO: 24 shows the amino acid sequence of heavy chain variable receptor accession number BAC01986.1.

[0106] SEQ ID NO: 25 is the heavy chain variable receptor IMGT accession number IGHV1-69-2 * The amino acid sequence of 01 is shown below.

[0107] SEQ ID NO: 26 is the heavy chain variable receptor IMGT accession number IGKJ1 * The amino acid sequence of 01 is shown.

[0108] SEQ ID NO: 27 is the light chain variable receptor IMGT accession number IGKV2-30 * The amino acid sequence of 02 is shown.

[0109] SEQ ID NO: 28 is the light chain variable receptor IMGT accession number IGKJ2 * The amino acid sequence of 01 is shown below.

[0110] SEQ ID NO: 29 shows the amino acid sequence of light chain variable receptor accession number AAZ09048.1.

[0111] SEQ ID NO: 30 shows the nucleic acid sequence encoding the heavy chain variable region of the murine 3D6 antibody.

[0112] SEQ ID NO: 31 shows the nucleic acid sequence encoding the light chain variable region of the murine 3D6 antibody.

[0113] SEQ ID NO: 32 shows the amino acid sequence of Kabat CDR-H1 of the murine 3D6 antibody.

[0114] SEQ ID NO: 33 shows the amino acid sequence of Chothia CDR-H1 of the mouse 3D6 antibody.

[0115] SEQ ID NO: 34 shows the amino acid sequence of Chothia CDR-H2 of the mouse 3D6 antibody.

[0116] SEQ ID NO: 35 shows the amino acid sequence of AbM CDR-H2 of the mouse 3D6 antibody.

[0117] SEQ ID NO: 36 shows the amino acid sequence of Contact CDR-L1 of mouse 3D6 antibody.

[0118] SEQ ID NO: 37 shows the amino acid sequence of Contact CDR-L2 of mouse 3D6 antibody.

[0119] SEQ ID NO: 38 shows the amino acid sequence of Contact CDR-L3 of mouse 3D6 antibody.

[0120] SEQ ID NO: 39 shows the amino acid sequence of Contact CDR-H1 of the mouse 3D6 antibody.

[0121] SEQ ID NO: 40 shows the amino acid sequence of Contact CDR-H2 of the mouse 3D6 antibody.

[0122] SEQ ID NO: 41 shows the amino acid sequence of Contact CDR-H3 of mouse 3D6 antibody.

[0123] SEQ ID NO: 42 shows the amino acid sequence of another Kabat-Chothia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3, hu3D6VHv1e, and hu3D6VHv1f).

[0124] SEQ ID NO: 43 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv5 and hu3D6VHv1bA11B6H3).

[0125] SEQ ID NO: 44 shows the consensus amino acid sequence in the heavy chain variable region of murine 3D6 and selected humanized 3D6 antibodies (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) (labeled "Majority" in Figure 2 of PCT / IB2017 / 052544).

[0126] SEQ ID NO: 45 shows the consensus amino acid sequence between the light chain variable regions of murine 3D6 and selected humanized 3D6 antibodies (labeled "Majority" in Figure 3 of PCT / IB2017 / 052544).

[0127] SEQ ID NO: 46 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1bA11B6G2.

[0128] SEQ ID NO: 47 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1bA11B6H3.

[0129] SEQ ID NO: 48 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1c.

[0130] SEQ ID NO: 49 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1d.

[0131] SEQ ID NO: 50 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1e.

[0132] SEQ ID NO: 51 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv1f.

[0133] SEQ ID NO: 52 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv3.

[0134] SEQ ID NO: 53 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv3b.

[0135] SEQ ID NO: 54 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv3c.

[0136] SEQ ID NO: 55 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv4.

[0137] SEQ ID NO: 56 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv4b.

[0138] SEQ ID NO: 57 shows the amino acid sequence of the heavy chain variable region of humanized 3D6 antibody hu3D6VHv4c.

[0139] SEQ ID NO: 58 shows the amino acid sequence of another Kabat-Chocia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VH1c).

[0140] SEQ ID NO: 59 shows the amino acid sequence of another Kabat-Chotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c).

[0141] SEQ ID NO: 60 shows the amino acid sequence of another Kabat-Chotia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHv3b and hu3D6VHv4b).

[0142] SEQ ID NO: 61 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1bA11B6G2).

[0143] SEQ ID NO: 62 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b).

[0144] SEQ ID NO: 63 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c).

[0145] SEQ ID NO: 64 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHv1e).

[0146] SEQ ID NO: 65 shows the amino acid sequence of another Kabat CDR-H3 of the humanized 3D6 antibody (similar to hu3D6VHv1f).

[0147] SEQ ID NO: 66 shows the amino acid sequence of the heavy chain variable region of the murine 6A10 antibody.

[0148] SEQ ID NO: 67 shows the amino acid sequence of the Kabat / Chothia combination CDR-H1 of the murine 6A10 antibody.

[0149] SEQ ID NO: 68 shows the amino acid sequence of the Kabat CDR-H2 of the murine 6A10 antibody.

[0150] SEQ ID NO: 69 shows the amino acid sequence of the Kabat CDR-H3 of the murine 6A10 antibody.

[0151] SEQ ID NO: 70 shows the amino acid sequence of the VH region of a murine antibody (pdb code 1CR9) used as a structural template for heavy chain humanization.

[0152] SEQ ID NO: 71 shows the consensus amino acid sequence in the heavy chain variable region of selected humanized 3D6 antibodies (VHv1, VHv1b, VHv1bA11, VHv1bA11B6G2, VHv1bA11B6H3, VHv1c, VHv1d, VHv1e, VHv1f, VHv2, VHv3, VHv3b, VHv3c, VHv4, VHv4b, VHv4c, and VHv5) (labeled "Majority" in Figures 4A and 4B of PCT / IB2017 / 052544).

[0153] SEQ ID NO: 72 shows the amino acid sequence of the heavy chain of the chimeric 3D6 antibody.

[0154] SEQ ID NO: 73 shows the amino acid sequence of the light chain of the chimeric 3D6 antibody.

[0155] SEQ ID NO: 74 shows the amino acid sequence of the heavy chain variable structure model accession number 5MYX-VH_mSt.

[0156] SEQ ID NO: 75 shows the amino acid sequence of heavy chain variable receptor accession number 2RCS-VH_huFrwk.

[0157] SEQ ID NO: 76 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb1 of the humanized 3D6 antibody.

[0158] SEQ ID NO: 77 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb2 of the humanized 3D6 antibody.

[0159] SEQ ID NO: 78 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb3 of the humanized 3D6 antibody.

[0160] SEQ ID NO: 79 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb4 of the humanized 3D6 antibody.

[0161] SEQ ID NO: 80 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb5 of the humanized 3D6 antibody.

[0162] SEQ ID NO: 81 shows the amino acid sequence of the light chain variable structure model accession number 5MYX-VL_mSt.

[0163] SEQ ID NO: 82 shows the amino acid sequence of light chain variable receptor accession number ARX71335-VL_huFrwk.

[0164] SEQ ID NO: 83 shows the amino acid sequence of the light chain variable region hu3D6VLvb1 of the humanized 3D6 antibody.

[0165] SEQ ID NO: 84 shows the amino acid sequence of the light chain variable region hu3D6VLvb2 of the humanized 3D6 antibody.

[0166] SEQ ID NO: 85 shows the amino acid sequence of the light chain variable region hu3D6VLvb3 of the humanized 3D6 antibody.

[0167] SEQ ID NO: 86 shows the amino acid sequence of another Kabat-Chocia combination CDR-H1 of the humanized 3D6 antibody (similar to hu3D6VHvb4 and hu3D6VHvb5).

[0168] SEQ ID NO: 87 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb3 and hu3D6VHvb4).

[0169] SEQ ID NO: 88 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb5).

[0170] SEQ ID NO: 89 shows the amino acid sequence of another Kabat CDR-L1 of the humanized 3D6 antibody (similar to hu3D6VLvb3).

[0171] SEQ ID NO: 90 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb6 of the humanized 3D6 antibody.

[0172] SEQ ID NO: 91 shows the amino acid sequence of the heavy chain variable region hu3D6VHvb7 of the humanized 3D6 antibody.

[0173] SEQ ID NO: 92 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to hu3D6VHvb6 and hu3D6VHvb7).

[0174] SEQ ID NO: 93 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54D.

[0175] SEQ ID NO: 94 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54G.

[0176] SEQ ID NO: 95 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L45N.

[0177] SEQ ID NO: 96 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54E.

[0178] SEQ ID NO: 97 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50E.

[0179] SEQ ID NO: 98 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54Q.

[0180] SEQ ID NO: 99 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50D.

[0181] SEQ ID NO: 100 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54K.

[0182] SEQ ID NO: 101 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54R.

[0183] SEQ ID NO: 102 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54T.

[0184] SEQ ID NO: 103 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50G.

[0185] SEQ ID NO: 104 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant I48G.

[0186] SEQ ID NO: 105 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant I48D.

[0187] SEQ ID NO: 106 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47G.

[0188] SEQ ID NO: 107 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant Y49E.

[0189] SEQ ID NO: 108 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54V.

[0190] SEQ ID NO: 109 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54S.

[0191] SEQ ID NO: 110 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant S52G.

[0192] SEQ ID NO: 111 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47N.

[0193] SEQ ID NO: 112 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47D.

[0194] SEQ ID NO: 113 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47E.

[0195] SEQ ID NO: 114 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47P.

[0196] SEQ ID NO: 115 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47T.

[0197] SEQ ID NO: 116 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47S.

[0198] SEQ ID NO: 117 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47A.

[0199] SEQ ID NO: 118 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50V.

[0200] SEQ ID NO: 119 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R.

[0201] SEQ ID NO: 120 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G.

[0202] SEQ ID NO: 121 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54G.

[0203] SEQ ID NO: 122 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R.

[0204] SEQ ID NO: 123 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54T.

[0205] SEQ ID NO: 124 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D.

[0206] SEQ ID NO: 125 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54R.

[0207] SEQ ID NO: 126 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54G.

[0208] SEQ ID NO: 127 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54D.

[0209] SEQ ID NO: 128 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G.

[0210] SEQ ID NO: 129 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D.

[0211] SEQ ID NO: 130 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54T.

[0212] SEQ ID NO: 131 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G.

[0213] SEQ ID NO: 132 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54G.

[0214] SEQ ID NO: 133 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54R.

[0215] SEQ ID NO: 134 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54G.

[0216] SEQ ID NO: 135 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54R.

[0217] SEQ ID NO: 136 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R_G100Q.

[0218] SEQ ID NO: 137 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G_G100Q.

[0219] SEQ ID NO: 138 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R_G100Q.

[0220] SEQ ID NO: 139 shows the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D_G100Q.

[0221] SEQ ID NO: 140 shows the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50D_L54G_G100Q.

[0222] SEQ ID NO: 141 shows the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50D_L54R_G100Q.

[0223] SEQ ID NO: 142 shows the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50V_L54D_G100Q.

[0224] SEQ ID NO: 143 shows the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q.

[0225] SEQ ID NO: 144 shows the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant G100Q.

[0226] SEQ ID NO: 145 shows the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L54E.

[0227] SEQ ID NO: 146 shows the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant D60E, also known as h3D6VHvb8.

[0228] SEQ ID NO: 147 shows the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant L82cV.

[0229] SEQ ID NO: 148 shows the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant D60E_L80M_Q81E_L82cV_T83R, also known as h3D6VHvb9.

[0230] SEQ ID NO: 149 shows the amino acid sequence of another Kabat CDR-H2 of the humanized 3D6 antibody (similar to h3D6VHvb8 and h3D6VHvb9).

[0231] SEQ ID NO: 150 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54D and hu3D6VLv2L37Q_L54D).

[0232] SEQ ID NO: 151 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54G and hu3D6VLv2L37Q_L54G).

[0233] SEQ ID NO: 152 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54N).

[0234] SEQ ID NO: 153 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54E and hu3D6VLv2L37Q_L54E).

[0235] SEQ ID NO: 154 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50E).

[0236] SEQ ID NO: 155 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54Q).

[0237] SEQ ID NO: 156 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50D and hu3D6VLv2L37Q_L50D).

[0238] SEQ ID NO: 157 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54K).

[0239] SEQ ID NO: 158 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54R and hu3D6VLv2L37Q_L54R).

[0240] SEQ ID NO: 159 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54T and hu3D6VLv2L37Q_L54T).

[0241] SEQ ID NO: 160 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50G and hu3D6VLv2L37Q_L50G).

[0242] SEQ ID NO: 161 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54V).

[0243] SEQ ID NO: 162 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L54S).

[0244] SEQ ID NO: 163 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2S52G and hu3D6VLv2L37Q_S52G).

[0245] SEQ ID NO: 164 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2L50V).

[0246] SEQ ID NO: 165 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50G_L54R and hu3D6VLv2 L37Q_L50G_L54R_G100Q).

[0247] SEQ ID NO: 166 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50G_L54G and hu3D6VLv2 L37Q_L50G_L54G_G100Q).

[0248] SEQ ID NO: 167 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54G).

[0249] SEQ ID NO: 168 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54R and hu3D6VLv2 L37Q_S52G_L54R_G100Q).

[0250] SEQ ID NO: 169 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54T).

[0251] SEQ ID NO: 170 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_S52G_L54D and hu3D6VLv2 L37Q_S52G_L54D_G100Q).

[0252] SEQ ID NO: 171 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50D_L54G and hu3D6VLv2 L37Q_L50D_L54G_G100Q).

[0253] SEQ ID NO: 172 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50D_L54R and hu3D6VLv2 L37Q_L50D_L54R_G100Q).

[0254] SEQ ID NO: 173 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50E_L54G).

[0255] SEQ ID NO: 174 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50E_L54R).

[0256] SEQ ID NO: 175 shows the amino acid sequence of another Kabat CDR-L2 of the humanized 3D6 antibody (similar to hu3D6VLv2 L37Q_L50V_L54D_G100Q).

[0257] SEQ ID NO: 176 shows the amino acid sequence of the heavy chain constant region (IgG1: allotype G1m17,1).

[0258] SEQ ID NO: 177 shows the amino acid sequence of the light chain constant region (kappa).

[0259] SEQ ID NO: 178 shows the amino acid sequence of the mature heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype).

[0260] SEQ ID NO: 179 shows the amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa).

[0261] SEQ ID NO: 180 shows the amino acid sequence of the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0262] SEQ ID NO: 181 shows the amino acid sequence of the light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0263] SEQ ID NO: 182 shows the nucleotide sequence encoding the heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0264] SEQ ID NO: 183 shows the nucleotide sequence encoding the light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4 kappa) with a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0265] SEQ ID NO: 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] SEQ ID NO: 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] SEQ ID NO: 186 shows the amino acid sequence of the region of tau microtubule-binding repeat 3 (amino acid residues 317-333 of SEQ ID NO: 1).

[0268] SEQ ID NO: 187 shows the amino acid sequence of the region of tau microtubule-binding repeat 4 (amino acid residues 349-365 of SEQ ID NO: 1).

[0269] SEQ ID NO: 188 shows the amino acid sequence of the tau core motif in MBTR1 bound by 3D6.

[0270] SEQ ID NO: 189 shows the amino acid sequence of the tau sequence N-terminal to the tau core motif in MBTR1 bound by 3D6.

[0271] SEQ ID NO: 190 shows the amino acid sequence of the tau sequence C-terminal to the tau core motif in MBTR1 bound by 3D6.

[0272] SEQ ID NO: 191 shows the amino acid sequence of the epitope of 3D6.

[0273] SEQ ID NO: 192 shows the amino acid sequence of the tau core motif in MBTR2 bound by 3D6.

[0274] SEQ ID NO: 193 shows the amino acid sequence of the tau core motif in MBTR3 bound by 3D6.

[0275] SEQ ID NO: 194 shows the amino acid sequence of the tau core motif in MBTR4 bound 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 from interfering proteins and other contaminants resulting from its production or purification, but does not exclude the possibility that the monoclonal antibody may be combined with an excess of pharmaceutically acceptable carrier(s) or other vehicles intended to facilitate use. Sometimes, the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95%, or 99% w / w pure from interfering proteins and other contaminants resulting from its production or purification. An isolated monoclonal antibody or other biological entity is often the predominant macromolecular species remaining after its purification.

