Antibodies that recognize tau

Abstract

The present invention provides antibodies that specifically bind to tau. The antibody inhibits or delays the pathology associated with tau and the associated symptom deterioration.

Description

[0001] Divisional Application Instructions

[0002] This application is a divisional application of an invention patent application with an application date of March 2, 2020, application number 202080018265.8, and invention name “Antibodies recognizing tau”.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to the following patent applications: U.S. Provisional Application No. 62 / 813,126 filed on March 3, 2019, U.S. Provisional Application No. 62 / 813,137 filed on March 3, 2019, and U.S. Provisional Application No. 62 / 838,159 filed on April 24, 2019, each of which is incorporated by reference in its entirety for all purposes.

[0005] Reference to a sequence listing

[0006] This application includes an electronic sequence listing with the file name 50887-0030CN2_SL_ST26.XML, which was created on October 8, 2024 (the actual sequence content was created on February 24, 2020) and contains 200,650 bytes, which is hereby incorporated by reference in its entirety for all purposes. Background Art

[0007] Tau is a well-known human protein that can exist in a phosphorylated form (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). It is reported that Tau plays a role in stabilizing microtubules, especially in the central nervous system. Total tau (t-tau, i.e. phosphorylated and non-phosphorylated forms) and phosphorylated-tau (p-tau, i.e. phosphorylated tau) are released by the brain in response to neuronal damage and neurodegeneration, and are reported to be present at increased levels in the CSF of Alzheimer's patients relative to the general population (Jack et al., Lancet Neurol 9:119-28 (2010)).

[0008] Tau is the main component of neurofibrillary tangles, which together with plaques are the hallmarks of Alzheimer's disease. Tangles consist of abnormal fibrils with diameters of 10 nm, which appear in pairs, winding in a spiral manner, with a regular periodicity of 80 nm. Tau in neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated), in which the phosphate group is attached to a specific site on the molecule. Severe involvement of neurofibrillary tangles is observed in the deep layers (III layer, V layer and surface layer VI) of the II layer neurons of the entorhinal cortex, the CA1 of the hippocampus and the subunciate region, the amygdala and the neocortex in Alzheimer's disease. It has also been reported that hyperphosphorylated tau interferes with microtubule assembly, which may promote the destruction of neuronal networks.

[0009] Tau inclusions are part of the defining neuropathology of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and Pick's disease. Summary of the invention

[0010] In one aspect, the present invention provides an isolated antibody that specifically binds to human tau, comprising a mature heavy chain variable region and a mature light chain variable region, the mature heavy chain variable region comprising a CDR-H1 having SEQ ID NO:8, a CDR-H2 having SEQ ID NO:9 or SEQ ID NO:149, and a CDR-H3 having SEQ ID NO:10, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO:18; the mature light chain variable region comprising a CDR-L1 having SEQ ID NO:12, a CDR-L2 having SEQ ID NO:150, 151, 153, 156, 158, 159, 160, 163, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 174, and a CDR-L3 having SEQ ID NO:14, wherein the light chain variable region is at least 90% identical to SEQ ID NO:122.

[0011] In some antibodies, at least one of positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81 and H91 can 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 can be occupied by V, P, L, Q, R, R, D and Q, respectively.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] In another aspect, the present invention provides an antibody that specifically binds to human tau, comprising a mature heavy chain variable region and a mature light chain variable region, the mature heavy chain variable region comprising CDR H1, CDR H2 and CDR H3 having SEQ ID NOs: 8, 9 and 10, respectively, except that position H28 can be occupied by N or T, H54 can be occupied by N or D, H56 can be occupied by D or E, position H58 can be occupied by V or I, and position H60 can be occupied by D or E, the mature light chain variable region comprising CDR L1, CDR L2 and CDR L3 having SEQ ID NOs: 12, 13 and 14, respectively. L3, except that position L24 can be occupied by K or R, position L50 can be occupied by L, E, D, G, or V, position L52 can be occupied by S or G, and position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by 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, H57 is occupied by E 8 is occupied by I, H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V or S, L83 is occupied by L, L86 is occupied by H, L100 is occupied by Q, and L106 is occupied by L.

[0017] In another aspect, the invention provides an isolated monoclonal antibody that binds to human tau, comprising three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibody characterized by having a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:11, except that position H27 can be occupied by F or Y, position H28 can be occupied by N or T, position H29 can be occupied by I or F, position H30 can be occupied by K or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, position H60 can be occupied by D, A, or E, position H61 can be occupied by P or E, position H102 can be occupied by F or Y, position L50 can be occupied by L, E, D, G, or V, and position L52 can be occupied by S or G. The amino acids in the formula (a) and (b) are represented by the amino acids in the table below: 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.

[0018] 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 one 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 one of SEQ ID NOs: 150-175.

[0019] Some antibodies are humanized, veneered, or chimeric.

[0020] Some antibodies comprise a humanized mature heavy chain variable region and a humanized mature light chain variable region, wherein the humanized mature heavy chain variable region has an amino acid sequence at least 95% identical to any one of SEQ ID NOs:76-80 and SEQ ID NOs:90-91, and the humanized mature light chain variable region has an amino acid sequence at least 90% identical to any one of SEQ ID NOs:83-85.

[0021] Some antibodies comprise a humanized mature heavy chain variable region and a humanized mature light chain variable region, wherein the humanized mature heavy chain variable region has an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 18 and 146-148, and the humanized mature light chain variable region has an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 93-145.

[0022] 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.

[0023] In some 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 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.

[0024] 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.

[0025] In some antibodies, position H58 in the VH region is occupied by I.

[0026] 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, H67 is occupied by V. In some antibodies, positions H28 and H67 in the VH region are occupied by T and V, respectively.

[0027] In some antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H54 is occupied by D, H56 is occupied by E. In some antibodies, positions H54 and H56 in the VH region are occupied by D and E, respectively.

[0028] In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H17 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, H54 is occupied by N or D, and H56 is occupied by D or E , H58 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.

[0029] In some antibodies, at least one of the following positions in the VH region is occupied by the specified 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, 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, H82a 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.

[0030] In some antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In some 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 antibodies, positions L7, L10, L15, L83, L86, and L106 are occupied by S, S, L, V, Y, and Y, respectively.

[0035] In some antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L7 is T or S, L10 is T or S, L15 is I or L, L17 is Q or E, L24 is K or R, L37 is L or Q, L45 is K or R, L47 is L, G, N, D, E, P, T, S or A, L48 is I, G, or D, L49 is Y or E, L50 is L, E, D, G or V, L52 is S or G, L54 is L, D, G, N, E, Q, K, R, T, V or S, L83 is L or V, L86 is H or Y, L100 is A or Q, and L106 is L or I.

[0036] In some antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is V or I, L7 is S or T, L12 is P or S, L15 is L or I, L36 is L, L37 is L or Q, L45 is R or K, L47 is L, G, N, D, E, P, T, S or A, L48 is I, G or D, L49 is Y or E, L50 is L, E, D, G or V, L52 is S or G, L54 is L, D, G, N, E, Q, K, R, T, V, L60 is D or S, and L100 is G or Q.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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, 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.

[0042] 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.

[0043] 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.

[0044] In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 76 to 80 and SEQ ID NOs: 90 to 91, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 83 to 85. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 18 and SEQ ID NOs: 146-148, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 93-145.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[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: 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] For example, the antibody can be a chimeric antibody, a veneered antibody, or a humanized antibody.

[0058] The antibody may be a complete chimeric, veneered or humanized antibody or a 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 other antibodies may have a human IgG2 or IgG4 isotype. Some antibodies have a mature light chain variable region fused to a light chain constant region and a mature heavy chain variable region fused to a heavy chain constant region. The heavy chain constant region of some antibodies is a mutant form of a natural human heavy chain constant region, and the mutant form of the natural human heavy chain constant region has reduced binding to Fcγ receptors relative to the natural human heavy chain constant region.

[0059] In some antibodies, the heavy chain constant region has an 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 an amino acid sequence of SEQ ID NO: 178. Some antibodies also include a signal peptide fused to the mature heavy chain variable region and / or the mature light chain variable region. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 180. In some antibodies, the light chain constant region has an 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 an amino acid sequence of SEQ ID NO: 179. In some antibodies, the light chain has an amino acid sequence of SEQ ID NO: 181. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 178, and the light chain has an amino acid sequence of SEQ ID NO: 179. In some antibodies, the heavy chain has an amino acid sequence of SEQ ID NO: 180, and the light chain has an amino acid sequence of SEQ ID NO: 181.

[0060] Some antibodies may have at least one mutation in the constant region, such as a mutation that reduces complement fixation or activation of the constant region, for example, a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 according to 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.

[0061] In another aspect, the present invention provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharmaceutically acceptable carrier.

[0062] In another aspect, the present invention provides a nucleic acid encoding the heavy chain and / or light chain of any antibody disclosed herein, a recombinant expression vector comprising the nucleic acid, and a host cell transformed with the recombinant expression vector.

[0063] 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.

[0064] Also provided are methods of producing antibodies, such as humanized, chimeric or veneered antibodies, e.g., humanized, chimeric or veneered forms of 3D6. In such methods, cells transformed with nucleic acids encoding the heavy and light chains of the antibody are cultured so that the cells secrete the antibody. The antibody can then be purified from the cell culture medium.

[0065] Cell lines producing any of the antibodies disclosed herein can be generated by introducing vectors encoding the heavy and light chains of the antibodies and a selectable marker into cells, allowing the cells to proliferate under conditions that select for cells with an increased copy number of the vector, isolating single cells from the selected cells; and banking cells cloned from single cells selected based on antibody production.

[0066] 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, the nucleic acid being operably connected to one or more regulatory sequences to achieve expression of any antibody disclosed herein in a mammalian cell. In some vectors, the expressed antibody is a scFv or Fab fragment. In some vectors, 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 also encodes a signal peptide fused to the mature heavy chain variable region and the mature light chain variable region. In some vectors, the nucleic acid is codon-optimized for expression in a host cell. In some vectors, one or more regulatory sequences include a eukaryotic promoter. In some vectors, the nucleic acid also encodes a selective gene.

[0067] In yet another aspect, the present invention provides a method for expressing an antibody in a mammalian cell, comprising incorporating the nucleic acid disclosed herein into the genome of a transgenic animal, thereby expressing the antibody.

[0068] In yet another aspect, the present invention provides a first vector and a second vector, which respectively contain nucleic acids encoding mature heavy chain variable regions and nucleic acids encoding mature light chain variable regions, each of which is operably linked to one or more regulatory sequences to achieve expression of any antibody disclosed herein, and a host cell comprising the nucleic acids. In some first vectors and second vectors, the nucleic acids also encode a heavy chain constant region fused to a mature heavy chain variable region and a light chain constant region fused to a mature light chain variable region, respectively. In some first vectors and second vectors, the heavy chain constant region has a sequence of SEQ ID NO: 176, which has or does not have a C-terminal lysine, and the light chain constant region has a sequence of SEQ ID NO: 177.

[0069] In yet another aspect, the present invention provides a method for expressing an antibody in a mammalian cell, comprising incorporating any of the nucleic acids disclosed herein into the genome of a transgenic animal, thereby expressing the antibody.