[0277] Specific binding of an antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 M -1 Specific binding refers to the affinity and / or avidity of an antibody. Specific binding is binding that is detectably high in magnitude and distinguishable from nonspecific binding that occurs with at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or specific spatial fits (e.g., lock-and-key type), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an 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-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. This variable region, linked to a cleavable signal peptide, is first expressed. The variable region without the signal peptide is often referred to as the mature variable region. Thus, for example, the mature variable region of a light chain refers to the light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0279] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. (See, for a review, Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7, which is incorporated by reference in its entirety into this application.)

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

[0281] The assignment of amino acids to the VL and VH domains, respectively, is consistent with any of the conventional definitions of CDRs, including those by Kabat (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), Chothia (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989), the Chothia-Kabat CDR combination, where CDR-H1 is the combination of the Chothia and Kabat CDRs, and Oxford Molecular's antibody modeling. These include the AbM definition used by the software; and the contact definition by Martin et al. (bioinfo.org.uk / abs) (see Table 1). Kabat provides a widely used numbering scheme (Kabat numbering) in which corresponding residues between different heavy chains or different light chains are assigned the same number. When an antibody is said to contain a CDR according to a particular CDR definition (e.g., Kabat), that definition specifies the minimum number of CDR residues (i.e., Kabat CDRs) present in the antibody. It does not exclude that other residues that fall within other conventional CDR definitions but are outside of that specified definition may also be present. For example, an antibody containing a CDR defined by Kabat includes, among other possibilities, an antibody in which the CDR contains Kabat CDR residues but no other CDR residues, and an antibody in which the CDR H1 is the combined Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues but no additional CDR residues according to other definitions. [Table 1]

[0282] The term "antibody" includes intact antibodies and their binding fragments. Fragments, typically including isolated heavy and light chains, Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv, compete with the intact antibody from which they were derived for specific binding to a target. Fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific and / or humanized antibodies. Bispecific or 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 specific for a different epitope on tau than that bound by 3D6.

[0283] In some bispecific antibodies, one heavy chain / light chain pair is the humanized 3D6 antibody further disclosed below, and the other heavy chain / light chain pair is derived from an antibody that binds to a receptor expressed on the blood-brain barrier, such as an insulin receptor, an insulin-like growth factor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, or a transferrin receptor (Friden et al., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such bispecific antibodies can be transported across the blood-brain barrier by receptor-mediated transcytosis. Brain uptake of bispecific antibodies can be further enhanced by engineering specific antibodies to reduce their affinity for blood-brain barrier receptors. The reduced affinity for the receptor results in a more widespread distribution in the brain (see, e.g., Atwal et al., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

[0284] Representative bispecific antibodies may be: (1) dual variable domain antibodies (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) Tandabs, which are the fusion of two single-chain diabodies to generate tetravalent bispecific antibodies with two binding sites for each target antigen. (3) flexibodies, which combine scFvs with diabodies to produce multivalent molecules; (4) so-called "dock and lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fabs, can result in trivalent bispecific binding proteins consisting of two identical Fab fragments bound to different Fab fragments; or (5) so-called scorpion molecules, which, for example, contain two scFvs 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-engineered IgG1 (Xencor), or DuoBody (based on Fab arm exchange, Genmab).

[0285] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by one or more tertiary folding structures of a protein. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three or more amino acids, usually at least five or eight to ten, in a unique spatial conformation. Methods for determining the spatial conformation 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 demonstrating the ability of one antibody to compete with the binding of another antibody to a target antigen. An antibody's epitope can also be determined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

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

[0288] The term "pharmaceutically acceptable" means the carrier, diluent or excipient or auxiliary material must be compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

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

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

[0291] The term "biological sample" refers to a sample of biological material within or obtainable from a biological source, e.g., a human or mammalian subject. Such samples may be organs, organelles, tissues, tissue sections, body fluids, peripheral blood, plasma, serum, cells, molecules such as proteins and peptides, and any portion or combination thereof derived therefrom. The term "biological sample" may also encompass any material obtained by processing a sample. Derived materials may include cells or their progeny. Processing of a biological sample may include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, etc.

[0292] The term "control sample" refers to a biological sample that is not known or suspected to contain a tau-related disease region, or at least not known or suspected to contain a disease region of a given type. A control sample can be obtained from an individual not affected by a tau-related disease. Alternatively, a control sample can be obtained from a patient not affected by a tau-related disease. Such a sample can be obtained at the same time or at a different time that the biological sample is suspected to contain a tau-related disease. Both the biological sample and the control sample can be obtained from the same tissue. Preferably, the control sample consists essentially or entirely of a healthy region and can be used for comparison with a biological sample suspected to contain a tau-related disease region. Preferably, the tissue of the control sample is of the same type as the tissue in the biological sample. Preferably, the tau-related disease-affected cells suspected to be present in the biological sample originate from the same cell type (e.g., neuron or glia) as the cell type in the control sample.

[0293] The term "disease" means any abnormal condition that impairs physiological function. This term is used broadly to encompass any disorder, disease, abnormality, pathology, pathological condition, state, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

[0294] The term "symptom" means subjective evidence of disease, such as a change in gait, that is noticed by a subject. "Sign" means objective evidence of disease, such as that recognized by a physician.

[0295] The term "positive response to treatment" refers to a more favorable response or average response in an individual patient in a patient population compared to the average response in a control population not receiving treatment.

[0296] For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0297] Percent sequence identity is determined using maximally aligned antibody sequences according to the Kabat numbering convention. After alignment, when a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody regions, divided by the total number of positions in the two aligned regions (not counting gaps), and multiplied by 100 to convert to a percentage.

[0298] A composition or method that "comprising" or "including" one or more described elements may include other elements not specifically recited. For example, a composition that "comprises" or "includes" an antibody may include the antibody alone or the antibody in combination with other components. Where the disclosure refers to a feature that comprises the specified elements, it should be understood that the disclosure instead refers to a feature that consists essentially of or consists of the specified elements.

[0299] The representation of a range of values ​​includes every integer within or defining that range, and every subrange defined by an integer within that range.

[0300] Unless otherwise clear from the context, the term "about" encompasses minor variations, such as the standard tolerance of error (eg, SEM) of measurement for the stated value.

[0301] Statistical significance means p<0.05.

[0302] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0303] Detailed Description I. Overview The present invention provides antibodies that bind to tau. Some antibodies specifically bind to one or more epitopes within the region of 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 at 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 of SEQ ID NO:1, respectively). Some antibodies bind within residues 259-268 and / or 290-299 and / or 321-330 and / or 353-362 of SEQ ID NO:1. For 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 are useful for preventing or slowing tau-associated disease progression and tau-associated pathological progression. While understanding the mechanism is not necessary for practicing the present invention, reduced toxicity may result from, among other mechanisms, stabilizing non-toxic conformations, inhibiting the intercellular or intracellular spread of pathogenic tau forms, inhibiting tau phosphorylation, interfering with tau binding to cells, or inducing tau proteolytic cleavage, resulting in antibodies inducing tau phagocytosis, inhibiting tau inter- or intramolecular aggregation, or inhibiting tau binding to other molecules. Antibodies or agents of the present invention that induce such antibodies can be used in methods for treating or effectively preventing Alzheimer's disease and other tau-associated diseases.

[0304] II.Target molecule Unless otherwise clear from the context, reference to tau refers to native human tau, including all isoforms, regardless of the presence or absence of post-translational modifications (e.g., phosphorylation, glycosylation, or acetylation). Six major isoforms (splice variants) of tau occur in the human brain. The longest of these variants has 441 amino acids, with the first methionine residue truncated. Residues are numbered according to the 441 isoform. Thus, for example, reference to phosphorylation at position 404 refers to position 404 in the 441 isoform, or the corresponding position in any other isoform when maximally aligned with the 441 isoform. The amino acid sequences and Swissprot numbers of the isoforms are shown below. P10636-8 (SEQ ID NO: 1) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEDVTAPLV DEGAPGKQAA 100 110 120 AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 130 140 150 HVTQARMVSK SKDGTGSDDK KAKGADGKTK 160 170 180 IATPRGAAPP GQKGQANATR IPAKTPPAPK 190 200 210 TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS 220 230 240 RTPSLPTPPT REPKKVAVVR TPPKSPSSAK 250 260 270 SRLQTAPVPM PDLKNVKSKI GSTENLKHQP 280 290 300 GGGKVQIINK KLDLSNVQSK CGSKDNIKHV 310 320 330 PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH 340 350 360 KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 370 380 390 THVPGGGNKK IETHKLTFRE NAKAKTDHGA 400 410 420 EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 430 440 DSPQLATLAD EVSASLAKQG L P10636-7 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA 100 110 120 GHVTQARMVS KSKDGTGSDD KKAKGADGKT 130 140 150 KIATPRGAAP PGQKGQANAT RIPAKTPPAP 160 170 180 KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 190 200 210 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA 220 230 240 KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 250 260 270 PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH 280 290 300 VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 310 320 330 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN 340 350 360 ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 370 380 390 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM 400 410 VDSPQLATLA DEVSASLAKQ GL P10636-6 (4R0N human tau) (SEQ ID NO: 3) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 70 80 90 AGHVTQARMV SKSKDGTGSD DKKAKGADGK 100 110 120 TKIATPRGAA PPGQKGQANA TRIPAKTPPA 130 140 150 PKTPPSSGEP 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 (SEQ ID NO: 5) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA 100 110 120 GHVTQARMVS KSKDGTGSDD KKAKGADGKT 130 140 150 KIATPRGAAP PGQKGQANAT RIPAKTPPAP 160 170 180 KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 190 200 210 SRTPSLPTPP TREPKKVAVV RTPPKSPSSA 220 230 240 KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 250 260 270 PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH 280 290 300 KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 310 320 330 THVPGGGNKK IETHKLTFRE NAKAKTDHGA 340 350 360 EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV 370 380 DSPQLATLAD EVSASLAKQG L P10636-2 (Sequence number 6) 10 20 30 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT 40 50 60 MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 70 80 90 AGHVTQARMV SKSKDGTGSD DKKAKGADGK 100 110 120 TKIATPRGAA PPGQKGQANA TRIPAKTPPA 130 140 150 PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS 160 170 180 RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 190 2,00 210 AKSRLQTAPV PMPDLKNVKS KIGSTENLKH 220 230 240 QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 250 260 270 HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN 280 290 300 ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 310 320 330 AEIVYKSPVV SGDTSPRHLS NVSSTGSIDM 340 350 VDSPQLATLA DEVSASLAKQ GL

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

[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, reference to tau or a fragment thereof includes the naturally occurring human amino acid sequence, including isoforms, mutants, and allelic variants thereof.

[0307] III. Antibodies A. Binding Specificity and Functional Properties The present invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within KXXSXXNX(K / H)H (SEQ ID NO: 191). Some antibodies bind to a peptide consisting essentially of, or consisting of, amino acid residues 259-268 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide consisting essentially of, or consisting of, amino acid residues 290-299 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide consisting essentially of, or consisting of, amino acid residues 321-330 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies bind to a peptide consisting essentially of, or consisting of amino acid residues 353-362 of the 441-amino acid tau protein (SEQ ID NO: 1). Some antibodies specifically bind to an epitope 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 an epitope 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 to 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 to 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 an epitope including residues 259, 262, 265, 267, 268, residues 290, 293, 296, 298, 299, residues 321, 324, 327, 329, 330, or residues 353, 356, 359, 362 of the 441-amino acid tau protein SEQ ID NO: 1. Some antibodies bind to tau regardless of its phosphorylation state. Some antibodies bind to an epitope that does not include residues that are subject to phosphorylation. These antibodies can be obtained by immunization with tau polypeptides purified from natural sources or recombinantly expressed. Antibodies can be screened for binding to unphosphorylated tau and to forms of tau phosphorylated at one or more residues susceptible to phosphorylation. Such antibodies preferably bind with indistinguishable affinity to unphosphorylated tau, or bind to phosphorylated tau within a factor of at least 1.5, 2, or 3 (i.e., are "pan-specific") compared to unphosphorylated tau. 3D6 is an example of a pan-specific monoclonal antibody. The present invention also provides antibodies that bind to the same epitope as any of the above-mentioned antibodies, for example, the epitope of 3D6. Also included are antibodies that compete with any of the above-mentioned antibodies for binding to tau, for example, those that compete with 3D6.

[0308] The above-described antibodies can be generated de novo by immunizing with a peptide comprising, consisting essentially of, or consisting of residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or with a peptide comprising, consisting essentially of, or consisting of residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO:1, or with a full-length tau polypeptide or a fragment thereof comprising such residues, and screening for specific binding to peptides comprising such residues. Such peptides are preferably conjugated to a heterologous conjugate molecule that helps elicit an antibody response to the peptide. Conjugation can be direct or via a spacer peptide or amino acid. Cysteine ​​is used as a spacer amino acid because its free sulfhydryl group facilitates attachment of a carrier molecule. Polyglycine linkers (e.g., 2-6 glycines) with or without cysteine ​​residues between the glycine and the peptide can also be used. Carrier molecules serve to provide T cell epitopes that help elicit antibody responses to the peptide. Several carriers are commonly used, particularly keyhole limpet hemocyanin (KLH), ovalbumin, and bovine serum albumin (BSA). Peptide spacers can be added to peptide immunogens as part of solid-phase peptide synthesis. Carriers are typically added by chemical cross-linking.Some examples of chemical cross-linkers that can be used include cross-N-maleimido-6-aminocaproyl ester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see, e.g., Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1988; Sinigaglia et al., Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47 (1993); Hammer et al., Cell, 74:197-203 (1993); Falk K. et al., Immunogenetics, 39:230-242 (1994); WO 98 / 23635; and Southwood et al. (See, e.g., J. Immunology, 160:3363-3373 (1998)). Carriers and spacers, if present, can be attached to either end of the immunogen.

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

[0310] The present invention provides monoclonal antibodies that bind to an epitope within tau. The antibody designated 3D6 is one such representative murine antibody. Unless otherwise clear from the context, reference to 3D6 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. This antibody has been deposited under [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 (corresponding to amino acid residues 257-271 and / or 320-334, respectively, of SEQ ID NO: 1) of the 383-amino acid 4R0N human tau protein (SEQ ID NO: 3). The antibody specifically binds to amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, and any combination of two, three, or all four thereof. The antibody is further characterized by its ability to bind to both phosphorylated and non-phosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded / aggregated forms of tau. The antibody designated 6A10 is one such representative murine antibody. Unless otherwise clear from the context, reference to 6A10 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. The Kabat / Chothia combination CDRs of the heavy chain of 6A10 are set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the Kabat CDRs of the light chain of 6A10 are set forth in SEQ ID NOs: 12, 13, and 14, respectively. Murine 6A10 shares 82.1% VH and 100% VL sequence identity with the murine 3D6 VH and VL chains, respectively.

[0311] Some antibodies of the invention bind to the same or overlapping epitopes as the antibody designated 3D6. The sequences of the heavy and light chain mature variable regions of this antibody are set forth in SEQ ID NOs: 7 and 11, respectively. Other antibodies with such binding specificity can be generated by immunizing mice with tau or a portion thereof that consists essentially of or consists of an epitope including the desired epitope (e.g., 199-213 and / or 262-276 of SEQ ID NO: 3, which correspond to residues 257-271 and / or 320-334 of SEQ ID NO: 1, respectively, 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 thereof), and screening the resulting antibodies for binding to tau, optionally in competition with an antibody having the variable region of mouse 3D6 (IgG1 kappa). Fragments of tau containing the desired epitope can be linked to a carrier that helps elicit an antibody response to the fragment and / or mixed with an adjuvant that helps elicit such a response. Such antibodies can be screened for differential binding to tau or its fragments compared to mutants of specified residues. Screening against such mutants allows for more precisely defining binding specificity, identifying antibodies whose binding is inhibited by mutagenesis of specific residues, and antibodies that may share the functional properties of other exemplified antibodies. Mutations can be systematic replacement of one residue at a time, or over a wider spatial distance, with alanine (or serine, if alanine is already present), across the entire target or segment where the epitope is known to reside. If the same set of mutations significantly reduces binding of two antibodies, the two antibodies bind to the same epitope.

[0312] Antibodies having the binding specificity of a selected murine antibody (e.g., 3D6) can also be generated using a variant of the phage display method. See Winter, International Publication No. 92 / 20791. This method is particularly suitable for generating human antibodies. In this method, either the heavy or light chain variable region of a selected murine antibody is used as the starting material. For example, if the light chain variable region is selected as the starting material, a phage display is constructed in which members display the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. 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 a fragment thereof (e.g., at least 10 8 , preferably at least 10 9 M -1 ) are selected. The heavy chain variable region from this phage then serves as the starting material for constructing an additional phage library. In this library, each phage displays 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, phage that show strong specific binding to tau or a fragment thereof are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

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

[0314] Table 2 shows the 3D6 CDRs defined by Kabat, Chothia, the combination of Chothia and Kabat (also referred to herein as the "Kabat / Chotia combination"), AbM, and Contact. [Table 2]

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

[0316] The present invention also provides antibodies having some or all (e.g., three, four, five, and six) CDRs derived entirely or substantially from 3D6. Such antibodies can 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. The antibody can include both a heavy chain and a light chain. The CDRs are substantially derived from the corresponding 3D6 CDRs, except that CDR-H2 (as defined by Kabat) can have no more than six, five, four, three, two, or one substitutions, insertions, or deletions, if any. Such antibodies may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity in the amino acid sequence of the mature heavy and / or light chain variable regions to 3D6 and retain its functional properties, and / or may differ from the 3D6 antibodies by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0317] Some antibodies identified by such assays can bind to monomeric, misfolded, aggregated, phosphorylated, or non-phosphorylated forms of tau, etc. Similarly, some antibodies are immunoreactive with non-pathological and pathological forms and conformations of tau.

[0318] B. Non-human antibodies The generation of other non-human antibodies, e.g., antibodies in mice, guinea pigs, primates, rabbits, or rats, against tau or a fragment thereof (e.g., amino acid residues 199-213 or 262-276 of SEQ ID NO: 3, which correspond to amino acid residues 257-271 or 320-334 of SEQ ID NO: 1, respectively, or amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1) can be achieved, for example, by immunizing the animal with tau or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference). Such immunogens can be obtained from natural sources by peptide synthesis or by recombinant expression. Optionally, the immunogen can be fused to or otherwise conjugated with a carrier protein and administered. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used, as described below. Complete Freund's adjuvant followed by incomplete adjuvant is preferred for immunizing laboratory animals. Rabbits or guinea pigs are typically used to generate polyclonal antibodies. Mice are typically used to generate monoclonal antibodies. Antibodies are screened for specific binding to tau or an epitope within tau (e.g., an epitope comprising one or more of amino acid residues 199-213 or 262-276 of SEQ ID NO: 3, which correspond to amino acid residues 257-271 or 320-334 of SEQ ID NO: 1, respectively, or an epitope comprising one or more of amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1).Such screening can be accomplished by determining binding of the antibody to a collection of tau variants, such as tau variants comprising amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, respectively, of SEQ ID NO:1), or tau variants comprising 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 assessed, for example, by Western blot, FACS, or ELISA.

[0319] C. Humanized Antibodies Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having at least three, four, five, or all of the CDRs, fully or substantially, derived from the donor antibody, as well as variable region framework sequences and, if present, constant regions, fully or substantially, derived from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody heavy chain, and, if present, a heavy chain variable region framework sequence and heavy chain constant region derived substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody light chain, and, if present, a light chain variable region framework sequence and light chain constant region derived substantially from human light chain variable region framework and constant region sequences. In addition to nanobodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. CDRs in a humanized antibody are derived from corresponding CDRs in a substantially non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (defined by any conventional method, but preferably by Kabat) are identical between the respective CDRs. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical.To be classified as humanized according to the 2014 World Health Organization (WHO) International Nonproprietary Name (INN) definition for humanized antibodies, an antibody must share at least 85% identity with the human germline antibody sequence (i.e., before somatic hypermutation). A mixed antibody is one in which one antibody chain (e.g., the heavy chain) meets the threshold and the other chain (e.g., the light chain) does not. If neither chain meets the threshold, the antibody is classified as chimeric, even if the variable framework regions of both chains are substantially human with some backmutations. See Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1, 1-9, DOI:10.1080 / 19420862.2015.1114320. Also available 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, which is incorporated herein by reference. For the avoidance of doubt, the term "humanized," as used herein, is not intended to be limited to the 2014 WHO INN definition of a humanized antibody. Some humanized antibodies provided herein have at least 85% sequence identity to human germline sequences, and some humanized antibodies provided herein have less than 85% sequence identity to human germline sequences. The heavy chains of some of the humanized antibodies provided herein have about 60% to 100% sequence identity to human germline sequences, such as in the range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89% sequence identity.Some heavy chains fall below the 2014 WHO INN definition, e.g., have about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identity to human germline sequences, while other heavy chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or more sequence identity to human germline sequences. Some light chains of the humanized antibodies provided herein have sequence identities of about 60% to 100% to human germline sequences, such as in the range of 80% to 84%, or 85% to 89%. Some light chains fall below the 2014 WHO INN definition, for example, having about 81%, 82%, 83%, or 84% sequence identity to human germline sequences, while other light chains meet the 2014 WHO INN definition, having about 85%, 86%, 87%, 88%, 89% or more sequence identity to human germline sequences. Some humanized antibodies provided herein that are "chimeric" according to the 2014 WHO INN definition have a heavy chain that is less than 85% identical to human germline sequences paired with a light chain that is less than 85% identical to human germline sequences. For example, some humanized antibodies provided herein that have a heavy chain that is at least 85% identical to human germline sequences paired with a light chain that is less than 85% identical to human germline sequences are "mixed" according to the 2014 WHO INN definition, or vice versa. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized" and have a heavy chain with at least 85% identity to human germline sequences paired with a light chain with at least 85% identity to human germline sequences. Additional humanized antibodies of the invention meet the 2014 WHO INN definition of "mixed."

[0320] Humanized antibodies often include all six CDRs from the murine antibody (defined by any conventional method, but preferably as defined by Kabat), although humanized antibodies can also be made with fewer than all CDRs from the murine antibody (e.g., at least three, four, or five CDRs) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., J. Immunol., 164:1432-1441, 2000).

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

[0322] Human acceptor antibody sequences can be selected from among many known human antibody sequences, if desired, to provide a high degree of sequence identity (e.g., 65-85% identity) between the variable region frameworks of the human acceptor sequence and the corresponding variable region frameworks of the donor antibody chain.

[0323] Some humanized and chimeric antibodies have the same (within experimental error) or improved functional properties as the murine antibodies from which they were derived, such as binding affinity to human tau and inhibition of tau internalization into neurons, as described in the Examples. For example, some humanized and chimeric antibodies have binding affinity within 3-fold, 2-fold, or 1-fold of the murine antibodies from which they were derived, or indistinguishable affinity within experimental error. Some humanized and chimeric antibodies inhibit tau internalization into neurons within 3-fold, 2-fold, or 1-fold of the murine antibodies from which they were derived, as described in the Examples, or inhibit tau internalization within experimental error in the same way as the murine antibodies from which they were derived. Several humanized antibodies exhibited reduced immunogenicity, increased affinity, increased thermal stability, and / or improved expression compared to previously described humanized versions of the 3D6 antibody (see WO 2017 / 191560), with hu3D6VHv1bA11 / L2-DIM4 demonstrating improved affinity over the parental hu3D6VHv1bA11 / hu3D6VLv2, as indicated by on-rate, off-rate, and Kd numbers. hu3D6VHv1bA11 / L2-DIM4 also demonstrated higher thermal stability and potency than the parental 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. An example of a heavy chain receptor sequence is IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25). IMGT No. IGHV1-69-2* 01 (SEQ ID NO: 25) shares the canonical mouse 3D6 heavy chain CDR-H1 and H2. IMGT No. IGHV1-69-2 * 01 (SEQ ID NO: 25) belongs to human heavy chain subgroup 1. An example of a light chain receptor sequence is the human mature light chain variable region of human antibody ARX71335VL (SEQ ID NO: 82) with PDB accession code. The variable light chain domains of the 3D6 and ARX71335VL antibodies also share identical lengths for the CDR-L1, L2, and L3 loops. An example of a light chain receptor sequence is IMGT number IGKV2-30 * 02 (SEQ ID NO: 27). IMGT No. IGKV2-30 * 02 (SEQ ID NO: 27) has the same canonical class of CDR-L1, CDR-L2 and L3 as mouse 3D6. IMGT No. IGKV2-30 * 02 (SEQ ID NO: 27) belongs to human kappa subgroup 2.

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

[0326] Specific amino acids from the human variable region framework residues can be selected for substitution based on their possible effect on CDR conformation and / or binding to antigen, with such possible effects being studied by modeling, examining the characteristics of the amino acid at a particular position, or empirical observation of the effects of substituting or mutagenesing specific amino acids.

[0327] For example, if an amino acid differs between the murine variable region framework residue and the selected human variable region framework residue, the human framework amino acid can be substituted with the equivalent framework amino acid from the murine antibody, where the amino acid is: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR region or within the CDR as defined by Chochia but not Kabat; (3) otherwise interacts with a CDR region (e.g., is within about 6 Å of a CDR region), (e.g., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain), or (4) Residues involved in the VL-VH interface: It is reasonable to assume that.

[0328] In one embodiment, humanized sequences are generated using a two-step PCR protocol that allows for the introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682].

[0329] As defined by U.S. Patent No. 5,530,101 to Queen, framework residues of classes (1) to (3) are sometimes referred to alternately as canonical and vernier residues. Framework residues that help determine the conformation of CDR loops are sometimes referred to as canonical residues (Chothia & Lesk, J. Mol. Biol. 196, 901-917 (1987), Thornton & Martin, J. Mol. Biol., 263:800-815 (1996)). Framework residues that support the conformation of antigen-binding loops and play a role in fine-tuning the fit of the antibody to the antigen are sometimes referred to as vernier residues (Foote & Winter, J. Mol. Biol. 224:487-499 (1992)).

[0330] Other framework residues that are candidates for substitution create potential glycosylation sites. Still other substitution candidates are recipient human framework amino acids that are unusual for that position in human immunoglobulins. These amino acids can be substituted with amino acids from the equivalent positions in the mouse donor antibody or with amino acids from the equivalent positions in more typical human immunoglobulins.

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

[0332] An exemplary humanized antibody is a humanized version of murine 3D6 designated Hu3D6.

[0333] Murine antibody 3D6 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NOs: 7 and 11, respectively. The present invention provides ten exemplary humanized mature heavy chain variable regions (hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6VHvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)) and 56 exemplified 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 (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 Humanized versions of the murine 3D6 antibody are provided, including hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).

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

[0335] For reasons such as their potential impact on CDR conformation and / or binding to antigen, mediating interactions between heavy and light chains, interactions with constant regions, being sites for desirable or undesirable post-translational modifications, being an unusual residue for its position in a human variable region and therefore potentially immunogenic, acquiring aggregation potential, and other reasons, as further demonstrated by the examples, the following 35 variable region framework positions were considered as potential substitutions in 56 exemplary human mature light chain variable regions and 10 exemplary 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 positions were considered as substitution candidates in the 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions, as further demonstrated by the 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, the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:87.In some humanized 3D6 antibodies, the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 149. In some humanized 3D6 antibodies, the Kabat-Chotia combination CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87. In some humanized 3D6 antibodies, the Kabat-Chotia combination CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 88. In some humanized 3D6 antibodies, the Kabat-Chotia combination CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 92. In some humanized 3D6 antibodies, the Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89. In some humanized 3D6 antibodies, the Kabat CDR-L2 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.

[0336] Here, as elsewhere, the first-mentioned residue is the residue of the humanized antibody formed by grafting the Kabat CDR, or in the case of CDR-H1, the combined Chothia-Kabat CDR, onto a human acceptor framework, and the second-mentioned residue is the residue being considered to replace such residue. Thus, the first-mentioned residue in the variable region framework is human, and the first-mentioned residue in the CDR is murine.

[0337] Exemplary antibodies include any permutation or combination of the following exemplary mature heavy and light chain variable regions: VHvb1 / VLvb1, VHvb1 / VLvb2, VHvb1 / VLvb3, VHvb2 / VLvb1, VHvb2 / VLvb2, VHvb2 / VLvb3, VHvb3 / VLvb1, VHvb3 / VLvb2, VHvb3 / VLvb3 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 regions hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), and hu3D6VHv1bA11 D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) and humanized 3D6VL light chain variable 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 (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2L37Q_S52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).

[0338] The exemplified antibodies include the exemplified mature heavy chain variable regions hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6Hvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), and hu3D6VHv1bA11 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 include 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), hu3D6VLv2 L54T (SEQ ID NO: 102), hu3D6VLv2 L50G (SEQ ID NO: 103), hu3D6VLv2 I48G (SEQ ID NO: 104), hu3D6VLv2 I48D (SEQ ID NO: 105), hu3D6VLv2 L47G (SEQ ID NO: 106), hu3D6VLv2 Y49E (SEQ ID NO: 107), hu3D6VLv2 L54V (SEQ ID NO: 108), hu3D6VLv2 L54S (SEQ ID NO: 109), hu3D6VLv2 S52G (SEQ ID NO: 110), hu3D6VLv2 L47N (SEQ ID NO: 111), hu3D6VLv2 L47D (SEQ ID NO: 112), hu3D6VLv2 L47E (SEQ ID NO: 113), hu3D6VLv2L47P (SEQ ID NO: 114), hu3D6VLv2 L47T (SEQ ID NO: 115), hu3D6VLv2 L47S (SEQ ID NO: 116), hu3D6VLv2 L47A (SEQ ID NO: 117), hu3D6VLv2 L50V (SEQ ID NO: 118), hu3D6VLv2 L37Q_L50G_L54R (SEQ ID NO: 119), hu3D6VLv2 L37Q_L50G_L54G (SEQ ID NO: 120), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_L54R (SEQ ID NO: 125), hu3D6VLv2 L37Q_L54G (SEQ ID NO: 126), hu3D6VLv2 L37Q_L54D (SEQ ID NO: 127), hu3D6VLv2 L37Q_L50G (SEQ ID NO: 128), hu3D6VLv2 L37Q_L50D (SEQ ID NO: 129), hu3D6VLv2 L37Q_L54t (SEQ ID NO: 130), hu3D6VLv2 L37Q_S52G (SEQ ID NO: 131), hu3D6VLv2 L37Q_L54E (SEQ ID NO: 145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO: 132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO: 133), hu3D6VLv2 L37Q_L50E_L54G (SEQ ID NO: 134), hu3D6VLv2 L37Q_L50E_L54R (SEQ ID NO: 135), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_s52G_L54R_G100Q (SEQ ID NO: 138), hu3D6VLv2 L37Q_s52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2G100Q (SEQ ID NO: 144) and the humanized 3D6 heavy chain variable regions hu3D6VHv1 (SEQ ID NO: 15); hu3D6VHv2 (SEQ ID NO: 16); hu3D6VHv1b (SEQ ID NO: 17); hu3D6VHv1bA11 (SEQ ID NO: 18); hu3D6VHv5 (SEQ ID NO: 19); hu3D6VHv1bA11b6G2 (SEQ ID NO: 46); hu3D6VHv1bA11b6H3 (SEQ ID NO: 47); hu3D6VHv1c (SEQ ID NO: 48); This includes any permutation or combination with any of hu3D6VHv1d (sequence number 49); hu3D6VHv1e (sequence number 50); hu3D6VHv1f (sequence number 51); hu3D6VHv3 (sequence number 52); hu3D6VHv3b (sequence number 53); hu3D6VHv3c (sequence number 54); hu3D6VHv4 (sequence number 55); hu3D6VHv4b (sequence number 56); and hu3D6VHv4c (sequence number 57).

[0339] The present invention provides antibodies that combine 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 antibodies that combine 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 antibodies that combine 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 antibodies that combine 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 antibodies that combine 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 antibodies that combine 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 that combines the humanized heavy chain variable region hu3D6VHv1bA11, also known as h3D6Hu5 (SEQ ID NO: 18), with the humanized light chain variable region hu3D6VLv2 L50G (L2-DIM22, SEQ ID NO: 103).