[0070] Also provided are methods for producing antibodies (such as humanized antibodies, chimeric antibodies 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 so that the cells secrete the antibodies. The antibodies can then be purified from the cell culture medium.

[0071] Cell lines producing any of the antibodies disclosed herein can be produced by introducing vectors encoding the heavy and light chains of the antibodies of claim 1 and a selective marker into cells, allowing these cells to proliferate under conditions for selecting cells with an increased copy number of the vector, isolating single cells from the selected cells, and banking cells cloned from single cells selected based on antibody production.

[0072] Some cells can be propagated under selective conditions and screened to naturally express and secrete at least 100 mg / L / 10 6 Cell lines that can be cloned from single cells can be constructed based on the desired properties, such as the yield of antibodies. An exemplary cell line is a cell line expressing 3D6 or a humanized version of 3D6.

[0073] The present invention also provides a method of inhibiting or reducing tau aggregation in a subject having tau-mediated amyloidosis or at risk of developing tau-mediated amyloidosis, comprising administering to the subject an effective regimen of an antibody disclosed herein, thereby inhibiting or reducing tau aggregation in the subject. Exemplary antibodies include humanized versions of 3D6.

[0074] Also provided is a method for treating tau related diseases or achieving its prevention in a subject, including applying an effective scheme of an antibody disclosed herein and thereby treating a disease or achieving its prevention. The example of this disease is Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, C-type Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant (LBVAD) of Alzheimer's disease, chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some methods, tau related diseases are Alzheimer's disease. In some methods, the patient is an ApoE4 carrier.

[0075] Also provided are methods of reducing abnormal propagation of tau comprising administering an effective regimen of an antibody disclosed herein and thereby reducing the propagation of tau.

[0076] Also provided are methods of inducing phagocytosis of tau comprising administering an effective regimen of an antibody disclosed herein and thereby inducing phagocytosis of tau.

[0077] Also provided are methods of inhibiting tau aggregation or deposition, comprising administering an effective regimen of an antibody disclosed herein, thereby inhibiting tau aggregation or deposition.

[0078] Also provided are methods of inhibiting tau tangle formation comprising administering an effective regimen of an antibody disclosed herein.

[0079] The present invention also provides a method for detecting tau protein deposits in a subject suffering from a disease associated with tau aggregation or deposition or having a risk of the disease, including administering an antibody disclosed herein to a subject, and detecting an antibody bound to tau in a subject. Examples of such diseases are Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitis Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, C-type Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant (LBVAD) of Alzheimer's disease, chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). In some embodiments, the antibody is administered to a subject 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).

[0080] The present invention also provides a method for measuring the efficacy of a treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising measuring a first level of tau deposits in the subject before treatment by administering an antibody disclosed herein to the subject, and detecting a first amount of the antibody that binds to tau in the subject; administering the treatment to the subject; measuring a second level of tau deposits in the subject after treatment by administering the antibody to the subject, and detecting the antibody that binds to tau in the subject; wherein a decrease in the level of tau deposits indicates a positive response to the treatment.

[0081] The present invention also provides a method for measuring the efficacy of a treatment in a subject being treated for a disease associated with tau aggregation or deposition, comprising measuring a first level of tau deposits in the subject before treatment by administering an antibody disclosed herein to the subject, and detecting a first amount of the antibody that binds to tau in the subject; administering the treatment to the subject; measuring a second level of tau deposits in the subject after treatment by administering the antibody to the subject, and detecting a second amount of the antibody that binds to tau in the subject; wherein no change in the level of tau deposits or a small increase in tau deposits indicates a positive response to the treatment.

[0082] In another aspect, the present invention provides a method for producing an antibody that specifically binds 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 produce antibodies, and screening the produced antibodies for antibodies that specifically bind within the motif. In another aspect, the present invention provides a method for producing an antibody that specifically binds to a peptide consisting of residues KXXSXXNX(K / H)H (SEQ ID NO: 191) or KIGSLDNITH (SEQ ID NO: 194), comprising immunizing an animal with human tau or a fragment thereof to produce antibodies, and screening the produced antibodies for antibodies that specifically bind to the peptide. In another aspect, the present invention provides a method for producing an antibody that specifically binds to an epitope comprising KXXSXXNX(K / H)H (SEQ ID NO: 191), comprising immunizing an animal with tau or a fragment thereof, and screening the produced antibodies for antibodies that specifically bind to the epitope.

[0083] In some methods, animals are immunized with human tau (4RON) containing 383 amino acids. In some methods, the human tau contains a P301S mutation. In some methods, the human tau is a recombinant tau with a His tag at the N-terminus.

[0084] In some methods, the screening measures specific binding between the antibody and one or more peptides of up to 15 amino acids, the one or more peptides comprising KIGSTENLKH (SEQ ID NO: 188), KCGSKDNIKH (SEQ ID NO: 192), KCGSLGNIHH (SEQ ID NO: 193), or any other consensus motif represented by KXXSXXNX(K / H)H (SEQ ID NO: 191), respectively. 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, the animal is immunized with a tau fragment of up to 15 amino acids linked to a carrier, the tau fragment comprising KXXSXXNX(K / H)H (SEQ ID NO: 191). In some methods, the peptide is KIGSTENLKH (SEQ ID NO: 188) or KCGSKDNIKH (SEQ ID NO: 192) or KCGSLGNIHH (SEQ ID NO: 193). BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Depicted are the results of an experiment designed to map the epitope bound by the murine 3D6 monoclonal antibody.

[0086] Figure 2 Depicted is an alignment of the heavy chain variable regions of the mouse 3D6 antibody (SEQ ID NO:7) and humanized versions of the 3D6 antibody (hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7) with the human germline heavy chain variable region sequence IGHV1-69-2*01 (SEQ ID NO:25) and the human acceptor heavy chain variable region sequence 2RCSVH 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. The CDRs as defined by the Kabat / Chothia complex are shown in bold.

[0087] Figure 3 Depicted is an alignment of the light chain variable regions of the mouse 3D6 antibody (SEQ ID NO: 11) and humanized versions of the 3D6 antibody (hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3) with 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.

[0088] Figure 4A , Figure 4B and Figure 4C Depicted are the results of an ELISA screening assay of selected mouse monoclonal anti-tau antibodies.

[0089] Figure 5 The binding kinetics of selected mouse monoclonal anti-tau antibodies to recombinant human tau are depicted.

[0090] Figure 6Depicted are the results of functional blocking assays of selected mouse monoclonal anti-tau antibodies.

[0091] Figure 7 Depicted are the results of disaggregation assays with selected mouse monoclonal anti-tau antibodies.

[0092] Figure 8 Depicted are the results of an experiment showing 3D6 and 5G8 immunocapture of tau from human Alzheimer's disease tissue.

[0093] Fig. 9A and Fig. 9B Shown is an alignment of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) and 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 acceptor 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 residues of the heavy chain variable region of murine 3D6 (SEQ ID NO: 7) are marked with a ".". CDRs as defined by the Kabat / Chothia complex are shown in bold.

[0094] Fig. 10A , Fig. 10B , Fig. 10C and Fig. 10DAlignment of the light chain variable regions of the following humanized forms of the 3D6 antibody is shown: 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 IDNO: 141), hu3D6VLv2L37Q_L50G_L54G_G100Q (SEQ ID NO: 137), hu3D6VLv2L37Q_L50G_L54R_G100Q (SEQ ID NO: 136), hu3D6VLv2L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), and hu3D6VLv2L37Q_S52G_L54R_G100Q (SEQ ID NO: 138). Residues identical to residues in the light chain variable region of hu3D6VLv2 (SEQ ID NO: 21) are marked with a ".". The CDRs as defined by Kabat are shown in bold.

[0095] Fig.11 Shown are the results of tau internalization assays for selected humanized 3D6 variants.

[0096] Fig. 12A and Fig. 12B Shown are the results of a displacement mapping microarray experiment. Fig. 12A A diagram showing the effect of substitution, and Fig. 12B An alignment of portions of the tau microtubule binding repeat sequence is shown, with important residues for binding to 3D6 highlighted. aa 255-271 is SEQ ID NO: 184, aa 286-302 is SEQ ID NO: 185, aa 317-333 is SEQ ID NO: 186, and aa 349-365 is SEQ ID NO: 187.

[0097] Fig.13 Depicted are the results of immunohistochemistry experiments showing 3D6 binding to tau in normal tissue (upper panels) and to tau in Alzheimer's disease tissue (middle and lower panels).

[0098] Fig.14 Shown are the results of mass spectrometry experiments used to assess the binding stoichiometry of 3D6.

[0099] Fig.15 3D6 was shown to disrupt tau seeding in an in vivo disease model of Alzheimer's disease.

[0100] Fig.16 The 3D6 humanized variants hu3D6VHv1bA11 / hu3D6VLv2 and hu3D6VHv1bA11 / L2-DIM4 were shown to block the interaction between tau and heparin.

[0101] Fig.17The 3D6 humanized variants hu3D6VHv1bA11 / hu3D6VLv2 and hu3D6VHv1bA11 / L2-DIM4 were shown to bind to the fibrillar form of tau.

[0102] A brief description of the sequence

[0103] SEQ ID NO: 1 sets forth the amino acid sequence of an isoform of human tau (Swiss-Prot P10636-8).

[0104] SEQ ID NO: 2 sets forth the amino acid sequence of an isoform of human tau (Swiss-Prot P10636-7).

[0105] SEQ ID NO: 3 sets forth the amino acid sequence of an isoform of human tau (Swiss-Prot P10636-6) (4R0N human tau).

[0106] SEQ ID NO:4 sets forth the amino acid sequence of an isoform of human tau (Swiss-Prot P10636-5)

[0107] SEQ ID NO: 5 sets forth the amino acid sequence of an isoform of human tau (Swiss-Prot P10636-4).

[0108] SEQ ID NO: 6 sets forth the amino acid sequence of an isoform of human tau (Swiss-Prot P10636-2).

[0109] SEQ ID NO:7 lists the amino acid sequence of the heavy chain variable region of the mouse 3D6 antibody.

[0110] SEQ ID NO:8 sets forth the amino acid sequence of the Kabat / Chothia complex CDR-H1 of the mouse 3D6 antibody.

[0111] SEQ ID NO:9 sets forth the amino acid sequence of Kabat CDR-H2 of the mouse 3D6 antibody.

[0112] SEQ ID NO: 10 sets forth the amino acid sequence of Kabat CDR-H3 of the mouse 3D6 antibody.

[0113] SEQ ID NO: 11 lists the amino acid sequences of the light chain variable regions of the mouse 3D6 antibody and the mouse 6A10 antibody.

[0114] SEQ ID NO: 12 sets forth the amino acid sequence of Kabat CDR-L1 of mouse 3D6 antibody and mouse 6A10 antibody.

[0115] SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-L2 of mouse 3D6 antibody and mouse 6A10 antibody.

[0116] SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-L3 of mouse 3D6 antibody and mouse 6A10 antibody.

[0117] SEQ ID NO: 15 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1 of the humanized 3D6 antibody.

[0118] SEQ ID NO: 16 lists the amino acid sequence of the heavy chain variable region hu3D6VHv2 of the humanized 3D6 antibody.

[0119] SEQ ID NO: 17 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1b of the humanized 3D6 antibody.

[0120] SEQ ID NO: 18 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1bA11 of the humanized 3D6 antibody.

[0121] SEQ ID NO: 19 lists the amino acid sequence of the heavy chain variable region hu3D6VHv5 of the humanized 3D6 antibody.

[0122] SEQ ID NO: 20 lists the amino acid sequence of the light chain variable region hu3D6VLv1 of the humanized 3D6 antibody.

[0123] SEQ ID NO: 21 lists the amino acid sequence of the light chain variable region hu3D6VLv2 of the humanized 3D6 antibody.

[0124] SEQ ID NO: 22 lists the amino acid sequence of the light chain variable region hu3D6VLv3 of the humanized 3D6 antibody.

[0125] SEQ ID NO: 23 lists the amino acid sequence of the light chain variable region hu3D6VLv4 of the humanized 3D6 antibody.

[0126] SEQ ID NO:24 sets forth the amino acid sequence of the heavy chain variable receptor Acc.#BAC01986.1.

[0127] SEQ ID NO: 25 sets forth the amino acid sequence of the heavy chain variable receptor Acc.#IMGT#IGHV1-69-2*01.

[0128] SEQ ID NO:26 sets forth the amino acid sequence of the heavy chain variable receptor Acc.#IMGT#IGKJ1*01.

[0129] SEQ ID NO:27 lists the amino acid sequence of the light chain variable receptor Acc.#IMGT#IGKV2-30*02.

[0130] SEQ ID NO: 28 lists the amino acid sequence of the light chain variable receptor Acc.#IMGT#IGKJ2*01.

[0131] SEQ ID NO:29 sets forth the amino acid sequence of light chain variable receptor Acc.#AAZ09048.1.

[0132] SEQ ID NO:30 lists the nucleic acid sequence encoding the heavy chain variable region of the mouse 3D6 antibody.

[0133] SEQ ID NO:31 lists the nucleic acid sequence encoding the light chain variable region of the mouse 3D6 antibody.

[0134] SEQ ID NO:32 sets forth the amino acid sequence of Kabat CDR-H1 of the mouse 3D6 antibody.

[0135] SEQ ID NO:33 sets forth the amino acid sequence of Chothia CDR-H1 of the mouse 3D6 antibody.

[0136] SEQ ID NO:34 sets forth the amino acid sequence of Chothia CDR-H2 of the mouse 3D6 antibody.

[0137] SEQ ID NO:35 sets forth the amino acid sequence of AbM CDR-H2 of the mouse 3D6 antibody.

[0138] SEQ ID NO:36 lists the amino acid sequence of Contact CDR-L1 of mouse 3D6 antibody.

[0139] SEQ ID NO:37 lists the amino acid sequence of Contact CDR-L2 of mouse 3D6 antibody.

[0140] SEQ ID NO:38 lists the amino acid sequence of Contact CDR-L3 of mouse 3D6 antibody.

[0141] SEQ ID NO:39 lists the amino acid sequence of Contact CDR-H1 of mouse 3D6 antibody.

[0142] SEQ ID NO:40 lists the amino acid sequence of Contact CDR-H2 of mouse 3D6 antibody.

[0143] SEQ ID NO:41 lists the amino acid sequence of Contact CDR-H3 of mouse 3D6 antibody.

[0144] SEQ ID NO:42 sets forth the amino acid sequence of an alternative Kabat-Chothia complex CDR-H1 of a humanized 3D6 antibody (such as in hu3D6VHv5, hu3D6VHv1bA11B6G2, hu3D6VHv1bA11B6H3, hu3D6VHv1e, and hu3D6VHv1f).

[0145] SEQ ID NO:43 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHv5 and hu3D6VHv1bA11B6H3).

[0146] SEQ ID NO: 44 lists the consensus amino acid sequence in the heavy chain variable regions (VHv1, VHv2, VHv1b, VHv1bA11, and VHv5) of mouse 3D6 and selected humanized 3D6 antibodies (in PCT / IB2017 / 052544). Figure 2 Marked as "Majority".

[0147] SEQ ID NO: 45 lists the consensus amino acid sequence between the light chain variable regions of mouse 3D6 and selected humanized 3D6 antibodies (in PCT / IB2017 / 052544 Figure 3 “Majority” in the

[0148] SEQ ID NO:46 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1bA11B6G2 of the humanized 3D6 antibody.

[0149] SEQ ID NO: 47 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1bA11B6H3 of the humanized 3D6 antibody.

[0150] SEQ ID NO: 48 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1c of the humanized 3D6 antibody.

[0151] SEQ ID NO: 49 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1d of the humanized 3D6 antibody.

[0152] SEQ ID NO:50 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1e of the humanized 3D6 antibody.

[0153] SEQ ID NO:51 lists the amino acid sequence of the heavy chain variable region hu3D6VHv1f of the humanized 3D6 antibody.

[0154] SEQ ID NO:52 lists the amino acid sequence of the heavy chain variable region hu3D6VHv3 of the humanized 3D6 antibody.

[0155] SEQ ID NO:53 lists the amino acid sequence of the heavy chain variable region hu3D6VHv3b of the humanized 3D6 antibody.

[0156] SEQ ID NO:54 lists the amino acid sequence of the heavy chain variable region hu3D6VHv3c of the humanized 3D6 antibody.

[0157] SEQ ID NO:55 lists the amino acid sequence of the heavy chain variable region hu3D6VHv4 of the humanized 3D6 antibody.

[0158] SEQ ID NO:56 lists the amino acid sequence of the heavy chain variable region hu3D6VHv4b of the humanized 3D6 antibody.

[0159] SEQ ID NO:57 lists the amino acid sequence of the heavy chain variable region hu3D6VHv4c of the humanized 3D6 antibody.

[0160] SEQ ID NO:58 sets forth the amino acid sequence of an alternative Kabat-Chothia complex CDR-H1 of the humanized 3D6 antibody (as in hu3D6VH1c).

[0161] SEQ ID NO:59 sets forth the amino acid sequence of an alternative Kabat-Chothia complex CDR-H1 of a humanized 3D6 antibody (as in hu3D6VHv1d, hu3D6VHv3c, and hu3D6VHv4c).

[0162] SEQ ID NO:60 sets forth the amino acid sequence of an alternative Kabat-Chothia complex CDR-H1 of the humanized 3D6 antibody (as in hu3D6VHv3b and hu3D6VHv4b).

[0163] SEQ ID NO:61 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (as in hu3D6VHv1bA11B6G2).

[0164] SEQ ID NO:62 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHv1c, hu3D6VHv3b, and hu3D6VHv4b).

[0165] SEQ ID NO:63 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (such as in hu3D6VHv1d, hu3D6VHv1f, hu3D6VHv3c, and hu3D6VHv4c).

[0166] SEQ ID NO:64 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHv1e).

[0167] SEQ ID NO:65 sets forth the amino acid sequence of an alternative Kabat CDR-H3 of a humanized 3D6 antibody (as in hu3D6VHv1f).

[0168] SEQ ID NO:66 sets forth the amino acid sequence of the heavy chain variable region of the mouse 6A10 antibody.

[0169] SEQ ID NO:67 sets forth the amino acid sequence of the Kabat / Chothia complex CDR-H1 of the mouse 6A10 antibody.

[0170] SEQ ID NO:68 sets forth the amino acid sequence of Kabat CDR-H2 of the mouse 6A10 antibody.

[0171] SEQ ID NO:69 sets forth the amino acid sequence of Kabat CDR-H3 of the mouse 6A10 antibody.

[0172] SEQ ID NO:70 sets forth the amino acid sequence of the VH region of a mouse antibody (pdb code 1CR9) used as a structural template for heavy chain humanization.

[0173] SEQ ID NO: 71 lists 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) (in PCT / IB2017 / 052544). Figure 4A and Figure 4B “Majority” in the

[0174] SEQ ID NO:72 sets forth the amino acid sequence of the heavy chain of the chimeric 3D6 antibody.

[0175] SEQ ID NO:73 sets forth the amino acid sequence of the light chain of the chimeric 3D6 antibody.

[0176] SEQ ID NO:74 lists the amino acid sequence of the heavy chain variable structure model Acc.#5MYX-VH_mSt.

[0177] SEQ ID NO:75 lists the amino acid sequence of the heavy chain variable receptor Acc.#2RCS-VH_huFrwk.

[0178] SEQ ID NO:76 sets forth the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb1.

[0179] SEQ ID NO:77 sets forth the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb2.

[0180] SEQ ID NO:78 sets forth the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb3.

[0181] SEQ ID NO:79 lists the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb4.

[0182] SEQ ID NO:80 sets forth the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb5.

[0183] SEQ ID NO:81 lists the amino acid sequence of the light chain variable structure model Acc.#5MYX-VL_mSt.

[0184] SEQ ID NO:82 lists the amino acid sequence of the light chain variable receptor Acc.#ARX71335-VL_huFrwk.

[0185] SEQ ID NO:83 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLvb1.

[0186] SEQ ID NO:84 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLvb2.

[0187] SEQ ID NO:85 sets forth the amino acid sequence of the light chain variable region of the humanized 3D6 antibody hu3D6VLvb3.

[0188] SEQ ID NO:86 sets forth the amino acid sequence of an alternative Kabat-Chothia complex CDR-H1 of the humanized 3D6 antibody (as in hu3D6VHvb4 and hu3D6VHvb5).

[0189] SEQ ID NO:87 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of the humanized 3D6 antibody (as in hu3D6VHvb3 and hu3D6VHvb4).

[0190] SEQ ID NO:88 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb5).

[0191] SEQ ID NO:89 sets forth the amino acid sequence of an alternative Kabat CDR-L1 of a humanized 3D6 antibody (as in hu3D6VLvb3).

[0192] SEQ ID NO:90 lists the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb6.

[0193] SEQ ID NO:91 lists the amino acid sequence of the heavy chain variable region of the humanized 3D6 antibody hu3D6VHvb7.

[0194] SEQ ID NO:92 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (as in hu3D6VHvb6 and hu3D6VHvb7).

[0195] SEQ ID NO:93 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54D.

[0196] SEQ ID NO:94 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54G.

[0197] SEQ ID NO:95 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L45N.

[0198] SEQ ID NO:96 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54E.

[0199] SEQ ID NO:97 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50E.

[0200] SEQ ID NO:98 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54Q.

[0201] SEQ ID NO:99 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50D.

[0202] SEQ ID NO: 100 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54K.

[0203] SEQ ID NO: 101 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54R.

[0204] SEQ ID NO:102 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54T.

[0205] SEQ ID NO:103 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50G.

[0206] SEQ ID NO:104 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant I48G.

[0207] SEQ ID NO:105 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant I48D.

[0208] SEQ ID NO:106 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47G.

[0209] SEQ ID NO: 107 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant Y49E.

[0210] SEQ ID NO:108 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54V.

[0211] SEQ ID NO:109 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L54S.

[0212] SEQ ID NO: 110 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant S52G.

[0213] SEQ ID NO:111 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47N.

[0214] SEQ ID NO: 112 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47D.

[0215] SEQ ID NO:113 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47E.