[0340] The present invention provides antibodies combining any one of the exemplified humanized heavy chain variable regions 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 (hu3D6VHv1bA11 IgG1 G1m17 allotype) having a bovine alpha-lactalbumin signal peptide at the N-terminus. The present invention provides antibodies combining any one of the exemplified humanized light chain variable regions with a light chain constant region. A representative light chain constant region is provided as SEQ ID NO: 177 (kappa). For example, SEQ ID NO: 179 shows the amino acid sequence of the mature light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4kappa). For example, SEQ ID NO: 181 shows the amino acid sequence of the light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4kappa) having a bovine alpha-lactalbumin signal peptide at the N-terminus.

[0341] The present invention provides variants of the 3D6 humanized antibody, in which the humanized mature heavy chain variable region is selected from the group consisting of 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), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), or hu3D6VHv1bA11 and a humanized mature light chain variable region exhibiting at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu3D6VHvb9 (h3D6VHvb9, SEQ ID NO: 148), and the humanized mature light chain variable region is selected from the group consisting of 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 (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), or hu3D6VLv2Variants of the 3D6 humanized antibody are provided that exhibit at least 90%, 95%, 96%, 97%, 98%, or 99% identity to 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 backmutations or other mutations in SEQ ID NOs: 76-80, 90-91, 146-148, 83-85, and 93-145.

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

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

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

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

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

[0349] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by a designated amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H17 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, and H54 is occupied by N or D. H56 is occupied by D or E, H58 is occupied by V or I, H66 is occupied by K or R, H67 is occupied by A or V, H75 is occupied by S or T, H76 is occupied by N or D, H80 is occupied by L or M, H81 is occupied by Q or E, H83 is occupied by T or R, H91 is occupied by F or Y, H93 is occupied by S, H94 is occupied by T, H108 is occupied by T or L, and H109 is occupied by L or V.

[0350] In some humanized 3D6 antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively, as in huVHvb1. In some humanized 3D6 antibodies, positions H1, H5, H11, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T, L, and V, respectively, as in huVHvb2. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, R, G, K, I, R, T, D, M, E, R, S, T, L, and V, respectively, similar to huVHvb3. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, similar to huVHvb4. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L, and V, respectively, 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 above-referenced antibodies can be modified to further reduce immunogenicity. For example, in some of the 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 the specified amino acid: L7 is occupied by S, L10 is occupied by S, L15 is occupied by L, L83 is occupied by V, L86 is occupied by Y, and L106 is occupied by I. In some humanized 3D6 antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.

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

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

[0357] In some humanized 3D6 antibodies, position L54 is occupied by D, as in hu3D6VLv2 L54D. In some humanized 3D6 antibodies, position L54 is occupied by G, as in hu3D6VLv2 L54G. In some humanized 3D6 antibodies, position L54 is occupied by N, as in hu3D6VLv2 L54N. In some humanized 3D6 antibodies, position L54 is occupied by E, as in hu3D6VLv2 L54E. In some humanized 3D6 antibodies, position L50 is occupied by E, as in hu3D6VLv2 L50E. In some humanized 3D6 antibodies, position L54 is occupied by Q, as in hu3D6VLv2 L54Q. In some humanized 3D6 antibodies, position L50 is occupied by D, as in hu3D6VLv2 L50D. In some humanized 3D6 antibodies, position L54 is occupied by K, as in hu3D6VLv2 L54K. In some humanized 3D6 antibodies, position L54 is occupied by R, as in hu3D6VLv2 L54R. In some humanized 3D6 antibodies, position L54 is occupied by T, as in hu3D6VLv2 L54T. In some humanized 3D6 antibodies, position L50 is occupied by G, as in hu3D6VLv2 L50G. In some humanized 3D6 antibodies, position L48 is occupied by G, as in hu3D6VLv2 I48G. In some humanized 3D6 antibodies, position L48 is occupied by D, as in hu3D6VLv2 I48D. In some humanized 3D6 antibodies, position L47 is occupied by G, as in hu3D6VLv2 L47G. In some humanized 3D6 antibodies, position L49 is occupied by E, as in hu3D6VLv2 Y49E. In some humanized 3D6 antibodies, position L54 is occupied by V, as in hu3D6VLv2 L54V. In some humanized 3D6 antibodies, position L54 is occupied by S, as in hu3D6VLv2 L54S. In some humanized 3D6 antibodies, position L52 is occupied by G, as in hu3D6VLv2 S52G. In some humanized 3D6 antibodies, position L47 is occupied by N, as in hu3D6VLv2 L47N.In some humanized 3D6 antibodies, position L47 is occupied by D, as in hu3D6VLv2 L47D. In some humanized 3D6 antibodies, position L47 is occupied by E, as in hu3D6VLv2 L47E. In some humanized 3D6 antibodies, position L47 is occupied by P, as in hu3D6VLv2 L47P. In some humanized 3D6 antibodies, position L47 is occupied by T, as in hu3D6VLv2 L47T. In some humanized 3D6 antibodies, position L47 is occupied by S, as in hu3D6VLv2 L47S. In some humanized 3D6 antibodies, position L47 is occupied by A, as in hu3D6VLv2 L47A. In some humanized 3D6 antibodies, position L50 is occupied by V, as in 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_L50G_L54R. In some humanized 3D6 antibodies, positions L37, L50, and L54 are occupied by Q, G, and T, respectively, similar to hu3D6VLv2 L37Q_S52G_L54T. In some humanized 3D6 antibodies, positions L37, L52, and L54 are occupied by Q, G, and D, respectively, similar to hu3D6VLv2 L37Q_S52G_L54D.

[0359] In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and R, respectively, as in hu3D6VLv2 L37Q_L54R. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q_L54G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q_L54D. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and G, respectively, as in hu3D6VLv2 L37Q_L50G. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and D, respectively, as in hu3D6VLv2 L37Q_L50D. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and T, respectively, 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, similar to 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, similar to 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, similar to 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, similar to 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, similar to 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, similar to 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, such as in hu3D6VLv2 L37Q. In some humanized 3D6 antibodies, position L100 is occupied by Q, such as in hu3D6VLv2 G100Q.

[0363] Some humanized 3D6 antibodies comprise a mature heavy chain variable region comprising CDRs H1, H2, and H3, which comprise SEQ ID NOs: 8, 9, and 10, respectively, except that position H28 may be occupied by N or T, H54 may be occupied by N or D, H56 may be occupied by D or E, position H58 may be occupied by V or I, and position H60 may be designated D or E; and CDRs H1, H2, and H3, which comprise SEQ ID NOs: 12, 13, and 14, respectively, 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. and a mature light chain variable region comprising 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 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 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S, or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G, or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V, or S, L83 is occupied by L, L86 is occupied by H, L100 is occupied by Q, and L106 is occupied by L.

[0364] Some humanized 3D6 antibodies comprise the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is selected from the group consisting of: 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; position L52 may be occupied by S or G; position L and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 11, except that 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 to human sequences. In some humanized 3D6 antibodies, the light chain variable region has ≥ 85% identity to human sequences. In some humanized 3D6 antibodies, each of the heavy and light chain variable regions has ≥ 85% identity to human germline sequences. In some humanized 3D6 antibodies, the three heavy chain CDRs are as defined by the Kabat / Chothias combination (SEQ ID NOs: 8, 9, and 10) and the three light chain CDRs are as defined by the Kabat / Chothias combination (SEQ ID NOs: 12, 13, and 14), with the proviso that position H28 is occupied by N or T, position H54 is occupied by N or D, position H56 is occupied by D or E, position H58 is occupied by V or I, position H60 is occupied by D or E, position L24 is occupied by K or R, position L50 is occupied by L, E, D, G, or V, position L52 is occupied by S or G, and position L54 is occupied by L, D, G, N, E, Q, K, R, T, V, or S. In some humanized 3D6 antibodies, the Kabat / Chothias combination CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86. In some humanized 3D6 antibodies, Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 92, or SEQ ID NO: 149. In some humanized 3D6 antibodies, Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89. In some humanized 3D6 antibodies, Kabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 150-175.

[0366] The CDR regions of such humanized antibodies may be identical or substantially identical to the CDR regions of 3D6. The CDR regions may be defined by any conventional definition (e.g., Chothia, or a combination of Chothia and Kabat), but are preferably defined by Kabat.

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

[0368] One possibility for further diversity in humanized 3D6 variants is additional backmutations in the variable region framework. Many of the framework residues in the humanized mAb that do not contact the CDRs are amenable to substitution with amino acids at corresponding positions in the donor mouse mAb or other mouse or human antibodies, and many potential CDR-contact residues are also susceptible to substitution. Even amino acids within the CDRs can be changed, for example, with residues found at corresponding positions in the human acceptor sequence used to provide the variable region framework. Furthermore, for example, different human acceptor sequences can be used for the heavy and / or light chains. If different acceptor sequences are used, one or more of the recommended backmutations above may not be performed, since the corresponding donor and acceptor residues already contain no backmutations.

[0369] Preferably, the substitutions or backmutations in the humanized 3D6 variants (whether conservative or not) do not substantially affect the binding affinity or potency of the humanized mAb, ie, its ability to bind to tau.

[0370] The humanized 3D6 antibody is further characterized for its ability to bind to both phosphorylated and non-phosphorylated tau, as well as misfolded / aggregated forms of tau.

[0371] D. Chimeric and Veneer Antibodies The present invention further provides chimeric and veneered versions of non-human antibodies, particularly the 3D6 antibody of the Examples.

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

[0373] A veneered antibody is a form of humanized antibody that retains some, and usually all, of the CDRs and some of the non-human variable region framework residues of the 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 in a 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 the substitutions. Veneered 3D6 antibodies are encompassed by the present invention.

[0374] E. Human antibodies Human antibodies against tau or fragments thereof (e.g., amino acid residues 199-213 and / or 262-276 of SEQ ID NO: 3, which correspond to amino acid residues 257-271 and / or 320-334 of SEQ ID NO: 1, respectively, or amino acid residues 259-268, 290-299, 321-330, or 353-362 of SEQ ID NO: 1, or any combination of two, three, or all four thereof) are provided by various techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, supra, 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 particular epitope specificity by using as the target antigen only a fragment of tau, such as a tau fragment comprising only amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334, 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 a collection of tau variants, such as tau variants comprising different 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.

[0375] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666; the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., al., WO 93 / 12227 (1993); U.S. 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, WO 91 / 10741 (1991)), phage display (see, e.g., Dower et al., WO 91 / 17271; McCafferty et al. al., WO 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 methods described in WO 2008 / 081008 (e.g., immortalizing memory B cells isolated from humans, e.g., with EBV, screening for desired properties, and cloning and expressing recombinant forms).

[0376] F. Constant Region Selection The heavy and light chain variable regions of chimeric, veneer, or humanized antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa. Numbering 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, supra).

[0377] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized to remove part or all of the molecule. Substitutions may be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, for example, U.S. Patent No. 5,624,821 to Winter et al., U.S. Patent No. 5,834,597 to Tso et al., and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions to increase antibody half-life include Gln at position 250 and / or Leu at position 428 (EU numbering of the constant region is used in this paragraph). Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., U.S. Patent No. 6,624,821). Alanine substitutions at positions 234, 235, and 237 of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. Optionally, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, e.g., U.S. Patent No. 5,624,821). Some antibodies use mutations at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 of human IgG1 (EU numbering). In some antibodies, mutations at one or more of positions 318, 320, and 322 of human IgG1, EU numbering, are used. In some antibodies, positions 234 and / or 235 are substituted with alanine and / or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4.

[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 mature variable domains of the heavy and light chains are linked via a spacer.

[0379] Human constant regions exhibit allotypic and isoallotypic variations between different individuals. That is, constant regions may differ between different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that serum that recognizes the isoallotype binds to the non-polymorphic regions of one or more other isotypes. Thus, for example, another heavy chain constant region is IgG1 G1m3 with or without a C-terminal lysine. Reference to a human constant region includes any natural allotype or constant region with any permutation of residues occupying positions in natural allotypes. A representative heavy chain constant region is SEQ ID NO: 176 with or without a C-terminal lysine, and a representative light chain constant region is SEQ ID NO: 177.

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

[0381] Light chain variable VL regions can be cloned in a similar manner. In one approach, a consensus primer set is designed to amplify the VL region, using a 5' primer designed to hybridize to a 3' primer specific for the VL region encompassing the translation initiation codon and the Ck region downstream of the VJ joining region. In a second approach, 5' RACE RT-PCR is used to clone the VL-encoding cDNA. Representative primers are described in Schenk (see above). The cloned sequence is then combined with sequences encoding human (or other non-human) constant regions.

[0382] In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of their respective VDJ or VJ junctions and inserted into mammalian expression vectors such as pCMV-hγ1 for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human γ1 and Ck constant regions as exon fragments downstream of the inserted variable region cassettes. After sequence verification, the heavy and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Culture supernatants are collected 48 hours after transfection and analyzed for antibody production by Western blot analysis or antigen binding by ELISA. The chimeric antibodies are then humanized as described above.

[0383] Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences operably linked to antibody chain coding sequences, including naturally associated or heterologous expression control sequences, e.g., promoters. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. After the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences and the collection and purification of cross-reacting antibodies.

[0384] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selectable markers, such as ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the DNA sequences of interest.

[0385] Escherichia coli is one type of prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is a yeast host with suitable vectors containing expression control sequences, an origin of replication, and termination sequences, as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization, among others.

[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 intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas, including SP2 / 0 and NS0. The cells can be non-human. Expression vectors for these cells can include expression control sequences such as replication origins, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary signal processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

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

[0388] Vectors containing the desired DNA segment can be introduced into host cells using methods appropriate for the host cell type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene guns, or viral transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. For the generation of transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs), the nuclei of which can be introduced into enucleated oocytes.

[0389] Vectors encoding antibody heavy and light chains are introduced into cell cultures, and cell pools can be screened for growth productivity and product quality in serum-free medium. The highest-producing cell pools can then be subjected to FACS-based single-cell cloning to generate monoclonal lines. Specific productivities of greater than 50 pg or 100 pg per cell per day, corresponding to product titers of greater than 7.5 g / L of culture, can be used. Antibodies produced by single-cell clones can also be tested for turbidity, filtration characteristics, PAGE, IEF, UV scanning, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. Selected clones can then be placed in multiple vials and cryopreserved for later use.

[0390] Once expressed, antibodies can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, etc. (For a review, see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0391] Methodologies for commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell preservation, use of selectable markers for copy number amplification, CHO terminators, or improved protein titer (see, e.g., U.S. Pat. Nos. 5,786,464, 6,114,148, 6,063,598, 7,569,339, WO 2004 / 050884, WO 2008 / 012142, WO 2008 / 012142, WO 2005 / 019442, WO 2008 / 107388, and WO 2009 / 027471, and U.S. Pat. No. 5,888,809).

[0392] IV. Active immunogens Agents used in active immunization serve to induce in the patient the same types of antibodies described in connection with passive immunization above. The agent used for active immunization can be the same type of immunogen used to generate monoclonal antibodies in laboratory animals, for example, a peptide of 3 to 15, or 3 to 12, or 5 to 12, or 5 to 8 contiguous amino acids from a region of tau corresponding to 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), such as a peptide 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 a region of tau corresponding to residues 259-268, or 290-299, or 321-330, or 353-362 of SEQ ID NO:1, such as a peptide including residues 259-268, or 290-299, or 321-330, or 353-362 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 testing binding to a series of overlapping peptides across tau). Fragments of tau that consist of, contain, or overlap the epitope can then be used as immunogens. Such fragments can typically be used in unphosphorylated form.