[0216] SEQ ID NO: 114 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47P.

[0217] SEQ ID NO: 115 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47T.

[0218] SEQ ID NO: 116 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47S.

[0219] SEQ ID NO: 117 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L47A.

[0220] SEQ ID NO:118 sets forth the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L50V.

[0221] SEQ ID NO:119 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R.

[0222] SEQ ID NO:120 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G.

[0223] SEQ ID NO:121 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54G.

[0224] SEQ ID NO:122 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R.

[0225] SEQ ID NO:123 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54T.

[0226] SEQ ID NO:124 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D.

[0227] SEQ ID NO:125 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54R.

[0228] SEQ ID NO:126 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54G.

[0229] SEQ ID NO:127 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54D.

[0230] SEQ ID NO:128 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G.

[0231] SEQ ID NO:129 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D.

[0232] SEQ ID NO:130 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L54T.

[0233] SEQ ID NO:131 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G.

[0234] SEQ ID NO:132 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54G.

[0235] SEQ ID NO:133 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50D_L54R.

[0236] SEQ ID NO:134 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54G.

[0237] SEQ ID NO:135 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50E_L54R.

[0238] SEQ ID NO:136 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54R_G100Q.

[0239] SEQ ID NO:137 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_L50G_L54G_G100Q.

[0240] SEQ ID NO:138 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54R_G100Q.

[0241] SEQ ID NO:139 lists the amino acid sequence of the light chain variable region of hu3D6VLv2 variant L37Q_S52G_L54D_G100Q.

[0242] SEQ ID NO:140 lists the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50D_L54G_G100Q.

[0243] SEQ ID NO:141 lists the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50D_L54R_G100Q.

[0244] SEQ ID NO:142 lists the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L50V_L54D_G100Q.

[0245] SEQ ID NO:143 lists the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q.

[0246] SEQ ID NO: 144 lists the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant G100Q.

[0247] SEQ ID NO:145 lists the amino acid sequence of the light chain variable region of Hu3D6VLv2 variant L37Q_L54E.

[0248] SEQ ID NO: 146 sets forth the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant D60E (also referred to as h3D6VHvb8).

[0249] SEQ ID NO: 147 sets forth the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant L82cV.

[0250] SEQ ID NO: 148 sets forth the amino acid sequence of the heavy chain variable region of hu3D6VHv1bA11 variant D60E_L80M_Q81E_L82cV_T83R (also referred to as h3D6VHvb9).

[0251] SEQ ID NO: 149 sets forth the amino acid sequence of an alternative Kabat CDR-H2 of a humanized 3D6 antibody (as in h3D6VHvb8 and h3D6VHvb9).

[0252] SEQ ID NO: 150 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54D and hu3D6VLv2 L37Q_L54D).

[0253] SEQ ID NO: 151 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54G and hu3D6VLv2 L37Q_L54G).

[0254] SEQ ID NO: 152 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54N).

[0255] SEQ ID NO: 153 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54E and hu3D6VLv2 L37Q_L54E).

[0256] SEQ ID NO: 154 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50E).

[0257] SEQ ID NO: 155 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54Q).

[0258] SEQ ID NO: 156 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50D and hu3D6VLv2 L37Q_L50D).

[0259] SEQ ID NO: 157 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54K).

[0260] SEQ ID NO: 158 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54R and hu3D6VLv2 L37Q_L54R).

[0261] SEQ ID NO: 159 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54T and hu3D6VLv2 L37Q_L54T).

[0262] SEQ ID NO: 160 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50G and hu3D6VLv2 L37Q_L50G).

[0263] SEQ ID NO: 161 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54V).

[0264] SEQ ID NO: 162 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L54S).

[0265] SEQ ID NO: 163 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 S52G and hu3D6VLv2 L37Q_S52G).

[0266] SEQ ID NO: 164 sets forth the amino acid sequence of an alternative Kabat CDR-L2 of a humanized 3D6 antibody (as in hu3D6VLv2 L50V).

[0267] SEQ ID NO: 165 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0268] The amino acid sequences of alternative Kabat CDR-L2 of hu3D6VLv2 (L37Q_L50G_L54R and hu3D6VLv2 L37Q_L50G_L54R_G100Q).

[0269] SEQ ID NO: 166 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0270] The amino acid sequences of alternative Kabat CDR-L2 of hu3D6VLv2 (L37Q_L50G_L54G and hu3D6VLv2 L37Q_L50G_L54G_G100Q).

[0271] SEQ ID NO: 167 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0272] L37Q_S52G_L54G) in the amino acid sequence of an alternative Kabat CDR-L2.

[0273] SEQ ID NO: 168 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0274] The amino acid sequences of alternative Kabat CDR-L2 of hu3D6VLv2 L37Q_S52G_L54R and hu3D6VLv2 L37Q_S52G_L54R_G100Q).

[0275] SEQ ID NO: 169 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0276] L37Q_S52G_L54T) in the amino acid sequence of an alternative Kabat CDR-L2.

[0277] SEQ ID NO: 170 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0278] The amino acid sequences of alternative Kabat CDR-L2 of hu3D6VLv2 L37Q_S52G_L54D and hu3D6VLv2 L37Q_S52G_L54D_G100Q).

[0279] SEQ ID NO: 171 lists the humanized 3D6 antibody (such as in hu3D6VLv2

[0280] The amino acid sequences of alternative Kabat CDR-L2 of hu3D6VLv2 (L37Q_L50D_L54G and hu3D6VLv2 L37Q_L50D_L54G_G100Q).

[0281] SEQ ID NO: 172 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0282] The amino acid sequences of alternative Kabat CDR-L2 of hu3D6VLv2 (L37Q_L50D_L54R and hu3D6VLv2 L37Q_L50D_L54R_G100Q).

[0283] SEQ ID NO: 173 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0284] The amino acid sequence of an alternative Kabat CDR-L2 of (L37Q_L50E_L54G).

[0285] SEQ ID NO: 174 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0286] The amino acid sequence of an alternative Kabat CDR-L2 of (L37Q_L50E_L54R).

[0287] SEQ ID NO: 175 sets forth the humanized 3D6 antibody (as in hu3D6VLv2

[0288] The amino acid sequence of an alternative Kabat CDR-L2 of (L37Q_L50V_L54D_G100Q).

[0289] SEQ ID NO: 176 sets forth the amino acid sequence of the heavy chain constant region (IgG1: allotype G1m17,1).

[0290] SEQ ID NO: 177 sets forth the amino acid sequence of the light chain constant region (κ).

[0291] SEQ ID NO: 178 sets forth the amino acid sequence of the mature heavy chain of the 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype).

[0292] SEQ ID NO: 179 lists the amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4κ).

[0293] SEQ ID NO: 180 sets forth the amino acid sequence of the heavy chain of a humanized variant of 3D6 (hu3D6VHv1bA11 IgG1 G1m17 allotype) with a bovine α-lactalbumin signal peptide at the N-terminus.

[0294] SEQ ID NO: 181 sets forth the amino acid sequence of the light chain of a humanized variant of 3D6 (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4κ) with a bovine α-lactalbumin signal peptide at the N-terminus.

[0295] SEQ ID NO: 182 sets forth the nucleotide sequence encoding the heavy chain of a humanized variant of 3D6 (hu3D6VHv1bA11 IgG1 G1m17 allotype) having a bovine α-lactalbumin signal peptide at the N-terminus.

[0296] SEQ ID NO: 183 sets forth the nucleotide sequence encoding the light chain of a 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4κ) having a bovine α-lactalbumin signal peptide at the N-terminus.

[0297] SEQ ID NO: 184 sets forth the amino acid sequence of the region of the tau microtubule-binding repeat 1 (amino acid residues 255-271 of SEQ ID NO: 1).

[0298] SEQ ID NO: 185 sets forth the amino acid sequence of the region of the microtubule-binding repeat 2 of tau (amino acid residues 286-302 of SEQ ID NO: 1).

[0299] SEQ ID NO: 186 sets forth the amino acid sequence of the region of the microtubule-binding repeat 3 of tau (amino acid residues 317-333 of SEQ ID NO: 1).

[0300] SEQ ID NO: 187 sets forth the amino acid sequence of the region of the microtubule-binding repeat 4 of tau (amino acid residues 349-365 of SEQ ID NO: 1).

[0301] SEQ ID NO: 188 sets forth the amino acid sequence of the core motif of tau in MBTR 1 that is bound by 3D6.

[0302] SEQ ID NO: 189 sets forth the amino acid sequence from the N-terminus of the tau sequence to the core motif of tau in MBTR 1 bound by 3D6.

[0303] SEQ ID NO: 190 sets forth the amino acid sequence from the C-terminus of the tau sequence to the core motif of tau in MBTR 1 bound by 3D6.

[0304] SEQ ID NO: 191 lists the amino acid sequence of the 3D6 epitope.

[0305] SEQ ID NO: 192 sets forth the amino acid sequence of the core motif of tau in MBTR 2 that is bound by 3D6.

[0306] SEQ ID NO: 193 sets forth the amino acid sequence of the core motif of tau in MBTR 3 that is bound by 3D6.

[0307] SEQ ID NO: 194 sets forth the amino acid sequence of the core motif of tau in MBTR 3 bound by 4D6.

[0308] definition

[0309] Monoclonal antibodies or other biological entities are usually provided in isolated form. This means that the antibody or other biological entity is usually at least 50% w / w free of interfering proteins and other contaminants produced by its production or purification, but does not exclude the possibility of combining the monoclonal antibody with an excess of pharmaceutically acceptable carriers or other vehicles intended to facilitate its use. Sometimes the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95% or 99% w / w free of interfering proteins and contaminants produced by its production or purification. Usually, the isolated monoclonal antibody or other biological entity is the main macromolecular species remaining after its purification.

[0310] Specific binding of an antibody to its target antigen means that at least 10 6 , 10 7 , 10 8, 10 9 , 10 10 , 10 11 or 10 12 M -1 The magnitude of specific binding can be detectably higher and can be distinguished from nonspecific binding to at least one unrelated target. Specific binding can be the result of a bond or a specific spatial fit (e.g., lock-and-key type) between specific functional groups, while nonspecific binding is typically the result of van der Waals forces. However, specific binding does not necessarily mean that the antibody binds only one target.

[0311] The basic antibody structural unit is a tetramer of subunits. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. This variable region was originally expressed as being connected to a cleavable signal peptide. The variable region without a signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without a light chain signal peptide. The carboxyl terminal portion of each chain defines a constant region that is primarily responsible for effector function.

[0312] Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and define the isotype of antibodies as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Fundamental Immunology, Paul, W., ed., 2nd ed., Raven Press, NY, 1989, Chapter 7 (incorporated by reference in its entirety for all purposes).

[0313] The immunoglobulin light chain 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 region is used to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively; CDRs 1, 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 with R as the C-terminal residue, R can be considered as the N-terminal residue of the light chain constant region instead. Therefore, the application should also be understood to disclose a VL sequence without a C-terminal R.

[0314] The assignment of amino acids to each VL and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the complex of the Chothia Kabat CDR, in which CDR-H1 is a complex of the Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the Contact definition of Martin et al. (bioinfo.org.uk / abs) (see Table 1). Kabat provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. When an antibody is said to contain a CDR by a certain definition of CDR (e.g., Kabat), the definition specifies the CDR residues present in the antibody (i.e., the Kabat residues). The minimum number of CDRs is defined as defined in the Kabat CDR definition. This does not exclude the simultaneous presence of other residues that fall within another conventional CDR definition but are outside the specified definition. For example, antibodies comprising CDRs defined by Kabat include, among other possibilities, antibodies in which the CDRs contain Kabat CDR residues and no other CDR residues, and antibodies in which CDR H1 is the complex Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues and no additional CDR residues based on other definitions.

[0315] Table 1 Conventional definition of CDRs using Kabat numbering

[0316]

[0317] *CDR-H1 according to Chothia can end at H32, H33 or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places the insertion of additional residues at 35A and 35B, while Chothia numbering places them at 31A and 31B. If neither H35A nor H35B are present (Kabat numbering), the Chothia CDR-H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0318] The term "antibody" includes complete antibodies and binding fragments thereof. Typically, fragments compete with the complete antibodies from which they are derived for specific binding to the target, including individual heavy chains, light chain Fabs, Fab', F(ab')2, F(ab)c, Dab, nanobodies, and Fv. Fragments can be produced by recombinant DNA technology, or by enzymatic or chemical separation of complete immunoglobulins. The term "antibody" also includes bispecific antibodies and / or humanized antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies with 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 chain / light chain pairs include a humanized 3D6 heavy chain / light chain pair and a heavy chain / light chain pair specific for an epitope on tau that is different from that bound by 3D6.

[0319] In some bispecific antibodies, one heavy chain / light chain pair is a humanized 3D6 antibody as further disclosed below, and the other heavy chain / light chain pair is from an antibody that binds to a receptor expressed on the blood-brain barrier, such as the insulin receptor, insulin-like growth factor (IGF) receptor, leptin receptor, or lipoprotein receptor, or transferrin receptor (Friden et al., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such bispecific antibodies can be transferred across the blood-brain barrier by receptor-mediated transcytosis. Brain uptake of bispecific antibodies can be further enhanced by engineering bispecific 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).

[0320] Exemplary bispecific antibodies may also be: (1) dual variable domain antibodies (DVD-Ig), in which each light chain and heavy chain contains two variable domains linked in series via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, in: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) Tandab, which is a fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (3) flexibody, which is a combination of scFv and diabody, resulting in a multivalent molecule; (4) so-called "dock and lock" molecules, which are based on the "dimerization and docking domain" in protein kinase A, and when applied to Fab, can result in a trivalent bispecific binding protein consisting of two identical Fab fragments connected to different Fab fragments; or (5) so-called Scorpion molecules, which contain, for example, two scFvs fused to the two ends of a human Fc region. Examples of platforms that can be used to prepare bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

[0321] The term "epitope" refers to a site on an antigen that binds to an antibody. An epitope can be formed by continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are usually lost when treated with denaturing solvents. An epitope typically includes at least 3 and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0322] Antibodies that recognize the same or overlapping epitopes can be identified in simple immunoassays that show the ability of one antibody to compete with another antibody for binding to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography of the antibody in conjunction with its antigen to identify contact residues. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have overlapping epitopes.

[0323] Competition between antibodies is determined by an assay in which the test antibody inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits at least 50% of the binding of the reference antibody (as measured in a competitive binding assay). Some test antibodies inhibit at least 75%, 90%, or 99% of the binding of the reference antibody. Antibodies identified by competition 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 to produce steric hindrance.

[0324] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0325] The term "patient" includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0326] An individual is at increased risk for a disease if the subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure) that places the individual with the risk factor at a statistically significantly greater risk of developing the disease compared to individuals without the risk factor.

[0327] The term "biological sample" refers to a sample of biological material in or obtainable from a biological source (e.g., a human or mammalian subject). Such samples can be organs, organelles, tissues, tissue sections, body fluids, peripheral blood, plasma, serum, cells, molecules such as proteins and peptides, and any part or combination derived therefrom. The term biological sample may also include any material derived by processing the sample. Derived materials may include cells or their progeny. Processing of biological samples may include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, etc.

[0328] The term "control sample" refers to a biological sample that is not known or suspected to include a tau-related disease-affected area, or at least a biological sample that is not known or suspected to include a diseased area of ​​a given type. The control sample can be obtained from an individual who has never suffered from a tau-related disease. Alternatively, the control sample can be obtained from a patient with a tau-related disease. Such samples can be obtained simultaneously with biological samples that are believed to contain tau-related diseases or obtained on different occasions. Both biological samples and control samples can be obtained from the same tissue. Preferably, the control sample is substantially or completely composed of normal healthy areas and can be used for comparison with biological samples that are believed to contain tau-related disease-affected areas. Preferably, the tissue in the control sample is the same type as the tissue in the biological sample. Preferably, it is believed that the tau-related disease-affected cells in the biological sample are from the same cell type (e.g., neurons or glial cells) as the cell type in the control sample.

[0329] The term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, ailment, abnormality, pathology, discomfort, condition or syndrome in which physiological function is impaired, regardless of the nature of the cause.

[0330] The term "symptom" refers to subjective evidence of disease perceived by the subject, such as a change in gait. "Sign" refers to objective evidence of disease observed by a physician.

[0331] The term "positive response to treatment" refers to a more favorable response in an individual patient or the average response in a population of patients relative to the average response in a control population not receiving the treatment.

[0332] To classify amino acid substitutions as conservative or non-conservative, the amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions include substitutions between amino acids of the same class. Non-conservative substitutions include exchanging a member of one of these classes for a member of another class.

[0333] The percentage of sequence identity is determined using antibody sequences that are aligned to the maximum extent possible using the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the subject antibody region and the reference antibody region is the number of positions occupied by the same amino acid in the subject antibody region and the reference antibody region divided by the total number of aligned positions of the two regions, wherein gaps are not counted, multiplied by 100 to convert to a percentage.

[0334] A composition or method that "comprising" or "includes" one or more of the recited elements may include other elements not specifically recited. For example, a composition that "comprising" or "includes" an antibody may contain the antibody alone or in combination with other ingredients. When the disclosure is directed to features that include specified elements, the disclosure should alternatively be understood to be directed to features that consist essentially of or consist of the specified elements.

[0335] The specification of a range of values ​​includes all integers within or defining the range, and all sub-ranges defined by integers within the range.

[0336] Unless otherwise apparent from the context, the term "about" includes insubstantial variations, such as values ​​within the standard tolerance of measurement error (eg, SEM) of the specified value.

[0337] Statistical significance means p≤0.05.

[0338] Unless the context clearly dictates otherwise, the singular forms of the articles "a," "an," and "the" include plural referents. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. DETAILED DESCRIPTION

[0339] I. Summary

[0340] The present invention provides antibodies that bind to tau. Some antibodies specifically bind to one or more epitopes within the microtubule binding region (MTBR) of human tau, which is defined by residues approximately 244 to 372 of SEQ ID NO:1. Since the MTBR region contains sequence repeats, a single antibody can bind to multiple repeat sites within the region. Some antibodies specifically bind to the epitope of 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. In some antibodies, the binding to the MTBR 4 region at residues 353-362 is weaker than the binding to MTBR 1, MTBR 2, and MTBR 3. Some antibodies bind to tau, regardless of whether it is phosphorylated. Some antibodies of the present invention are used to inhibit or delay the pathology associated with tau and the associated symptom deterioration. Although the practice of the present invention does not require an understanding of the mechanism, due to the antibody-induced phagocytosis of tau, inhibition of intermolecular or intramolecular aggregation of tau or binding to other molecules, a reduction in toxicity may occur by stabilizing a non-toxic conformation, by inhibiting the intercellular or intracellular spread of pathogenic tau forms, by blocking tau phosphorylation, by preventing tau from binding to cells, or by inducing proteolytic cleavage of tau. The antibodies of the present invention or agents that induce such antibodies can be used to treat Alzheimer's disease and other diseases associated with tau or to achieve methods for preventing them.

[0341] II. Target molecules

[0342] Unless otherwise apparent from the context, reference to tau means the natural human form of tau, including all isoforms, regardless of whether there is post-translational modification (e.g., phosphorylation, glycosylation or acetylation). There are six major tau isoforms (splice variants) in the human brain. The longest of these variants has 441 amino acids, in which the initial met residue is cut. Residues are numbered according to the 441 isoforms. Therefore, for example, reference to phosphorylation at position 404 means position 404 of the 441 isoform, or the corresponding position of any other isoform when aligned to the 441 isoform to the maximum extent. The amino acid sequence and Swiss-Prot numbering of the isoforms are as indicated below.

[0343]

[0344]

[0345] References to tau include known natural variants, approximately 30 of which are listed in the Swiss-Prot database and permutations thereof, as well as mutations associated with tau pathologies such as dementia, Pick's disease, supranuclear palsy, etc. (see, e.g., the Swiss-Prot database and Poorkaj et al., Ann Neurol. 43:815-825 (1998)). Some examples of tau mutations numbered by 441 isoform are lysine to threonine mutation at amino acid residue 257 (K257T), isoleucine to valine mutation at amino acid position 260 (I260V); glycine to valine mutation at amino acid position 272 (G272V); asparagine to lysine mutation at amino acid position 279 (N279K); asparagine to histidine mutation at amino acid position 296 (N296H); proline to serine mutation at amino acid position 301 (P301S); proline to leucine mutation at amino acid position 301 (P301L); glycine to valine mutation at amino acid position 303 (G303V); serine to asparagine mutation at position 305 (S305N); glycine to serine mutation at amino acid position 335 (G335S); valine to methionine mutation at position 337 (V337M); glutamate to valine mutation at position 342 (E342V); lysine to isoleucine mutation at amino acid position 369 (K3691); glycine to arginine mutation at amino acid position 389 (G389R); and arginine to tryptophan mutation at amino acid position 406 (R406W).

[0346] Tau can 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 apparent from the context, reference to tau or a fragment thereof includes the native human amino acid sequence, including isoforms, mutants, and allelic variants thereof.