[0393] If used, heterologous carriers and adjuvants may be the same as those used to generate monoclonal antibodies, but may be selected for better pharmaceutical suitability for human use. Suitable carriers include serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin, or toxins or attenuated derivatives from other pathogenic bacteria, such as diphtheria (e.g., CRM197), E. coli, cholera, or H. pylori. T-cell epitopes are also suitable carrier molecules. Some conjugates can be formed by linking the agents of the invention to immunostimulatory polymer molecules (e.g., tripalmitoyl-S-glyceryl 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 the carrier with or without spacer amino acids (e.g., gly-gly). Additional carriers include virus-like particles. Virus-like particles (VLPs), also called pseudovirions or virus-derived particles, represent subunit structures composed of multiple copies of viral capsid and / or envelope proteins that can self-assemble into VLPs with defined spherical symmetry in vivo (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 majority of MHC class II molecules, such as the panDR epitope ("PADRE"). PADREs are described in U.S. Pat. No. 5,736,142, WO 95 / 07707, and Alexander J et al., Immunity, 1:751-761 (1994). Active immunogens can be provided in multimeric form, in which multiple copies of the immunogen and / or its carrier are provided as a single covalently linked molecule.

[0394] The fragments are often administered with a pharmaceutically acceptable adjuvant. The adjuvant increases the titer and / or binding affinity of the induced antibodies compared to the situation when the peptide is used alone. Various adjuvants can be used in combination with immunogenic fragments of tau to elicit an immune response. Preferred adjuvants enhance the intrinsic response to the immunogen without causing conformational changes in the immunogen that affect the nature and morphology of the response. Preferred adjuvants include aluminum salts such as aluminum hydroxide and aluminum phosphate, 3-O-deacylated monophosphoryl lipid A (MPL™) (UK Patent 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now 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, MA; now Antigenics, Inc., New York, NY). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally combined with immunostimulants such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), Pluronic polymers, and killed mycobacteria. Ribi adjuvant is an oil-in-water emulsion. Ribi contains a metabolizable oil (squalene) emulsified in saline containing Tween 80. Ribi also contains purified mycobacterial products that act as immunostimulants, and bacterial monophosphoryl lipid A. Another adjuvant is CpG (WO 98 / 40100).Adjuvants can be administered as a component of a therapeutic composition with the active agent, or can be administered separately, before, simultaneously with, or after administration of the therapeutic agent.

[0395] Analogs of natural fragments of tau that induce antibodies against tau can also be used. For example, one, more, or all of the L-amino acids can be replaced with D-amino acids in such peptides. Also, the order of amino acids can be reversed (retro peptides). Optionally, the peptide contains all of the D-amino acids in reverse order (retro-inverso peptides). Peptides and other compounds that do not necessarily have significant amino acid sequence similarity with tau peptides can still serve as mimics of tau peptides and induce similar immune responses. Anti-idiotypic antibodies against the above-mentioned monoclonal antibodies to tau can also be used. Such anti-Id antibodies mimic the antigen and generate 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 the peptides) can also be administered in the form of nucleic acids encoding the peptides and expressed in situ in patients. Nucleic acid segments encoding immunogens are typically linked to regulatory elements, such as promoters and enhancers, that allow expression of the DNA segment in the intended target cells of the patient. For expression in blood cells, which is desirable for inducing an immune response, promoter and enhancer elements from light and heavy chain immunoglobulin genes, or the CMV major-immediate-early promoter and enhancer, are suitable for directing expression. The linked regulatory elements and coding sequences are often cloned into vectors. Antibodies can also be administered in the form of nucleic acids encoding the heavy and / or light chains of the antibody. When both heavy and light chains are present, the chains are preferably linked as single-chain antibodies. Antibodies for passive administration can also be prepared, for example, by affinity chromatography from the serum of patients treated with peptide immunogens.

[0397] DNA can be delivered in naked form (i.e., without colloidal or encapsulating material) or by retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors, including vectors derived from retroviruses such as MMLV, HIV-1, and ALV (see, e.g., Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated viral vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)); lentiviral vectors, such as those based on the HIV or FIV gag sequence; poxvirus vectors, including vaccinia virus and avian poxvirus; and alphavirus vectors, such as those derived from Sindbis and Semliki Forest viruses (see, e.g., Dubensky et al., J. Exp. Med. 179, 1867 (1994)). Several viral vector systems can be used, including rhabdoviruses such as Venezuelan equine encephalitis virus (see U.S. Pat. No. 5,643,576) and vesicular stomatitis virus (see WO 96 / 34625), and papillomaviruses (see Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al., WO 94 / 12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).

[0398] The DNA encoding immunogen, or the DNA encoding antibody heavy and / or light chain, or the vector containing it can be packaged into liposome.Suitable lipids and related analogues are described in U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185.The vector and DNA encoding immunogen, or the antibody heavy and / or light chain, can also be adsorbed or bound to a microparticle carrier, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides) (see, for example, McGee et al., J. Micro Encaps. 1996).

[0399] Vectors or segments therefrom encoding antibody heavy and / or light chains can be incorporated into cells ex vivo, for example, into cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, which can then be reimplanted into the patient, typically after selection of cells that have incorporated the transgene (see, e.g., WO 2017 / 091512). Exemplary patient-derived cells include patient-derived pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal).

[0400] Vectors or segments thereof encoding antibody heavy and / or light chains can be introduced ex vivo into any region of interest in cells, such as the albumin gene or other safe harbor genes. Cells incorporating the vectors can be transplanted with or without prior differentiation. Cells can be transplanted into specific tissues, such as secretory tissues or locations of lesions, or systemically, such as by infusion into the blood. For example, cells can be transplanted into a patient's secretory tissue, such as the liver, optionally pre-differentiated into cells present in that tissue, such as hepatocytes in the case of the liver. Expression of the antibody in the liver results in the secretion of the antibody into the blood.

[0401] H. Antibody Screening Assays As noted above, antibodies can be initially screened for the desired binding specificity. Active immunogens can similarly be screened for the ability to induce antibodies with such binding specificity. In this case, the active immunogen is used to immunize a laboratory animal and the resulting sera are tested for the appropriate binding specificity.

[0402] Antibodies with the desired binding specificity can then be tested in cell and animal models. Neuronal cells should be the preferred cells for such screening. A cellular model of tau pathology has been reported in which neuroblastoma cells are genetically transfected with the 4-repeat domain of tau, optionally along with a mutation associated with tau pathology (e.g., Delta K280; see Khlistunova, Current Alzheimer Research 4, 544-546 (2007)). In another model, tau is induced in the neuroblastoma N2a cell line by adding doxycycline. This cellular model allows for the study of the toxicity of tau to cells in a soluble or aggregated state, the appearance of tau aggregates after switching on tau gene expression, the dissolution of tau aggregates after switching off gene expression, and the efficacy of antibodies in inhibiting the formation of tau aggregates or disaggregating them.

[0403] Antibodies or active immunogens can also be screened in transgenic animal models of tau-associated diseases. Such transgenic animals can contain a tau transgene (e.g., any of the human isoforms) and, optionally, a human APP transgene, such as a kinase that phosphorylates tau, ApoE, presenilin, or alpha-synuclein. Such transgenic animals are treated to develop at least one sign or symptom of a tau-associated disease.

[0404] A representative transgenic animal is the K3 strain of mice (Itner et al., Proc. Natl. Acad. Sci. USA 105(41):15997-6002(2008)). These mice carry a K369I mutation (associated with Pick's disease) and a human tau transgene with the Thy1.2 promoter. This model exhibits a rapid course of neurodegeneration, motor deficits, and degeneration of afferent fibers and cerebellar granule cells. Another representative animal is the JNPL3 strain of mice. These mice carry a P301L mutation (associated with frontotemporal dementia) and a human tau transgene with the Thy1.2 promoter (Taconic, Germantown, NY, Lewis, et al., Nat Genet. 25:402-405(2000)). These mice have a slower course of neurodegeneration. These mice develop neurofibrillary tangles in several brain regions and the spinal cord, which is incorporated herein by reference in its entirety. This makes them an excellent model for studying the consequences of tangle development and for screening therapeutics that can inhibit the formation of these aggregates. Another advantage of these animals is the relatively early onset of pathology. In homozygous strains, behavioral abnormalities associated with tau pathology can be observed as early as 3 months of age, but these animals remain relatively healthy until at least 8 months of age. That is, at 8 months of age, these animals are able to ambulate, feed themselves, and perform behavioral tasks sufficiently well to allow monitoring of treatment efficacy. Active immunization of these mice with AI wI KLH-PHF-1 for 6–13 months resulted in titers of approximately 1,000 and resulted in fewer neurofibrillary tangles, pSer422, and reduced weight loss compared to untreated control mice.

[0405] The activity of an antibody or active agent can be assessed by a variety of criteria, including reduction in the amount of total tau or phosphorylated tau, reduction in other pathological features such as Aβ amyloid deposits, and inhibition, delay, or behavioral deficits. Active immunogens can also be tested for the induction of antibodies in serum. Both passive and active immunogens can be tested for passage of antibodies across the blood-brain barrier into the brain of transgenic animals. Antibodies or antibody-inducing fragments can also be tested in non-human primates that naturally or inducedly develop symptoms of a disease characterized by tau. Testing of an antibody or active agent is usually performed simultaneously with a parallel control experiment in which the antibody or active agent is absent (e.g., replaced by vehicle). The reduction, delay, or suppression of signs or symptoms of the disease attributable to the antibody or active agent under test can then be assessed compared to the control.

[0406] V. Patients eligible for treatment The presence of neurofibrillary tangles has been observed in several diseases, including Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathies, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), and progressive supranuclear palsy (PSP). The present dosing regimen can also be used to treat or prevent any of these diseases. Due to the widespread relationship between neurological diseases and conditions and tau, this dosage regimen can be used to treat or prevent any subject who shows elevated levels of tau or phosphorylated tau (e.g., in CSF) compared to the average level of individuals without neurological disease. This dosage regimen can also be used to treat or prevent neurological disease in individuals with tau mutations associated with neurological disease. This method is particularly suitable for treating or preventing Alzheimer's disease, and in particular in patients.

[0407] Patients eligible for treatment include individuals at risk for the disease but who do not exhibit symptoms, as well as those currently exhibiting symptoms. Patients at risk include those with a known genetic risk for the disease. These individuals include those with relatives who have experienced 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 described above, as well as mutations in other genes associated with neurological disorders. For example, heterozygous and even homozygous ApoE4 alleles are associated with the risk of Alzheimer's disease. Other markers of risk for Alzheimer's disease include mutations in the APP gene, particularly mutations at position 717 and mutations at positions 670 and 671, known as the Hardy and Swedish mutations; mutations in PS1 and PS2 of the presenilin gene; and a family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by PET imaging due to the presence of characteristic dementia and the risk factors described above. Furthermore, several diagnostic tests are available to identify individuals with AD. These include measuring CSF tau or phosphorylated tau and Aβ42 levels. Elevated levels of tau or phosphorylated tau and decreased levels of Aβ42 indicate the presence of AD. Several mutations are associated with Parkinson's disease. Mutations in Ala30Pro or Ala53, or in other genes such as PARK8, a leucine-rich repeat kinase, are associated with Parkinson's disease. Individuals can also be diagnosed with any of the above-mentioned neurological disorders according to the DSM IV TR criteria.

[0408] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, 30). However, it is usually not necessary to begin treatment until the patient reaches 40, 50, 60, or 70 years of age. Treatment typically requires multiple doses over a period of time. Treatment can be monitored by assaying antibody levels over time. If the response decreases, booster doses are necessary. In patients with potential Down's syndrome, treatment can begin prenatally by administering a therapeutic agent to the mother or shortly after birth.

[0409] I. Nucleic acids The present invention further provides nucleic acids encoding any of the above heavy and light chains (e.g., SEQ ID NO:7, SEQ ID NO:11, SEQ ID 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 a heavy chain of the present invention is SEQ ID NO:182, and a representative nucleic acid encoding a light chain of the present invention is SEQ ID NO:183. Optionally, such nucleic acids may further encode a signal peptide and express the signal peptide linked to the variable region. The coding sequence of the nucleic acid may be operably linked to regulatory sequences that ensure expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, transcription termination signal, etc. Regulatory sequences may include a promoter, e.g., a prokaryotic or eukaryotic promoter. The nucleic acids encoding the heavy or light chains may be codon-optimized for expression in a host cell. The nucleic acids encoding the heavy and light chains may encode selectable genes. The nucleic acids encoding the heavy and light chains may occur in isolated form or may be inserted into one or more vectors. The nucleic acids can be synthesized, for example, by solid phase synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains can be linked, for example, as one contiguous nucleic acid in an expression vector, or can be inserted into separate, for example, their own, expression vectors.

[0410] J. Conjugated Antibodies Conjugated antibodies that specifically bind to antigens such as tau are useful for detecting the presence of tau; Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; monitoring and evaluating the efficacy of therapeutic agents used to treat patients diagnosed with corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP); and detecting tau aggregation. inhibiting or reducing tau fibril formation; reducing or clearing tau deposits; stabilizing non-toxic conformations of tau; or treating or preventing Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) in a patient. For example, such antibodies can be conjugated to other therapeutic moieties, other proteins, other antibodies, and / or detectable labels. See WO 03 / 057838; U.S. Patent No. 8,455,622.Such therapeutic moieties may be any agent that can be used to treat, combat, alleviate, prevent, or ameliorate an undesirable condition or disease in a patient, such as Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0411] The conjugated therapeutic moiety may comprise a cytotoxic drug, a cytostatic drug, a neurodifferentiator, a neuroprotective drug, a radiotherapeutic drug, an immunomodulator, or any bioactive agent that promotes or enhances the activity of the antibody. A cytotoxic drug may be any agent that is toxic to cells. A cytotoxic drug may be any agent that inhibits cell proliferation. A neurodifferentiator may be any agent, including a chemical or protein substance, that promotes neuronal maintenance, proliferation, or differentiation. A neuroprotective drug may be an agent, including a chemical or protein substance, that protects neurons from acute insults or degenerative processes. An immunomodulator may be any agent that stimulates or suppresses the development or maintenance of an immune response. A radiotherapeutic drug may be any molecule or compound that emits radiation. When such a therapeutic moiety is conjugated to a tau-specific antibody, such as the antibodies described herein, the conjugated therapeutic moiety will have specific affinity for cells affected by a tau-related disease over normal cells. Thus, administration of the conjugated antibody directly targets cancer cells with minimal damage to surrounding healthy tissue. This may be particularly useful when the therapeutic moieties are too toxic to be administered alone. Additionally, lower amounts of the therapeutic agent moiety can be used.

[0412] Some such antibodies can be modified to act as immunotoxins. See, e.g., U.S. Patent No. 5,194,594. For example, ricin, a plant-derived cytotoxin, can be conjugated to an antibody using the bifunctional reagents S-acetylmercaptosuccinic anhydride for the antibody and succinimidyl 3-(2-pyridyldithio)propionate for the ricin. See Pietersz et al., Cancer Res. 48(16):4469-4476 (1998). Conjugation reduces the binding activity of the ricin B chain, but does not impair either the toxic potential of the ricin A chain or the antibody activity. Similarly, saporin, an inhibitor of ribosome assembly, can be conjugated to an antibody via a disulfide bond between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

[0413] Some such antibodies can be linked to radioisotopes, such as yttrium. 90 (90Y) Indium 111 (111In), 131 I, 99 mTc, radioactive silver-111, radioactive silver-199, and bismuth 213 Radioisotopes can be linked to antibodies using conventional bifunctional chelates. For linking radioactive silver-111 and radioactive silver-199, sulfur-based linkers can be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linking silver radioisotopes may require reduction of immunoglobulin with ascorbic acid. For radioisotopes such as In and Y, ibritumomab tiuxetan can be used, reacting with such isotopes to form In-ibritumomab tiuxetan and Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0414] Some such antibodies can be linked to other therapeutic moieties. Such therapeutic moieties can be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective. For example, the antibody can be linked to a toxic chemotherapeutic agent such as a tubulin inhibitor, such as maytansine, geldanamycin, a tubulin binding agent (e.g., auristatin), or a minor groove binding agent, such as calicheamicin. Other representative therapeutic moieties include agents known to be useful in treating, managing, or ameliorating Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) in a patient.

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

[0416] The antibodies disclosed herein can also be bound or conjugated to one or more other antibodies (e.g., to form antibody heteroconjugates), which can bind to a different epitope within tau or can bind to a different target antigen.