[0347] III. Antibodies

[0348] A. Binding Specificity and Functional Properties

[0349] The present invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within KXXSXXNX(K / H)H(SEQ ID NO:191). Some antibodies bind to a peptide comprising amino acid residues 259-268 of a tau protein (SEQ ID NO:1) containing 441 amino acids, consisting essentially of these amino acid residues, or consisting of these amino acid residues. Some antibodies bind to a peptide comprising amino acid residues 290-299 of a tau protein (SEQ ID NO:1) containing 441 amino acids, consisting essentially of these amino acid residues, or consisting of these amino acid residues. Some antibodies bind to a peptide comprising amino acid residues 321-330 of a tau protein (SEQ ID NO:1) containing 441 amino acids, consisting essentially of these amino acid residues, or consisting of these amino acid residues. Some antibodies bind to a peptide comprising amino acid residues 353-362 of a tau protein (SEQ ID NO:1) containing 441 amino acids, consisting essentially of these amino acid residues, or consisting of these amino acid residues. Some antibodies bind to two, three or all four of these peptides. Some antibodies specifically bind to an epitope within residues 199-213 (corresponding to residues 257-271 of SEQ ID NO:1) of 4RON human tau protein (SEQ ID NO:3) containing 383 amino acids. Some antibodies specifically bind to an epitope within residues 262-276 (corresponding to residues 320-334 of SEQID NO:1) of 4RON human tau protein (SEQ ID NO:3) containing 383 amino acids. Some antibodies of the present invention specifically bind to a peptide consisting of residues 257-271 of a tau protein (SEQ ID NO:1) containing 441 amino acids. Some antibodies of the present invention specifically bind to a peptide consisting of residues 320-334 of a tau protein (SEQ ID NO:1) containing 441 amino acids. Some antibodies of the present invention specifically bind to a peptide consisting of residues 259-268 of tau protein SEQ ID NO: 1 containing 441 amino acids (i.e., KIGSTENLKH (SEQ ID NO: 188)). Some antibodies of the present invention specifically bind to a peptide consisting of residues 290-299 of tau protein SEQ ID NO: 1 containing 441 amino acids (i.e., KCGSKDNIKH (SEQ ID NO: 192)). Some antibodies of the present invention specifically bind to a peptide consisting of residues 321-330 of tau protein SEQ ID NO: 1 containing 441 amino acids (i.e., KCGSLGNIHH (SEQ ID NO: 193)).Some antibodies of the present invention specifically bind to a peptide consisting of residues 353-362 (i.e., KIGSLDNITH (SEQ ID NO: 194)) of a tau protein SEQ ID NO: 1 containing 441 amino acids. Some antibodies of the present invention specifically bind to a peptide consisting of a consensus motif KXXSXXNX(K / H)H (SEQ ID NO: 191). Some antibodies bind to an epitope comprising 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 a tau protein SEQ ID NO: 1 containing 441 amino acids. Some antibodies bind to tau regardless of the phosphorylation state. Some antibodies bind to an epitope that does not include a residue that is subject to phosphorylation. These antibodies can be obtained by immunizing with a tau polypeptide purified from a natural source or recombinantly expressed. Antibodies can be screened for binding to tau in a non-phosphorylated form as well as in a form in which one or more residues susceptible to phosphorylation are phosphorylated. Such antibodies preferably bind phosphorylated tau with an indistinguishable affinity or at least within a factor of 1.5, 2, or 3 times greater than non-phosphorylated tau (i.e., are pan-specific). 3D6 is an example of a pan-specific monoclonal antibody. The present invention also provides antibodies that bind to the same epitope as any of the aforementioned antibodies, such as an epitope of 3D6. Also included are antibodies that compete with any of the aforementioned antibodies for binding to tau, such as competing with 3D6.

[0350] The above-mentioned antibodies can be generated de novo by immunization 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 immunization 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 immunization with a full-length tau polypeptide or fragment thereof comprising such residues and screening for specific binding to a peptide comprising such residues. Such peptides are preferably attached to a heteroconjugate molecule that facilitates eliciting an antibody response to the peptide. Attachment can be direct or via a spacer peptide or amino acid. Cysteine ​​is used as a spacer amino acid because its free SH group facilitates attachment of a carrier molecule. Polyglycine linkers (e.g., 2 to 6 glycines) with or without cysteine ​​residues between the glycine and the peptide can also be used. This carrier molecule is used to provide T cell epitopes that help elicit an antibody response against the peptide. Several carriers are commonly used, specifically keyhole limpet hemocyanin (KLH), ovalbumin, and bovine serum albumin (BSA). Peptide spacers can be added to the peptide immunogen as part of solid phase peptide synthesis. Carriers are typically added by chemical cross-linking. Some examples of chemical crosslinkers that can be used include crosslinking-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 2002 / 05166). 98 / 23635; and Southwood et al., J. Immunology, 160:3363-3373 (1998). If present, the carrier and spacer can be attached to either end of the immunogen.

[0351] Peptides with optional spacers and carriers can be used to immunize laboratory animals or B cells, as described in more detail below. The ability of hybridoma supernatants to bind to phosphorylated forms and non-phosphorylated forms of one or more peptides and / or tau (such as full-length isoforms of tau with position 404 in phosphorylated form) can be tested, the peptides include 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), are essentially composed of or are composed of these residues, or include residues 259-268, 290-299, 321-330 or 353-362 of SEQ ID NO:1, are essentially composed of or are composed of these residues. The peptides can be attached to carriers or other tags to facilitate screening assays. In this case, the carrier or tag is preferentially different from the combination of spacers and carrier molecules used for immunity to eliminate antibodies that are specific to the spacer or carrier rather than the tau peptide. Any tau isotype can be used.

[0352] The present invention provides monoclonal antibodies that bind to epitopes within tau. The antibody named 3D6 is one such exemplary mouse antibody. Unless otherwise apparent from the context, reference to 3D6 should be understood to refer to any of the mouse, chimeric, facing and humanized forms of the antibody. The antibody has been deposited with [Accession Number]. The antibody specifically binds to the epitope of KXXSXXNX(K / H)H (SEQ ID NO: 191). The antibody specifically binds to amino acid residues 199-213 and / or 262-276 (corresponding to amino acid residues 257-271 and / or 320-334 of SEQ ID NO: 1) of the 4RON human tau protein (SEQ ID NO: 3) containing 383 amino acids. The antibody specifically binds within amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO: 1, and any combination of 2, 3 or all 4 thereof. The antibody is further characterized by its ability to bind phosphorylated and non-phosphorylated tau, non-pathological and pathological forms and conformations of tau, and misfolded / aggregated forms of tau. The antibody designated 6A10 is one such exemplary mouse antibody. Unless otherwise apparent from the context, reference to 6A10 should be understood to refer to any of the mouse, chimeric, veneered and humanized forms of the antibody. The Kabat / Chothia complex CDRs of the heavy chain of 6A10 are designated as SEQ ID NOs: 67, 68 and 69, respectively, and the Kabat CDRs of the light chain of 6A10 are designated as SEQ ID NOs: 12, 13 and 14, respectively. Mouse 6A10 has 82.1% VH sequence identity and 100% VL sequence identity with the VH chain and VL chain of mouse 3D6, respectively.

[0353] Some antibodies of the present invention bind to the same or overlapping epitopes as the antibody designated as 3D6. The sequences of the heavy chain mature variable region and the light chain mature variable region of the antibody are designated as SEQ ID NO:7 and SEQ ID NO:11, respectively. Other antibodies with such binding specificity can be produced by immunizing mice with tau or a portion thereof, wherein the tau or a portion thereof comprises a desired epitope (e.g., 199-213 and / or 262-276 of SEQ ID NO:3, corresponding to residues 257-271 and / or 320-334 of SEQ ID NO:1, respectively; or, for example, 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1, a combination of 2, 3 or all 4 thereof), consists essentially of the desired epitope or consists of the desired epitope, and screening the resulting antibodies to obtain antibodies that compete for binding to tau, optionally with antibodies having a variable region of mouse 3D6 (IgG1κ). The tau fragments including the desired epitopes can be connected to a carrier that helps to induce an antibody response to the fragment and / or combined with an adjuvant that helps to induce such a response. Such antibodies can be screened for the difference in binding to tau or its fragments compared to the mutants of the specified residues. The screening for such mutants more accurately defines the binding specificity to allow identification of antibodies whose binding is inhibited by the mutagenesis of specific residues and may share the functional properties of other exemplified antibodies. The mutation can be a systematic replacement substitution with alanine (or serine, if alanine already exists) in the entire target or in the entire segment of the target where the epitope is known to exist, one residue at a time, or spaced apart at a wider spacing. If the same set of mutations significantly reduces the binding of two antibodies, then the two antibodies bind to the same epitope.

[0354] Antibodies having the binding specificity of a selected murine antibody (e.g., 3D6) can also be produced using a variation of the phage display method. See Winter, WO 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, the heavy chain variable region or the light chain variable region of the 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 library is constructed in which the members display the same light chain variable region (i.e., the murine starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Select a phage library that is specific for tau or a fragment thereof (e.g., at least 10 8 , preferably at least 10 9 M -1) shows a phage with strong specific binding. Then, the heavy chain variable region from the phage is used as the starting material for constructing another phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and different light chain variable regions. The light chain variable region can be obtained, for example, from a library of rearranged human variable light chain regions. Once again, a phage that shows strong specific binding to tau or its fragments is selected. The resulting antibodies generally have the same or similar epitope specificity as the mouse starting material.

[0355] The Kabat / Chothia complex CDRs of the heavy chain of 3D6 are designated as SEQ ID NOs: 8, 9, and 10, respectively, and the Kabat CDRs of the light chain of 3D6 are designated as SEQ ID NOs: 12, 13, and 14, respectively.

[0356] Table 2 indicates the 3D6 CDRs as defined by Kabat, Chothia, the complex of Chothia and Kabat (also referred to herein as the "Kabat / Chothia complex"), AbM, and Contact.

[0357] Table 2: 3D6 CDRs as defined by Kabat, Chothia, a complex of Chothia and Kabat, AbM and Contact

[0358]

[0359] Table 2: 3D6 CDRs as defined by Kabat, Chothia, a complex of Chothia and Kabat, AbM and Contact

[0360]

[0361] Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of exemplary antibodies (such as 3D6). Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to 3D6 in the amino acid sequence of the mature heavy chain variable region and / or the mature light chain variable region and maintain their functional properties, and / or differ from the corresponding antibody by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions or insertions are also included in the present invention. Also included are monoclonal antibodies having at least one or all six CDRs, the CDRs being as defined by any conventional definition, but preferably Kabat, which have 90%, 95%, 99% or 100% identity to the corresponding CDRs of 3D6.

[0362] The present invention also provides antibodies having some or all (e.g., 3, 4, 5, and 6) CDRs that are completely or substantially derived from 3D6. Such antibodies may include a heavy chain variable region having at least two and typically all three CDRs that are completely or substantially derived from the heavy chain variable region of 3D6, and / or a light chain variable region having at least two and typically all three CDRs that are completely or substantially derived from the light chain variable region of 3D6. The antibody may include a heavy chain and a light chain. When a CDR contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, the CDR is substantially derived from the corresponding 3D6 CDR, except that CDR-H2 (when defined by Kabat) may have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies may be at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to 3D6 in the amino acid sequence of the mature heavy and / or light chain variable regions and maintain their functional properties, and / or differ from 3D6 by minor functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions or insertions.

[0363] Some antibodies identified by such assays can bind to monomeric, misfolded, aggregated, phosphorylated or non-phosphorylated forms or other forms of tau. Likewise, some antibodies are immunoreactive to non-pathological and pathological forms and conformations of tau.

[0364] B. Non-human Antibodies

[0365] The production of other non-human antibodies (e.g., mice, guinea pigs, primates, rabbits or rats) against tau or fragments thereof (e.g., amino acid residues 199-213 or 262-276 of SEQ ID NO:3, corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively; or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1) can be achieved, for example, by immunizing animals with tau or fragments thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such immunogens can be obtained from natural sources, by peptide synthesis or by recombinant expression. Optionally, the immunogen can be fused to a carrier protein or administered in combination in other ways. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used as described below. For the immunization of laboratory animals, complete Freund's adjuvant is preferred, followed by incomplete adjuvant. Rabbits or guinea pigs are generally used to prepare polyclonal antibodies. Mice are generally used to prepare monoclonal antibodies. Antibodies are screened for specific binding to tau or tau epitopes (e.g., epitopes comprising one or more residues in amino acid residues 199-213 or 262-276 of SEQ ID NO:3, which residues correspond to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively; or epitopes comprising one or more residues in amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1). Such screening can be achieved by determining the binding of the antibody to a collection of tau variants, such as a tau variant containing amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively), or a tau variant containing amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1, or a tau variant with 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.