[0417] The antibody may also be conjugated to a detectable label, and such an antibody can be used to diagnose and / or assess the efficacy of treatment for, for example, Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Such antibodies are particularly useful for making such determinations in subjects with or susceptible to Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), or in suitable biological samples obtained from such subjects. Representative detectable labels that can be bound or linked to an antibody include various enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as luminol; bioluminescent materials such as luciferase, luciferin, and aequorin; radioactive silver-111, radioactive silver-199, bismuth, etc. 213 , iodine ( 131 I, 125 I, 123 I, 121I), carbon ( 14 C), sulfur ( 5 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, 111 In), technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117 These include radioactive materials such as tin; positron-emitting metals using various positron emission tomography techniques; non-radioactive paramagnetic metal ions; and molecules that are radiolabeled or conjugated to specific radioisotopes.

[0418] Linking of radioisotopes to antibodies can be carried out using conventional bifunctional chelates. For linking radioactive silver-111 and radioactive silver-199, sulfur-based linkers can be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linking of silver radioisotopes may require reduction of immunoglobulin with ascorbic acid. For radioisotopes such as In and Y, ibritumomab tiuxetan can be used, reacting with such isotopes to form In-ibritumomab tiuxetan and Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0419] Therapeutic drug moieties, other proteins, other antibodies, and / or detectable labels can be attached or conjugated directly or indirectly through an intermediary (e.g., a linker) to an antibody of the invention. For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Hellstrom et al., “Antibodies For Drug Delivery,”; Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985), Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,”; Therapy,Baldwin et See, for example, "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in J. Chem. Soc., 1985, pp. 303-16 (Academic Press), and Thorpe et al., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers can be used that release the attached therapeutic agent moiety, protein, antibody, and / or detectable label upon exposure to specific proteases under acidic or reducing conditions, or under other defined conditions.

[0420] VI. Pharmaceutical Compositions and Methods of Use In prophylactic applications, the antibody, or an agent for inducing the antibody, or a pharmaceutical composition thereof, is administered to a patient susceptible to or at risk of a disease (e.g., Alzheimer's disease) in a dosage regimen (dosage, frequency, and route of administration) effective to reduce the risk of the disease, reduce the severity of the disease, or delay the onset of at least one sign or symptom of the disease. In particular, the dosage regimen is preferably effective to inhibit or delay the formation of tau or phosphorylated tau and filament pairs formed therefrom in the brain, and / or to inhibit or delay their toxic effects, and / or to inhibit or delay the onset of behavioral deficits. In therapeutic applications, the antibody, or an agent for inducing the antibody, is administered to a patient suspected of having or already suffering from a disease (e.g., Alzheimer's disease) in a dosage regimen (dosage, frequency, and route of administration) effective to improve at least one sign or symptom of the disease or at least prevent further deterioration. In particular, the dosing regimen is preferably effective to reduce or at least inhibit further increases in the levels of tau, phosphorylated tau, or filament pairs formed therefrom, and associated toxicity and / or behavioral impairments.

[0421] A dosing regimen is considered to be therapeutically or prophylactically effective if each treated patient achieves a better outcome than the average outcome in a control population of comparable patients not treated by the methods of the invention, or if a controlled clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial) demonstrates a better outcome for treated patients versus control patients at a p<0.05 or 0.01, or even 0.001 level.

[0422] The effective dose will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is an ApoE carrier, whether the patient is human or animal, whether other agents are administered, and whether the treatment is prophylactic or therapeutic.

[0423] Typical dosage ranges for antibodies are about 0.01-60 mg / kg, or about 0.1-3 mg / kg, or 0.15-2 mg / kg, or 0.15-1.5 mg / kg of patient body weight. Antibodies can be administered at such doses daily, every other day, weekly, biweekly, monthly, quarterly, or according to any other schedule determined by empirical analysis. Typical treatments involve administration in multiple doses over an extended period of time, e.g., for at least six months. Further typical dosing regimens involve administration every two weeks, monthly, or once every three to six months.

[0424] The amount of drug for active administration varies from 0.1 to 500 μg per patient, more usually 1 to 100 or 1 to 10 μg per injection in humans. The timing of injections can vary widely, from once daily to once a year to once every 10 years. A typical regimen consists of an immunization followed by booster injections at time intervals such as 6-week or 2-month intervals. Another regimen consists of an immunization followed by booster injections 1, 2, and 12 months later. Another regimen requires lifelong injections every 2 months. Alternatively, booster injections can be administered on an irregular basis, as indicated by monitoring the immune response.

[0425] Antibodies, or agents for inducing antibodies, are preferably administered via a peripheral route (i.e., a route by which the administered or induced antibody crosses the blood-brain barrier and reaches the intended site in the brain). Administration routes include topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, or intramuscular administration. Preferred routes of antibody administration are intravenous and subcutaneous. Preferred routes for active immunization are subcutaneous and intramuscular. This type of injection is most typically administered into the arm or leg muscles. In some methods, the agent is injected directly into a specific tissue where deposits have accumulated, e.g., intracranially.

[0426] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation will depend on the selected route of administration. For injection, the antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0427] The dosage regimen can be administered in combination with another agent effective in treating or preventing the disease being treated. For example, in the case of Alzheimer's disease, the dosage regimen can be combined with immunotherapy against Aβ (WO 2000 / 072880), cholinesterase inhibitors, or memantine, or in the case of immunotherapy for Parkinson's disease, with immunotherapy against α-synuclein (WO 2008 / 103472), levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, amantadine, or anticholinergics.

[0428] The antibody is administered in an effective dosing regimen, which refers to a dosing regimen including a dosage, route of administration, and frequency of administration that delays onset, reduces severity, prevents further deterioration, and / or ameliorates at least one sign or symptom of the disorder being treated. If the patient already suffers from the disorder, the dosing regimen may also be referred to as a therapeutically effective regimen. If the patient is at high risk for the disorder compared to the general population but is not yet symptomatic, the dosing regimen may also be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in an individual patient relative to historical controls or past experience of the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated during preclinical or clinical trials of a treated patient population compared to a control population of untreated patients.

[0429] Typical dosages of antibodies are 0.1-60 mg / kg (e.g., 0.5, 3, 10, 30, or 60 mg / kg), or 0.5-5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4, or 5 mg / kg), or as a fixed dose, 10-4000 mg or 10-1500 mg. Dosage will depend, among other things, on the patient's condition and response to previous treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

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

[0431] The frequency of administration depends, inter alia, on the half-life of the antibody in circulation, the patient's condition, and the route of administration. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals, depending on changes in the patient's condition or the progression of the disorder being treated. Typical frequencies for intravenous administration range from weekly to quarterly, depending on the cause of treatment, although more or less frequent administration is possible. For subcutaneous administration, typical frequencies range from daily to monthly, although more or less frequent administration is also possible.

[0432] The number of doses depends on whether the disorder is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often administered. Sometimes, for acute disorders or acute exacerbations of chronic disorders, a single rapid dose, in divided doses as needed, is sufficient. For recurrence of acute disorders or acute exacerbations, treatment can be repeated. For chronic disorders, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, every three months, or every six months, for at least 1, 5, or 10 years, or for the lifetime of the patient.

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

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

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

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

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

[0438] The signals detected in a PET scan can be represented as a multidimensional image. A multidimensional image can represent a cross-section of the brain in two dimensions, a three-dimensional image of the three-dimensional brain in three dimensions, or a four-dimensional image of the three-dimensional brain over time. A color scale can be created using different colors to indicate different amounts of label and the estimated amount of tau protein deposits detected. The results of the scan can also be presented numerically as a number related to the amount of label detected and the resulting amount of tau protein deposits. The label present in brain regions known to be associated with deposits in a particular tauopathy (e.g., Alzheimer's disease) can be compared with the label present in regions not known to be associated with deposits to provide a ratio representing the extent of deposits in the former region. This ratio for the same radiolabeled ligand provides a comparable measure of tau protein deposits and their variation among different patients.

[0439] In some methods, a PET scan is performed simultaneously with an MRI or CAT scan or at the same patient visit. An MRI or CAT scan provides more anatomical detail of the brain than a PET scan. However, images from a PET scan can be overlaid onto the MRI or CAT scan images, which more accurately shows the location of the PET ligand and putative tau deposits relative to anatomical structures in the brain. Some devices can perform both a PET scan and an MRI or CAT scan on a patient without the patient changing position between scans, facilitating image overlay.

[0440] Suitable PET ligands include radiolabeled antibodies of the invention (e.g., murine, humanized, chimeric, or veneered 3D6 antibodies). The radioisotopes used can be, for example, C 11 , N 13 , O 15 , F 18 , or I 123 The interval between administering the PET ligand and performing the scan may depend on the uptake and clearance rate of the PET ligand, particularly from the brain, and the half-life of the radiolabel.

[0441] PET scans can also be performed as a preventative measure in asymptomatic patients or those with symptoms of mild cognitive impairment but who have not been diagnosed with a tauopathy but are still at high risk for developing a tauopathy. In asymptomatic patients, scans are particularly useful for individuals considered at high risk for tauopathy due to family history, genetic or biochemical risk factors, or advanced age. Prophylactic scans can begin, for example, in patients between the ages of 45 and 75. In some patients, the first scan is performed at age 50.

[0442] Preventive scans can be performed, for example, at intervals of 6 months to 10 years, preferably 1 to 5 years. In some patients, preventive scans are performed annually. If a preventive PET scan shows abnormally high levels of tau protein deposits, immunotherapy can be initiated, and then PET scans can be initiated, as in patients diagnosed with tauopathy. If a preventive PET scan shows levels of tau protein deposits within normal levels, further PET scans can be performed, as described above, at intervals of 6 months to 10 years, preferably 1 to 5 years, or in response to the appearance of signs and symptoms of tauopathy or mild cognitive impairment. If above-normal levels of tau protein deposits are detected, by combining prophylactic scanning with administration of tau-targeted immunotherapy, the level of tau protein deposits can be reduced to or closer to normal levels, or at least prevented from increasing further, and the patient can remain tauopathy-free for a longer period of time (e.g., at least 5, 10, 15, or 20 years, or for the rest of the patient's life) than if the patient had not received prophylactic scanning and tau-targeted immunotherapy.

[0443] Normal levels of tau protein deposits can be determined by the amount of neurofibrillary tangles or tau inclusions in the brains of a representative sample of individuals from a population (e.g., a representative sample of disease-free individuals under the age of 50) who have not been diagnosed with a particular tauopathy (e.g., Alzheimer's disease) and are not considered at high risk for developing such a disease. Alternatively, normal levels can be recognized in an individual patient if the PET signal from the methods of the present invention in brain regions known to develop tau protein deposits does not differ (within the precision of measurement) from signals from brain regions known to typically develop such deposits. Elevated levels in an individual can be recognized by comparison with normal levels (e.g., levels outside the mean + standard deviation variance) or simply from elevated signals beyond experimental error in brain regions associated with tau protein deposits compared to regions not known to be associated with deposits. For the purpose of comparing the levels of tau protein deposits in individuals and populations, tau protein deposits should preferably be determined in the same brain region(s), including at least one region known to form tau protein deposits associated with a particular tauopathy (e.g., Alzheimer's disease). Patients with elevated levels of tau protein deposits are candidates for initiating immunotherapy.

[0444] A decrease in the level of tau protein deposits after initiating immunotherapy can be seen as an initial indication that the treatment is having the desired effect. The observed decrease can be, for example, within a range of 1-100%, 1-50%, or 1-25% of baseline values. Such an effect can be measured in one or more brain regions known to form deposits, or can be measured from the average values ​​of such regions. The overall effect of the treatment can be estimated by adding the percent decrease relative to baseline to the increase in tau protein deposits that would occur in the average untreated patient in the absence of treatment.

[0445] Maintenance of a near-constant level of tau protein deposits or even a small increase in tau protein deposits may be indicative of a response to treatment, albeit a suboptimal response. Such a response can be compared to the time course of tau protein deposit levels in untreated patients with a particular tauopathy (e.g., Alzheimer's disease) to determine whether immunotherapy is effective in inhibiting further increases in tau protein deposits.

[0446] Monitoring changes in tau protein deposits allows for adjustments to immunotherapy or other treatment regimens in response to the treatment. PET monitoring provides an indication of the nature and extent of the response to treatment. It can then be determined whether treatment adjustments should be made, and if necessary, the treatment can be adjusted in response to the PET monitoring. Thus, PET monitoring allows for adjustments to the administration regimen of tau-targeted immunotherapy or other treatments before other biomarkers, MRI, or cognitive measures show a detectable response. A significant change means that comparing the post-treatment parameter value to the baseline provides evidence that the treatment has or has not had a beneficial effect. In some cases, a change in the parameter value in the patient provides evidence that the treatment has or has not had a beneficial effect. In other examples, a change in the patient's value is compared with a change in the value, if any, in a representative control population of patients not receiving immunotherapy. The difference between a particular patient's response and the normal response of control patients (e.g., the mean plus the variance of the standard deviation) can also provide evidence that the immunotherapy regimen is or is not achieving a beneficial effect in the patient.

[0447] In some patients, monitoring shows a detectable reduction in tau protein deposits, but tau protein deposit levels remain above normal. In such patients, the treatment regimen can be continued at the same dosing frequency and / or dose, or, if not at the maximum recommended dose, at an increased dosing frequency and / or dose, in the absence of unacceptable side effects.

[0448] If monitoring indicates that the level of tau protein deposits in the patient has decreased to normal or near-normal, the immunotherapy regimen may be adjusted from an induction regimen (i.e., a regimen that reduces the level of tau protein deposits) to a maintenance regimen (i.e., a regimen that maintains tau protein deposits at a near-constant level). Such a regimen may be achieved by reducing the dose and / or frequency of administration of the immunotherapy.

[0449] In other patients, monitoring may indicate that immunotherapy has some beneficial effect but with suboptimal efficacy. An optimal effect may be defined as a percentage reduction in tau protein deposit levels within the upper half or quartile of change in tau protein deposits (measured or calculated across the whole brain or representative region(s) where tau protein deposits are known to form) in a representative sample of tauopathy patients receiving immunotherapy at a given time point after initiation of treatment. Patients whose tau protein deposits experience a small decrease, or whose tau protein deposits remain stable, or whose tau protein deposits increase but to a lesser extent than would be expected in the absence of immunotherapy (e.g., when extrapolated from a control group of patients not receiving immunotherapy) may be classified as experiencing a positive but suboptimal response. Such patients may be given an adjustment in their dosing regimen, increasing the drug dose and / or frequency of administration, as needed.

[0450] In some patients, tau protein deposits may increase to levels similar to or greater than those in patients not receiving immunotherapy. If such an increase continues over a period of time, such as 18 months or 2 years, even after the frequency or dose of the drug has been increased, immunotherapy may be discontinued in favor of other treatments, if necessary.

[0451] The foregoing discussion of diagnosing, monitoring, and adjusting treatment for tauopathies has focused primarily on the use of PET scans, however, any other technique for visualizing and / or measuring tau protein deposits that is amenable to the use of a tau antibody of the invention (e.g., a murine, humanized, chimeric, or veneered 3D6 antibody) can be used in place of PET scans to carry out such methods.

[0452] Also provided is a method for detecting an immune response to tau in a patient suffering from or susceptible to a tau-related disease. This method can be used to monitor the course of therapeutic and prophylactic treatment with the agents provided herein. The antibody profile after passive immunization typically shows an immediate peak in antibody concentration followed by an exponential decay. Without further administration, the decay approaches pre-treatment levels within a period of several days to several months, depending on the half-life of the administered antibody. For example, the half-life of some human antibodies is on the order of 20 days.

[0453] In some methods, a baseline measurement of the subject's tau antibody is taken before administration, a second measurement is taken shortly thereafter to measure the peak level of the antibody, and one or more additional measurements are taken at intervals to monitor the decay of the antibody level. When the antibody level falls to a predetermined percentage (50%, 25%, or 10%) of the baseline or baseline-subtracted peak, an additional dose of antibody is administered. In some methods, the peak or background-subtracted subsequent measured levels are compared to a predetermined reference level to generate a beneficial prophylactic or therapeutic treatment regimen for other subjects. If the measured antibody level is significantly lower than the reference level (e.g., less than the mean minus one standard deviation, preferably two standard deviations, of the reference value of the subject population that would benefit from treatment), an additional dose of antibody is required.

[0454] Also provided is a method for detecting tau in a subject, for example, by measuring tau in a sample from the subject or by in vivo imaging of tau in the subject.Such a method is useful for diagnosing or confirming the diagnosis of tau-related diseases or susceptibility to them.This method can also be used for asymptomatic subjects.The presence of tau indicates susceptibility to symptomatic disease in the future. 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-encephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0455] Biological samples from subjects with, suspected of, or at risk of having Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) can be contacted with an antibody disclosed herein to assess the presence of tau. For example, the tau levels in such subjects can be compared to the levels of tau present in healthy subjects. Alternatively, tau levels in such subjects undergoing treatment for a disease can be compared with tau levels in subjects who have not received treatment for Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Some such tests require tissue biopsies from such subjects. ELISA assays can also be useful methods, for example, to assess tau in fluid samples.

[0456] VII. Kit The present invention further provides kits (e.g., containers) that contain the antibodies and related materials disclosed herein, such as instructions for use (e.g., package inserts). The instructions may include, for example, instructions for administration of the antibody and, optionally, one or more additional agents. The antibody containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses.

[0457] Package insert means instructions customarily included in commercial packaging of therapeutic products containing information about the indications, uses, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0458] The kit may also include a second container containing a pharmaceutically acceptable buffer, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

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

[0460] Additionally, antibodies can be used to detect the presence of tau in biological samples to monitor and evaluate the efficacy of therapeutic agents being used to treat patients diagnosed with Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). A biological sample from a patient diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP) is evaluated to establish baseline antibody binding to the sample (i.e., baseline for the presence of tau in the sample) prior to initiating treatment with the therapeutic agent. In some cases, multiple biological samples from a patient are evaluated at multiple time points to establish both a baseline and a criterion for random fluctuations unrelated to treatment. A therapeutic agent is then administered according to a dosing regimen. The dosing regimen may include multiple administrations of the agent over a period of time. Optionally, antibody binding (i.e., the presence of tau) is assessed in multiple biological samples at multiple time points to establish a criterion for random fluctuations and indicate a trend toward response to immunotherapy. Various comparative assessments of antibody binding to the biological samples are then performed. If only two assessments are performed, a direct assessment is made between the two assessments to determine whether antibody binding (i.e., the presence of tau) increases, decreases, or remains the same between the two assessments.If three or more measurements are taken, the measurements can be analyzed as a time course beginning before treatment with the therapeutic agent and as a time course progressing through the course of treatment. If antibody binding (i.e., the presence of tau) to the biological sample is reduced in the patient, it can be concluded that the therapeutic agent is effective in treating Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). The reduction in antibody binding is statistically significant. In some cases, binding is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Assessment of opponent coupling can be performed in conjunction with the evaluation of signs and symptoms of Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis-Parkinson's disease / dementia complex; corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

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

[0462] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in their entirety into this application to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with accession numbers at different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date means a date earlier than the actual filing date or, if applicable, the filing date of the priority application for the accession number. Similarly, where different versions of a publication, website, etc. were published at different times, the most recently published version as of the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other unless otherwise indicated. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

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

[0464] Five-week-old female Balb / c mice were immunized intraperitoneally with 25 μg of immunogen A on day 0 and 10 μg of immunogen A on days 7, 14, 21, 27, 34, 48, 55, and 62. Mice were immunized with 10 μg of immunogen B on days 76 and 90. Mice were bled on days 43 and 98, and titers against immunogen A were measured; on day 101, animals with the highest titers were boosted with a terminal immunization of 50 μg of immunogen B. One-half of these boosts were delivered intraperitoneally and one-half intravenously. Fusion hybridomas were screened against both immunogens by ELISA, and those with the highest signals were epitope mapped (see Example 2). 3D6 reacted with peptides corresponding to amino acid residues 199-213 of SEQ ID NO:3 and amino acid residues 262-276 of SEQ ID NO:3 (according to the numbering of the longest CNS isoform of tau, which corresponds to amino acid residues 257-271 of SEQ ID NO:1 and amino acid residues 320-334 of SEQ ID NO:1).

[0465] Example 2. Epitope mapping of antibody 3D6 A series of overlapping biotinylated peptides spanning the 383aa 4R0N human tau protein were used to map the mouse 3D6 antibody, and additional peptides were used to model potential post-translational modifications at the C- and N-termini of the protein.

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

[0467] Example 3. Design of humanized 3D6 The starting or donor antibody for humanization was murine antibody 3D6. The heavy chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:7. The light chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:11. The heavy chain Kabat / Chothia combination CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs:8-10, respectively. The light chain Kabat CDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOs:12-14, respectively. Kabat numbering is used throughout.

[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]. The 16-residue ChotiCDR-L1 belongs to canonical class 4 of Vκ, the 7-residue ChotiCDR-L2 belongs to class 1, and the 9-residue ChotiCDR-L3 belongs to class 1 [Martin AC, and Thornton JM (1996) J. Mol. Biol. 263:800-15. [Martin & Thornton, 1996]. The 10-residue Chothia CDR-H1 belongs to class 1, and the 17-residue Chothia CDR-H2 belongs to class 2 [Martin & Thornton, 1996]. CDR-H3 does not have a canonical class. To find a structure that could provide a rough structural model of the 3D6 antibody, we searched against protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33:D233-7]. To build the Fv model of 3D6, we used the structure of mouse anti-pyroglutamate Abeta antibody Fab c number 24 (PDB code 5MYX) with a resolution of 1.4 Å [Piechotta, A. et al., 2017, J Biol Chem 292: 12713-12724], which retained the same canonical structure as the 3D6 loops.

[0469] The 3D6 VH framework 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 the same lengths for the CDR-H1 and H2 loops. Similarly, the 3D6 VL framework 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 the 3D6 and ARX71335 antibodies also share the same lengths for the CDR-L1, L2, and L3 loops. Therefore, the framework regions of 48G7 VH (2RCS-VH) and ARX71335 VL were selected as acceptor sequences for the CDRs of 3D6. A model of the 3D6 CDRs grafted onto each human framework of VH and VL was constructed and used as guidance for further backmutation.

[0470] The heavy and light chain variant sequences resulting from the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains as outlined in the WHO INN Committee guidelines. (WHO-INN: International Nonproprietary Nomenclature for Biological and Biotechnological Substances (INN) (Overview) (Internet) 2014, available at http: / / www.who.int / medicines / services / inn / BioRev2014.pdf) Where possible, residues were changed to correspond to the corresponding human germline sequences to enhance the degree of humanization and reduce potential immunogenicity. For the humanized VLvb2 and VLvb3 variants, the sequences were aligned to the human germline gene IGKV2-30. * Mutations were introduced to make VHvb2, VHvb3, VHvb4, VHvb5, VHvb6, and VHvb6 variants more similar to the human germline gene IGHV1-69-2 (SEQ ID NO: 27).* Mutations were introduced to make it more similar to 01 (SEQ ID NO: 25).

[0471] The hu3D6-VH and hu3D6-VL versions were designed to allow evaluation of various framework residues for their contribution to antigen binding, thermal stability, and immunogenicity, as well as for optimization of glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface exposed charged patches, deamination, and proteinase susceptibility. Positions considered for mutation included: - the positions that define the canonical CDR conformation (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.), - Position within the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99.) - localization at the VH / VL domain interface (summarized in Leger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. in Shepherd P and Dean C (eds). Monoclonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.), - positions susceptible to post-translational modifications, such as glycosylation or pyroglutamation, - positions occupied by residues predicted to clash with the CDRs according to a model of the 3D6 CDRs grafted onto the VH and VL frameworks, or - positions occupied by rare residues in sequenced human antibodies, where either the parental murine 3D6 residue or some other residue is much more prevalent in the human antibody repertoire; Examples include:

[0472] Sequence comparisons of murine 3D6 and various humanized antibodies are shown for the light chain variable region (Table 4 and Figure 3) and 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 containing different substitution permutations: hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, or hu3D6VHvb7 (SEQ ID NOS: 76-80 and 90-91, respectively), and hu3D6VLvb1, hu3D6VLvb2, or hu3D6VLvb3 (SEQ ID NOS: 83-85, respectively) (Tables 3 and 4). Representative humanized Vk and Vh designs with backmutations and other mutations based on selected human frameworks are shown in Tables 3 and 4, respectively. Bolded regions in Tables 3 and 4 indicate CDRs defined by the Kabat / Chothia combination. SEQ ID NOS: 76-80 and 90-91 contain backmutations and other mutations as shown in Table 5. The amino acids at the positions in hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 are shown in Table 6. The amino acids at the positions in hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3 are shown in Table 7. [Table 3] TIFF2025069265000005.tif239161TIFF2025069265000006.tif239161TIFF20250692650 00007.tif238160TIFF2025069265000008.tif239159TIFF2025069265000009.tif130161 [Table 4] TIFF2025069265000011.tif239162TIFF2025069265000012.tif238160TIFF2025069265000013.tif238162TIFF2025069265000014.tif209162 [Table 5] [Table 6] TIFF2025069265000017.tif128162 [Table 7]

[0474] Positions where canonical, Vernier, or interface residues differ between the mouse and human receptor sequences are candidates for substitution. Examples of canonical / CDR-interacting residues include Kabat residues H54 and H94 in Table 3. Examples of Vernier residues include Kabat residues H28, H67, H93, and H94 in Table 3. Examples of interface / packing (VH+VL) residues include Kabat residues H91 and H93 in Table 3.

[0475] The principles for selecting the positions shown in Table 3 in the heavy chain variable region as substitution candidates are as follows.

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

[0477] hu3D6VHvb2 - Restores all framework substitutions at positions that are important for defining the Chothia canonical class, that are part of the Vernier zone, that are located at the VH / VL domain interface, or that contribute to structural stability. 3D6-VH_vb2 incorporates backmutations or substitutions Q1E, Q5V, L11V, L20I, T23K, K38R, E42G, Q43K, K66R, S75T, N76D, Q81E, Y91F, A93S, S94T, T108L, and L109V, allowing for evaluation of the contribution of these positions to antigen-binding affinity and immunogenicity.

[0478] hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 consists of additional substitutions and increases antibody stability and / or optimizes glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface exposed charge patch, surface exposed charge patch, deamination and proteinase susceptibility.

[0479] Q1E is a stability-enhancing mutation that reduces the ability to form pyroglutamate (Liu, supra).

[0480] Q5V is a frequency-based mutation and a germline-aligning mutation. Val is the most frequently occurring mutation in human sequences at this position. Val is the most frequently occurring mutation in human germline genes at this position. IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25).

[0481] L11V is a germline mutation. Val is a mutation in the human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO: 25).

[0482] S17T is a germline mutation. Thr is located at this position in the human germline gene IMGT number IGHV1-69-2. * 01 (SEQ ID NO: 25).

[0483] L20I is a germline alignment mutation. Ile is a mutation in the human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO: 25).

[0484] T23K is a frequency-based and germline aligned mutation. Lys is the most commonly occurring at this position. Lys is present at this position in the human germline gene IMGT number IGHV1-69-2 * 01 (SEQ ID NO: 25).

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

[0486] K38R ​​is a frequency-based backmutation. Arg is the most commonly occurring at this position. Arg at this position is predicted to form one H-bond plus two H-bonds with Glu46, Asp86, and Tyr90, respectively, in the heavy chain; therefore, Arg may enhance stability over Lys at this position.

[0487] E42G is a frequency-based and germline-aligned mutation. Gly is present at this position in the human germline gene IMGT number IGHV1-69-2. * 01 (SEQ ID NO: 25). Gly is the most abundant at this position. Gly substitution is not predicted to affect stability.

[0488] Q43K: The Lys side chain at this position is predicted to form H-bonds with the main chain as well as the G42 side chain and with Gln39 and Arg40, which may make the Lys substitution more stable than Q at this position.

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

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

[0491] K66R: Arg at this position is predicted to form H-bonds with Ser82a and Thr83 in addition to forming H-bonds and salt bridges with Asp86.

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

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

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

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

[0497] T83R, which enhances thermostability and humanization. Arg is a germline alignment mutation. Arg is a human germline gene IMGT number IGHV1-69-2 at this position. * 01 (SEQ ID NO: 25). Arg is the most common at this position.

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

[0499] A93S is a back mutation of a Vernier zone and interface zone residue.

[0500] S94T is a backmutation of the canonical structural residue defined by Chothia and the Vernier residue.

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

[0502] L109V is a frequency-based mutation. Val is the most common at this position.

[0503] The principles for selecting the positions shown in Table 4 in the light chain variable region as substitution candidates are as follows.

[0504] Kappa light chain variable region

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

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

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

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

[0509] I15L is a germline alignment mutation. Leu is located 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 light chain residues, and is predicted to enhance antibody stability.

[0511] K24R is a mutation in a CDR residue. Both Lys and Arg are predicted to form H-bonds and salt bridges with Asp70 in the light chain. Arg is predicted to fit better in the conformation. Arg is also a germline alignment mutation. Arg is present in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO: 27).

[0512] L37Q: This is predicted to be a deeply buried residue, and Leu is not predicted to interact with surrounding residues, whereas Gln is predicted to form H-bonds with Q38 and Asp82 in the light chain. Gln is also a germline aligned mutation. Gln is present in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO: 27).

[0513] K45R:Lys is predicted to form H-bonds with S56 and Gly57; the predicted interactions of Arg with neighboring residues are much more extensive, as it is predicted to form a salt bridge with D55, an H-bond with Arg46, and a double H-bond with S56. Arg is also a germline aligned mutation. Arg is present in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO: 27).

[0514] L83V: This is a mutation based on the frequency of a residue predicted to be surface exposed. Val is also a germline aligned mutation. Val is a mutation found in the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO: 27).

[0515] H86Y: Mouse 3D6 VL has a Tyr at this position. Tyr is also the most common 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 mutation at this position and is also a germline alignment mutation. Gln is rare at this position in the human germline gene IGKV2-30. * 02 (SEQ ID NO: 27). Gln is predicted to form an H-bond with Ser7 to stabilize the intrachain.

[0517] L106I is a frequency-based and germline aligned mutation. Ile is the most common mutation at this position. Ile is the most common mutation at this position in the human germline gene IGKV2-30. * 02 (SEQ ID NO: 27).

[0518] Designs based on these human frameworks are shown below:

[0519] Heavy chain variable region >hu3D6VHvb1 (SEQ ID NO: 76) QVQLQQSGAELVKPGASVKLSCTASGFNIKDYYLHWVKQRPEQGLEWIGWIDPENGDTVYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYFCSTLDFWGQGTTLTVSS

[0520] >hu3D6VHvb2 (SEQ ID NO: 77) EVQLVQSGAEVVKPGASVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTVYDPKFQGRATITADTSTDTAYLELSSLTSEDTAVYFCSTLDFWGQGTLVTVSS

[0521] >hu3D6VHvb3 (SEQ ID NO: 78) EVQLVQSGAEVVKPGATVKISCKASGFNIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRATITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0522] hu3D6VHvb4 (SEQ ID NO: 79) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPENGDTIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0523] >hu3D6VHvb5 (SEQ ID NO: 80) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETIYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0524] >hu3D6VHvb6 (SEQ ID NO: 90) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYFCSTLDFWGQGTLVTVSS

[0525] >hu3D6VHvb7 (SEQ ID NO: 91) EVQLVQSGAEVVKPGATVKISCKASGFTIKDYYLHWVRQRPGKGLEWIGWIDPEDGETVYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCSTLDFWGQGTLVTVSS

[0526] Kappa light chain variable region hu3D6VLvb1 (SEQ ID NO: 83) DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVHYCWQGTHFPYTFGAGTKLELK

[0527] >hu3D6VLvb2 (SEQ ID NO: 84) DVVMTQSPLSLSVTLGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGAGTKLEIK

[0528] >hu3D6VLvb3 (SEQ ID NO: 85) DVVMTQSPLSLSVTLGEPASISCRSSQSLLDSDGKTYLNWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIK

[0529] Humanized sequences are generated using a two-step PCR protocol that allows for the introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682].