[0366] C. Humanized Antibodies

[0367] Humanized antibodies are genetically engineered antibodies in which CDRs from non-human "donor" antibodies are grafted into human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex 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 CDRs that are completely or substantially derived from a donor antibody, as well as variable region framework sequences and constant regions (if present) that are completely or substantially derived from human antibody sequences. Similarly, humanized heavy chains have at least one, two and usually all three CDRs from the heavy chain of a donor antibody, and heavy chain variable region framework sequences and heavy chain constant regions (if present) substantially from human heavy chain variable region frameworks and constant region sequences. Similarly, humanized light chains have at least one, two and usually all three CDRs from the light chain of a donor antibody, and light chain variable region framework sequences and light chain constant regions (if present) substantially from human light chain variable region frameworks and constant region sequences. In addition to nano antibodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. When at least 85%, 90%, 95% or 100% of the corresponding residues (as defined by any conventional definition, but preferably defined by Kabat) are identical between the corresponding CDRs, the CDRs in the humanized antibodies are substantially from the corresponding CDRs in non-human antibodies. When at least 85%, 90%, 95% or 100% of the corresponding residues defined by Kabat are identical, the variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively. In order to be classified as humanized according to the definition of humanized antibodies by the World Health Organization (WHO) International Nonproprietary Name (INN) in 2014, the antibody must have at least 85% identity with the human germline antibody sequence (i.e., before somatic hypermutation). A hybrid antibody is an antibody in which one antibody chain (e.g., heavy chain) meets the threshold but the other chain (e.g., light chain) does not meet the threshold. If neither chain meets the threshold, the antibody is classified as a chimera, even if the variable framework regions of the two chains are substantially human, with some mouse back mutations. See Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1, 1-9, DOI: 10.1080 / 19420862.2015.1114320.See also " WHO-INN:International nonproprietary names (INN) for biological and biotechnological substances (areview) " (Internet) 2014. Available from: http: / / www.who.int / medicines / services / inn / BioRev2014.pdf), which is incorporated herein by reference. For the avoidance of doubt, the term "humanization" as used herein is not intended to limit the 2014 WHO INN definition of humanized antibodies. Some humanized antibodies provided herein have at least 85% sequence identity with human germline sequences, and some humanized antibodies provided herein have less than 85% sequence identity with human germline sequences. Some heavy chains of humanized antibodies provided herein have about 60% to 100% sequence identity with human germline sequences, for example, in the range of about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89%. Some heavy chains do not meet the 2014 WHO INN definition and have, for example, 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 with human germline sequences, while other heavy chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or higher sequence identity with human germline sequences. Some light chains of the humanized antibodies provided herein have about 60% to 100% sequence identity with human germline sequences, for example, in the range of about 80% to 84%, or 85% to 89%. Some light chains do not meet the 2014 WHO INN definition and have, for example, about 81%, 82%, 83%, or 84% sequence identity with human germline sequences, while other light chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or higher sequence identity with human germline sequences. Some humanized antibodies provided herein as "chimeric" according to the 2014 WHO INN definition have a heavy chain with less than 85% identity to human germline sequences, and the heavy chain and the light chain with less than 85% identity to human germline sequences are paired. Some humanized antibodies provided herein as "mixed" according to the 2014 WHO INN definition, for example, have a heavy chain with at least 85% sequence identity to human germline sequences, and the heavy chain and the light chain with less than 85% sequence identity to human germline sequences are paired, 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% sequence identity to a human germline sequence, paired with a light chain with at least 85% sequence identity to a human germline sequence. Additional humanized antibodies of the present invention meet the 2014 WHO INN definition of "hybrid".

[0368] Although humanized antibodies typically incorporate all six CDRs from a mouse antibody (as defined by any conventional definition, but preferably by the Kabat definition), they can also be made with less than all CDRs from a mouse antibody (e.g., at least 3, 4, or 5 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).

[0369] In some antibodies, only a subset of CDR residues required for binding, referred to as SDR, is required to remain bound in humanized antibodies. Can be based on previous studies (e.g., residues H60-H65 in CDR H2 are generally not required) from the Kabat CDR region (Chothia, J.Mol.Biol.196:901, 1987) outside the Chothia hypervariable loop, by molecular modeling and / or empirically, or as Gonzales et al., Mol.Immunol.41:863, 2004, identifying CDR residues that do not contact antigen and are not in SDR. In such humanized antibodies, at the position where one or more donor CDR residues do not exist or omit the entire donor CDR, the amino acid occupying the position can be the amino acid occupying the corresponding position (by Kabat numbering) in the receptor antibody sequence. The number of such substitutions of the acceptor to the donor amino acid included in the CDR reflects the balance of competitive considerations. Such substitutions may help reduce the number of mouse amino acids in the humanized antibody, and therefore reduce potential immunogenicity and / or meet the WHO INN definition of "humanization". However, substitutions may also cause changes in affinity, and preferably avoid a significant reduction in affinity. The position for substitution in the CDR and the amino acid for substitution may also be selected empirically.

[0370] The human acceptor antibody sequence can optionally be selected from a number of known human antibody sequences to provide a high degree of sequence identity (eg, 65% to 85% identity) between the human acceptor sequence variable region framework and the corresponding variable region framework of the donor antibody chain.

[0371] Some humanized antibodies and chimeric antibodies have the same (within experimental error) or improved functional properties, such as binding affinity for human tau, inhibition of tau internalization into neurons, as described in the Examples, as the mouse antibodies from which they are derived. For example, some humanized antibodies and chimeric antibodies have binding affinities within 3-fold, 2-fold, or 1-fold of the mouse antibodies from which they are derived, or affinities that are indistinguishable within experimental error. Some humanized antibodies and chimeric antibodies inhibit tau internalization into neurons, as described in the Examples, at levels within 3-fold, 2-fold, or 1-fold of the mouse antibodies from which they are derived, or at the same levels as the mouse antibodies from which they are derived within experimental error. Compared to previously described humanized forms of the 3D6 antibody (see WO 2017 / 191560), some humanized antibodies exhibit reduced immunogenicity, increased affinity, increased thermal stability and / or improved expression hu3D6VHv1bA11 / L2-DIM4 exhibits improved affinity compared to the parental hu3D6VHv1bA11 / hu3D6VLv2, as demonstrated by on-rate, off-rate, and Kd numbers. hu3D6VHv1bA11 / L2-DIM4 exhibits higher thermal stability and titer than the parental hu3D6VHv1bA11 / hu3D6VLv2.

[0372] An example of a heavy chain acceptor sequence is the human mature heavy chain variable region of humanized 48G7 Fab, PDB accession code 2RCS-VH_huFrwk (SEQ ID NO: 75). The variable domains of 3D6 and 48G7 Fab also share the same length on the CDR-H1, H2 loops. An example of a heavy chain acceptor sequence is the human mature heavy chain variable region IMGT#IGHV1-69-2*01 (SEQ ID NO: 25). IMGT#IGHV1-69-2*01 (SEQ ID NO: 25) shares the canonical form of mouse 3D6 heavy chain CDR-H1 and H2. IMGT#IGHV1-69-2*01 (SEQ ID NO: 25) belongs to human heavy chain subgroup 1. An example of an acceptor sequence for a light chain is the human mature light chain variable region with human antibody PDB accession code ARX71335 VL (SEQ ID NO: 82). The variable light domains of 3D6 and ARX71335 antibodies also share the same length on the CDR-L1, L2 and L3 loops. An example of a light chain acceptor sequence is a human mature light chain variable region with IMGT#IGKV2-30*02 (SEQ ID NO: 27). IMGT#IGKV2-30*02 (SEQ ID NO: 27) has the same canonical classification of CDR-L1, CDR-L2 and L3 as mouse 3D6. IMGT#IGKV2-30*02 (SEQ ID NO: 27) belongs to human kappa subgroup 2.

[0373] If more than one human receptor antibody sequence is selected, a complex or hybrid of those receptors can be used, and the amino acids used at different positions in the humanized light chain and heavy chain variable regions can be taken from any human receptor antibody sequence used. For example, the human mature heavy chain variable region of IMGT#IGHV1-69-2*01 (SEQ ID NO: 25) and PDB accession code #2RCS-VH_huFrwk (SEQ ID NO: 75) are used as the humanized receptor sequence for the 3D6 mature heavy chain variable region. An example of a position where these two receptors differ is position H17 (T or S). The humanized version of the 3D6 heavy chain variable region can include any amino acid at this position. For example, the human mature light chain variable region of IMGT#IGKV2-30*02 (SEQ ID NO: 27) and PDB code #ARX71335-VL_huFrwk (SEQ ID NO: 82) are used as the humanized receptor sequence for the 3D6 mature light chain variable region. An example of a position where the two receptors differ is position L100 (Q or A). Humanized versions of the 3D6 light chain variable region can include either amino acid at this position.

[0374] Certain amino acids from human variable region framework residues can be selected for substitution based on their possible effects on CDR conformation and / or binding to antigen. Studies of such possible effects are achieved by modeling, examining the characteristics of the amino acids at specific positions, or empirically observing the effects of specific amino acid substitution or mutagenesis.

[0375] For example, when there is an amino acid difference between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid may be substituted with the equivalent framework amino acid from a mouse antibody when it is reasonable to expect that the amino acid has the following characteristics:

[0376] (1) Direct non-covalent binding to antigens;

[0377] (2) adjacent to a CDR region or within a CDR defined by Chothia rather than Kabat;

[0378] (3) interact with the CDR region in other ways (e.g., (e.g., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain); or

[0379] (4) are the residues involved in the VL-VH interface.

[0380] In one embodiment, the humanized sequence is generated using a two-stage PCR protocol that allows the introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682)].

[0381] Framework residues from categories (1) to (3) defined by Queen, US 5,530,101 are sometimes alternatively referred to as canonical residues and vernier residues. Framework residues that contribute to the conformation of the CDR loop are sometimes referred to as canonical residues (Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Thornton & Martin, J. Mol. Biol. 263: 800-815 (1996)). Framework residues that support the conformation of the antigen binding loop and play a role in finely tuning the coordination of the antibody with the antigen are sometimes referred to as vernier residues (Foote & Winter, J. Mol. Biol 224: 487-499 (1992)).

[0382] Other framework residues that are candidates for substitution are residues that create potential glycosylation sites. Other substitution candidates are acceptor human framework amino acids that are uncommon for human immunoglobulins at this position. These amino acids can be substituted with amino acids from the equivalent positions of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins.