[0530] Example 4. Mouse monoclonal antibodies bind to tau in an ELISA assay Methods: Indirect ELISA: 96-well polystyrene plates were coated with capture antibodies anti-6xHis (Figure 4A) or polyclonal anti-tau (Dako #A20024, Figure 4B) suspended in 1x PBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution was removed, and the plates were blocked with 1% BSA in 1x PBS for 1 hour, followed by incubation with human recombinant tau, either with (Figure 5A) or without (Figure 4B) a polyhistidine tag at the N-terminus of the protein. After washing, the plates were incubated with the indicated antibodies followed by incubation with an HRP-conjugated goat anti-mouse secondary antibody. The plates were developed with TMB and A 450 was measured using a plate reader.

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

[0532] Results: A series of hybridoma-produced antibodies were assayed for binding to tau using several different ELISA formats. Detection of tau was confirmed using an indirect format with tau protein immobilized by a polyhistidine tag fused to the N-terminus of tau (Figure 4A). Native, untagged protein was also confirmed (Figure 4B). To assess the affinity of the various antibodies, a sandwich ELISA format was used, using the tested hybridoma antibodies as capture reagents (Figure 4C).

[0533] Example 5. Affinity of mouse monoclonal antibodies to tau Methods: SPR analysis was performed using a Biacore T200 to measure the binding kinetics of mouse antibodies to recombinant human tau. To prepare the sensor surface, an anti-mouse antibody (GE Life Sciences) was immobilized on a sensor chip CM5 via amine coupling, capturing the antibody at a level ensuring maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 10 to 0.14 nM were passed over the capture ligand at a flow rate of 50 μL / min in running buffer (HBS + 0.05% P-20, 1 mg / mL BSA) for 180 s of binding and 900 s of dissociation. Data were double-referenced against both an unrelated sensor without antibody ligand and a 0 nM analyte concentration to capture dissociation of the ligand from the capture moiety. Data were then analyzed using a global 1:1 fit.

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

[0535] Example 6. Mouse monoclonal antibodies block binding of human tau to the surface of immortalized neuronal cells Methods: Inhibition of tau binding to B103 neuroblastoma cells by anti-tau monoclonal antibodies 1. 5x10 B103 cells 5 Resuspend cells at a concentration of 1000k cells / mL. Plate 50 μL of cell suspension per well in an MSD high-binding plate. This will yield 25k cells / well. Cover the plate and allow cells to attach for 2 hours at 37°C, 5% CO2. 2. After cell attachment, remove PBS from the wells by inverting the plate and gently tapping to remove excess buffer. 50 μl of 3% MSD Blocker A buffer or other suitable blocking buffer in PBS was added to each well and the plate was incubated at room temperature for 1 hour without shaking. 3. During the plate blocking step, co-incubate tau and anti-tau antibodies as follows: a. Starting with 2 mg / mL anti-tau antibody, seven further dilutions are made in 1:2 serial dilutions in PBS. b. Tau is diluted to 20 nM in PBS, so that the tau concentration is constant in each well. c. Mix tau and anti-tau antibody 1:1 for a final tau concentration of 10 nM and a starting anti-tau concentration of 1 mg / mL. d. The mixture is incubated at room temperature with shaking (600 rpm) for approximately 1 hour. 4. After blocking the plate in step 2, 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. Ensure that excess buffer is completely removed. Cool the plated cells to 4°C before adding the tau:anti-tau complex. 5. Add 50 μL of chilled complex from step 3 to the plated cells and incubate on ice for 30 minutes. 6. Wash plates twice with cold PBS as previously described. 7. For detection of cell surface-bound tau, add 50 μL of 16B5.SULFO-TAG per well. Incubate on ice for 30 minutes. 8. Wash the plate again twice with cold PBS as previously described. 9. Add 150 μL of 1× detergent-free Read Buffer T (diluted in H2O) per well and read immediately on the MSD SECTOR™ 600 instrument. Avoid introducing air bubbles when adding the Read Buffer. 10. Report the MSD signal vs. the concentration of tau.

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

[0537] Results: A decrease in sulfo-tag anti-tau signal with increasing antibody tested indicates functional blocking of tau binding to neurons. No blocking was observed with the isotype control, 16G7, or 3H9. Increasing amounts of functional blocking activity were observed with 4C5, 5G8, and 3D6. 3D6 exhibited the strongest blocking activity of the antibodies tested. See Figure 6.

[0538] Example 7. Disaggregation activity Methods: Recombinant tau - Purified recombinant tau with an N-terminal 6xHis tag was mixed with an equimolar amount of low molecular weight heparin in 1x PBS (pH 7.4) and incubated for 96 hours at 37°C using a nutator. Aggregation of the sample was confirmed by binding to thioflavin T. Antibody incubation - Antibodies were incubated with aggregated recombinant tau for 96 hours at 37°C without rotation or nutator. At the end of the experiment, aggregation was measured by incubating samples with 25mM Thioflavin T and measuring the emitted fluorescence (450 / 482 ex / em). Signal was background subtracted to buffer samples.

[0539] Results: As shown in Figure 7, 3D6 selectively degrades intact tau fibrils. Various molar ratios of 3D6 (triangles), isotype control (circles), and 16G7 (squares) were incubated with amyloid-containing tau fibrils for 96 hours. At the end of this period, the extent of aggregation was assessed by binding to Thioflavin T. 3D6 selectively reduces the Thioflavin T signal present in the sample compared to the isotype control antibody as well as 16G7, an anti-tau antibody that binds to a different region of tau.

[0540] Example 8. 3D6 and 5G8 immunocapture tau from human disease tissue. Methods: High-salt soluble protein fractions were prepared at 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 an additional hour to capture the antibody / antigen complex. The samples were washed thoroughly 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 a polyclonal anti-tau antibody (Dako, #A0024).

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

[0542] Example 9. Immunoreactivity of 3D6 by immunohistochemistry Frontotemporal cortex tissue was obtained from patients without neurodegenerative disease or with confirmed Alzheimer's disease at postmortem evaluation. Immunohistochemistry was performed on 10-μm frozen sections mounted on slides and lightly acetone-fixed. All staining steps were performed using a Leica BOND Rx automated immunostainer and Leica consumables. Mouse or human 3D6 was incubated with the tissue sections, followed by the addition of a species-appropriate secondary antibody conjugated to an HRP polymer. To prevent nonspecific binding of endogenous immunoglobulins when using humanized antibodies against human tissue, the antibody was noncovalently labeled in vitro with a biotin-conjugated anti-human monovalent Fab fragment prior to tissue incubation. Tissue labeled with the primary antibody-biotin Fab fragment complex was further amplified using an avidin-biotin amplification system (Vector Laboratories, Burlingame, CA). Staining was visualized using DAB chromogen, which produces a brown deposit. Negative controls were created by performing the entire immunohistochemical procedure on adjacent sections using an IgG isotype control antibody.

[0543] The antibodies tested were murine CD6, chimeric 3D6 (which contained VH and VL derived from a murine antibody with human constant regions, heavy chain SEQ ID NO: 72 and light chain SEQ ID NO: 73), and humanized variants hu3D6VHv5 / hu3D6VLv2.

[0544] Staining performed on mouse, chimeric, and humanized forms of 3D6 was qualitatively compared and evaluated for staining intensity and brightness, as well as the localization of immunoreactivity. Staining brightness was similar for the chimeric and humanized forms of 3D6 compared with the mouse form of the antibody, and they showed similar localization patterns. Tau was detected in neurofibrillary tangles, fibrils, thread-like structures, and degenerating axons. Prominent cell body staining was also detected.

[0545] Example 10. Affinity of humanized variants to tau Method: Indirect ELISA: 96-well polystyrene plates are coated with human recombinant tau suspended in 1x PBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution is removed and the plates are blocked with 1% BSA in 1x PBS for 1 hour. 1 μg / mL of humanized variant antibody in 0.1% BSA in 1x PBS is added to the plates, followed by a 1-hour hold, followed by washing, and the addition of HRP-conjugated goat anti-human antibody. The plates are developed with TMB and A 450 is measured using a plate reader.

[0546] Sandwich ELISA: 96-well polystyrene plates are coated with anti-human antibody in 1x PBS for 2 hours at room temperature or 16 hours at 4°C. The coating solution is removed and the plates are blocked with 1% BSA in 1x PBS for 1 hour. Various concentrations of humanized variant antibodies are added to the plates diluted in 0.1% BSA in 1x PBS, and allowed to stand for 1 hour, followed by washing and the addition of biotinylated recombinant human tau diluted in 0.1% BSA in 1x PBS. After washing, streptavidin-HRP is added, the plates are developed with TMB, and A 450 is measured using a plate reader.

[0547] SPR analysis was 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) was immobilized on a sensor chip CM5 via amine coupling, and a humanized variant antibody was captured at a level ensuring maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 10 to 0.14 nM were passed over the capture ligand at a flow rate of 50 μL / min in running buffer (HBS + 0.05% P-20, 1 mg / mL BSA) for 180 s of binding and 900 s of dissociation. Data were double-referenced against both an unrelated sensor without antibody ligand and a 0 nM analyte concentration to account for dissociation of the ligand from the capture moiety. Data were then analyzed using a 1:1 global fit.

[0548] Example 11. Immunogenicity of the hu3D6VLv2 light chain variable region 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 peptides that may be selected for deimmunization of hu3D6VLv2, i.e., areas where further substitutions may be made to reduce potential immunogenicity. [Table 8]

[0549] Based on the results of the analysis 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] TIFF2025069265000022.tif28162

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

[0551] The rationale for selecting positions L37Q and G100Q shown in Tables 12 and 13 in the light chain variable region as candidates for substitution 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 increases the degree of humanization of the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO: 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 increases the degree of humanization of the human germline gene IGKV2-30 at this position. * 02 (SEQ ID NO: 27). [Table 11] TIFF2025069265000024.tif107161

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

[0555] Figures 10A, 10B, 10C, and 10D show an alignment of the light chain variable region of humanized versions of the 3D6 antibody: 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 (SEQ ID NO: 132), hu3D6VLv2 L37Q_S52G_L54D (SEQ ID NO: 124), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO: 121), hu3D6VLv2 L37Q_S52G_L54T (SEQ ID NO: 123), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO: 122), hu3D6VLv2 L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2 L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2 L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2 L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2 L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2L37Q_S52G_L54D_G100Q (sequence number 139), and hu3D6VLv2 L37Q_S52G_L54R_G100Q (sequence number 138).

[0556] Example 12 Immunogenicity of hu3D6VHv1bA11 (heavy chain variable region) Additional heavy chain variable region variants of hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) were designed as shown in Table 14.

[0557] Figures 9A and 9B show the alignment of the heavy chain variable region of murine 3D6 (SEQ ID NO: 7) and the heavy chain variable regions of humanized versions of the 3D6 antibody (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, hu3D6VHvb7, hu3D6VHv1bA11, h3D6VHvb8, and h3D6VHvb9) with the human germline heavy chain variable region sequenc...

Claims

1. 1. An antibody or antigen-binding antibody fragment that specifically binds to human tau, 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; 4. An antibody or antigen-binding antibody fragment comprising:

2. 2. The antibody or antigen-binding antibody fragment of claim 1, wherein the mature heavy chain variable region comprises a sequence that is at least 90% identical to SEQ ID NO:

18.

3. 3. The antibody or antigen-binding antibody fragment of claim 2, wherein at least one of Kabat positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81, and H91 is occupied by V, K, T, A, R, Q, I, R, A, D, L, Q, and F, respectively.

4. The antibody or antigen-binding antibody fragment of any one of claims 1 to 3, wherein the mature light chain variable region comprises a sequence that is at least 90% identical to SEQ ID NO:

122.

5. 5. The antibody or antigen-binding antibody fragment of claim 4, wherein at least one of Kabat positions L2, L12, L15, L37, L39, L45, L60 and L100 is occupied by V, P, L, Q, R, R, D and Q, respectively.

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

7. The antibody or antigen-binding antibody fragment of any one of claims 1 to 6 which is an antibody.

8. The antibody or antigen-binding antibody fragment of any one of claims 1 to 6, which is an antigen-binding antibody fragment.

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

10. 8. The antibody or antigen-binding antibody fragment of claim 7, wherein the antibody has an isotype of human IgG1, human IgG2, or human IgG4.

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

12. 12. The antibody or antigen-binding antibody fragment of 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. 12. The antibody or antigen-binding antibody fragment of 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. 12. The antibody or antigen-binding antibody fragment of 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. 15. The antibody or antigen-binding antibody fragment of 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 of claim 11, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO:

177.

17. 12. The antibody or antigen-binding antibody fragment of claim 11 , wherein the mature light chain variable region fused to a light chain constant region comprises the amino acid sequence of SEQ ID NO:

179.

18. 18. The antibody or antigen-binding antibody fragment of claim 17, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

181.

19. 14. The antibody or antigen-binding antibody fragment of claim 13, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 178 with or without a C-terminal lysine, and the light chain comprises the amino acid sequence of SEQ ID NO:

179.

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

181.

21. 12. The antibody or antigen-binding antibody fragment of claim 11, comprising at least one mutation in the heavy chain constant region.

22. 22. The antibody or antigen-binding antibody fragment of claim 21 , wherein the at least one mutation reduces binding or activation of complement by the heavy chain constant region compared to a native human heavy chain constant region, or reduces binding to an Fcγ receptor.

23. 23. The antibody or antigen-binding antibody fragment of claim 22, wherein the at least one mutation is present at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 according to EU numbering, or is an alanine at positions 318, 320 and 322 according to EU numbering.

24. 24. The antibody or antigen-binding antibody fragment of any one of claims 1 to 23, conjugated to a therapeutic agent, a cytotoxic agent, a cytostatic agent, a neurotrophic agent, or a neuroprotective agent.

25. A pharmaceutical composition comprising the antibody or antigen-binding antibody fragment of any one of claims 1 to 24 and a pharma- ceutically acceptable carrier.

26. A nucleic acid encoding the heavy and light chains of an antibody according to any one of claims 1 to 7 and 10 to 23.

27. 27. The nucleic acid of claim 26, comprising SEQ ID NO: 182 encoding the heavy chain and SEQ ID NO: 183 encoding the light chain.

28. 1. A method for producing an antibody, comprising: (a) culturing a cell transformed with a nucleic acid according to claim 26 or 27, such that the cell secretes the antibody; and (b) purifying the antibody from the cell culture medium; A method comprising:

29. 1. A method for producing an antibody-producing cell line, comprising: (a) introducing into a cell a vector comprising the nucleic acid of claim 26 or 27 and a selectable marker; (b) growing the cells under conditions to select for cells having an increased copy number of the vector; (c) isolating single cells from the selected cells; and (d) banking the cloned cells from the single cells selected based on antibody yield; A method comprising:

30. 1. A method for producing an antibody, comprising: (a) culturing a cell comprising nucleic acid encoding the heavy and light chains of an antibody according to any one of claims 1 to 7 and 10 to 23, such that the cell secretes said antibody; and (b) purifying the antibody from the cell culture medium; A method comprising:

31. 1. A method for producing an antibody-producing cell line, comprising: (a) introducing into a cell a vector encoding the heavy and light chains of the antibody according to any one of claims 1 to 7 and 10 to 23 and a selection marker; (b) growing the cells under conditions to select for cells having an increased copy number of the vector; (c) isolating single cells from the selected cells; and (d) banking the cloned cells from the single cells selected based on antibody yield; A method comprising:

32. 26. The pharmaceutical composition of claim 25 for use in a method of inhibiting or reducing tau aggregation in a subject having or at risk of developing tau-mediated amyloidosis.

33. 33. The pharmaceutical composition of claim 32, wherein the antibody or antigen-binding antibody fragment comprises a mature heavy chain variable region comprising an amino acid sequence comprising SEQ ID NO: 18 and a mature light chain variable region comprising an amino acid sequence comprising SEQ ID NO:

122.

34. 26. The pharmaceutical composition of claim 25 for use in a method of treating a tau-associated disease or effecting prophylaxis of a tau-associated disease in a subject.

35. 26. The pharmaceutical composition of claim 25 for use in a method of reducing the abnormal propagation of tau, inducing phagocytosis of tau, inhibiting tau deposition or aggregation, or inhibiting the formation of tau tangles in a subject.

36. 26. The pharmaceutical composition of claim 25 for use in a method for determining the efficacy of treatment in a subject being treated for a disease associated with tau aggregation or deposition.

37. The pharmaceutical composition according to any one of claims 32 to 36, which is formulated for intravenous injection or infusion.

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