[0383] Other framework residues that are candidates for substitution are the N-terminal glutamine residue (Q), which can be replaced with glutamic acid (E) to minimize the possibility of pyroglutamic acid conversion [Y. Diana Liu et al., 2011, J. Biol. Chem., 286: 11211-11217]. The conversion of glutamic acid (E) to pyroglutamic acid (pE) occurs more slowly than from glutamine (Q). Due to the loss of primary amines in the conversion of glutamine to pE, the antibody becomes more acidic. Incomplete conversion produces heterogeneity of the antibody, which can be observed as multiple peaks using charge-based analytical methods. Differences in heterogeneity may indicate a lack of process control.

[0384] An exemplary humanized antibody is a humanized form of mouse 3D6, designated Hu3D6.

[0385] The mouse antibody 3D6 includes a mature heavy chain variable region and a mature light chain variable region, which have amino acid sequences including SEQ ID NO: 7 and SEQ ID NO: 11, respectively. The present invention provides a humanized form of the murine 3D6 antibody, which includes 10 exemplified humanized heavy chain mature variable regions (hu3D6VHvb1 (SEQ ID NO: 76), hu3D6VHvb2 (SEQ ID NO: 77), hu3D6VHvb3 (SEQ ID NO: 78), hu3D6VHvb4 (SEQ ID NO: 79), hu3D6VHvb5 (SEQ ID NO: 80), hu3D6VHvb6 (SEQ ID NO: 90), hu3D6VHvb7 (SEQ ID NO: 91), hu3D6VHv1bA11 D60E (h3D6VHvb8, SEQ ID NO: 146), hu3D6VHv1bA11 L82cV (SEQ ID NO: 147), hu3D6VHv1bA11 L92cV (SEQ ID NO: 148), hu3D6VHv1bA11 L10cV (SEQ ID NO: 149), hu3D6VHv1bA11 L20dV (SEQ ID NO: 150), hu3D6VHv1bA11 L30dV (SEQ ID NO: 151), hu3D6VHv1bA11 L40dV (SEQ ID NO: 153), hu3D6VHv1bA11 L50dV (SEQ ID NO: 154), hu3D6VHv1bA11 L60dV (SEQ ID NO: 155), hu3D6VHv1bA11 L70dV (SEQ ID NO: 156), hu3D6VH NO: 147) and hu3D6VHv1bA11D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148)), and 56 exemplified humanized light chain mature variable regions (hu3D6VLvb1 (SEQ ID NO: 83), hu3D6VLvb2 (SEQ ID NO: 84), hu3D6VLvb3 (SEQ ID NO: 85), hu3D6VLv2L54D (SEQ ID NO: 93), hu3D6VLv2 L54G (SEQ ID NO: 94), hu3D6VLv2L54N (SEQ ID NO: 95), hu3D6VLv2 L54E (SEQ ID NO: 96), hu3D6VLv2L50E (SEQ ID NO: 97), hu3D6VLv2 L54Q (SEQ ID NO: 98), ID NO:98), hu3D6VLv2L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO:100), hu3D6VLv2L54R (SEQ ID NO:101), hu3D6VLv2 L54T (SEQ ID NO:102), hu3D6VLv2L50G (SEQ ID NO:102) NO:103), hu3D6VLv2 I48G (SEQ ID NO:104), hu3D6VLv2I48D (SEQ ID NO:105), hu3D6VLv2 L47G (SEQ ID NO:106), hu3D6VLv2Y49E (SEQ ID NO:107), hu3D6VLv2 L54V (SEQ ID NO:108), hu3D6VLv2L54S (SEQ IDNO:109)、hu3D6VLv2 S52G(SEQ ID NO:110)、hu3D6VLv2L47N(SEQ ID NO:111)、hu3D6VLv2 L47D(SEQID 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 IDNO:119)、hu3D6VLv2L37Q_L50G_L54G(SEQ ID NO:120)、hu3D6VLv2 L37Q_S52G_L54G(SEQID NO:121)、hu3D6VLv2 L37Q_S52G_L54R(SEQ ID NO:122)、hu3D6VLv2 L37Q_S52G_L54T(SEQ ID NO:123)、hu3D6VLv2L37Q_S52G_L54D(SEQ ID NO:124)、hu3D6VLv2 L37Q_L54R(SEQ ID NO:125)、hu3D6VLv2 L37Q_L54G(SEQ ID NO:126)、hu3D6VLv2L37Q_L54D(SEQ IDNO:127)、hu3D6VLv2 L37Q_L50G(SEQ ID NO:128)、hu3D6VLv2 L37Q_L50D(SEQ ID NO:129)、hu3D6VLv2L37Q_L54T(SEQ ID NO:130)、hu3D6VLv2 L37Q_S52G(SEQ ID NO:131)、hu3D6VLv2 L37Q_L54E(SEQ ID NO:145)、hu3D6VLv2L37Q_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)、hu3D6VLv2L37Q_L50G_L54R_G100Q(SEQ IDNO:136), hu3D6VLv2L37Q_L50G_L54G_G100Q (SEQ ID NO:137), hu3D6VLv2L37Q_S52G_L54R_G100Q (SEQ ID NO:138), hu3D6VLv2L37Q_S52G_L54D_G100Q (SEQ ID NO:139), hu3D6VLv2L37Q_L50D_L54G_G100Q (SEQ ID NO:140), hu3D6VLv2L37Q_L50D_L54R_G100Q (SEQ ID NO:141), hu3D6VLv2L37Q_L50V_L54D_G100Q (SEQ ID NO:142), hu3D6VLv2 L37Q (SEQ ID NO:143) and hu3D6VLv2 G100Q (SEQ ID NO:144)).

[0386] Figure 2 and Figure 3 Alignments of the heavy and light chain variable regions of murine 3D6 and various humanized antibodies are shown, respectively. Fig. 9A and Fig. 9B Shown is an alignment of the heavy chain variable region of murine 3D6 with the heavy chain variable regions of various humanized antibodies. Fig. 10A , Fig. 10B , Fig. 10C and Fig. 10D Shown is an alignment of the light chain variable region of hu3D6VLv2 and the light chain variable regions of various humanized antibodies.

[0387] For reasons such as the potential to affect CDR conformation and / or binding to antigen, mediate interactions between the heavy and light chains, interactions with the constant region, be sites of desired or undesired post-translational modifications, be unusual residues for their location in human variable region sequences and therefore potentially immunogenic, acquire aggregation potential, and other reasons, the following 35 variable region framework positions were considered candidates for substitution in the 56 exemplified human mature light chain variable regions and the 10 exemplified human mature heavy chain variable regions, as further specified in the Examples: L7 (T7S), L10 (T10S), L15 (I15L), L17 (Q17E), L37 (L37Q), L45 (K45R), L47 (L47G, L47N, L47D, L47E, L47P, L47T, L47S, or L47A), L 48 (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), H 43(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 9 variable region CDR positions are considered as candidates for substitution in the 56 exemplified human mature light chain variable regions and 10 exemplified human mature heavy chain variable regions, as further specified in 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, Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 87. In some humanized 3D6 antibodies, Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 149. In some humanized 3D6 antibodies, Kabat-Chothia complex CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO:87.In some humanized 3D6 antibodies, the Kabat-Chothia complex CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 88. In some humanized 3D6 antibodies, the Kabat-Chothia complex CDR-H1 has an amino acid sequence comprising SEQ ID NO: 86, and the Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 92. In some humanized 3D6 antibodies, the Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 89. In some humanized 3D6 antibodies, the Kabat CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 150 to SEQ ID NO: 175.

[0388] Here, as elsewhere, the first mentioned residue is the residue of the humanized antibody formed by grafting the Kabat CDR or the complex Chothia-Kabat CDR (in the case of CDR-H1) into a human acceptor framework, and the second mentioned residue is the residue considered to replace this residue. Thus, within the variable region framework, the first mentioned residue is human, and within the CDR, the first mentioned residue is mouse.

[0389] The exemplified antibodies include any permutation or combination of the exemplified mature heavy chain variable regions and mature light chain variable regions, VHvb1 / VLvb1, VHvb1 / VLvb2, VHvb1 / VLvb3, VHvb2 / VLvb1, VHvb2 / VLvb2, VHvb2 / VLvb3, VHvb3 / VLvb1, VHvb3 / VLvb2, VHvb3 / VLvb3, VHvb4 / VLvb1, VHvb4 / VLvb2, VHvb4 / VLvb3, VHvb5 / VLvb1, VHvb5 / VLvb2, VHvb5 / VLvb3, VHvb6 / VLvb1, VHvb6 / VLvb2, VHvb6 / VLvb3, VHvb7 / VLvb1, VHvb7 / VLvb2, VHvb7 / VLvb3. Exemplary 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), hu3D6VHv1bA11 L82cV (SEQ ID NO: 148), hu3D6VHv1bA11 L82cV (SEQ ID NO: 149), hu3D6VHv1bA11 L82cV (SEQ ID NO: 150), hu3D6VHv1bA11 L82cV (SEQ ID NO: 151), hu3D6VHv1bA11 L82cV (SEQ ID NO: 152), hu3D6VHv1bA11 L82cV (SEQ ID NO: NO: 147) and hu3D6VHv1bA11D60E_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 L55A (SEQ ID NO: 100), hu3D6VLv2 L55C (SEQ ID NO: 101), hu3D6VLv2 L55D (SEQ ID NO: 102), hu3D6VLv2 L55E (SEQ ID NO: 103), hu3D6VLv2 L55G (SEQ ID NO: 104), hu3D6VLv2 L55N (SEQ ID NO: 105), hu3D6VLv2 L55E (SEQ ID NO: 105), hu3D6VLv2 L55 NO:98), hu3D6VLv2 L50D (SEQ ID NO:99), hu3D6VLv2 L54K (SEQ ID NO:100), hu3D6VLv2 L54R (SEQ IDNO:101)、hu3D6VLv2 L54T(SEQ ID NO:102)、hu3D6VLv2 L50G(SEQ ID NO:103)、hu3D6VLv2 I48G(SEQ ID NO:104)、hu3D6VLv2 I48D(SEQ ID NO:105)、hu3D6VLv2 L47G(SEQ ID NO:106)、hu3D6VLv2 Y49E(SEQ ID NO:107)、hu3D6VLv2 L54V(SEQ ID NO:108)、hu3D6VLv2 L54S(SEQ ID NO:109)、hu3D6VLv2 S52G(SEQ ID NO:110)、hu3D6VLv2 L47N(SEQ ID NO:111)、hu3D6VLv2 L47D(SEQ ID NO:112)、hu3D6VLv2 L47E(SEQ ID NO:113)、hu3D6VLv2 L47P(SEQ ID NO:114)、hu3D6VLv2 L47T(SEQ ID NO:115)、hu3D6VLv2 L47S(SEQ ID NO:116)、hu3D6VLv2 L47A(SEQ ID NO:117)、hu3D6VLv2 L50V(SEQ ID NO:118)、hu3D6VLv2L37Q_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)、hu3D6VLv2L37Q_S52G_L54T(SEQ IDNO:123)、hu3D6VLv2 L37Q_S52G_L54D(SEQ ID NO:124)、hu3D6VLv2 L37Q_L54R(SEQ IDNO:125)、hu3D6VLv2L37Q_L54G(SEQ ID NO:126)、hu3D6VLv2 L37Q_L54D(SEQ ID NO:127)、hu3D6VLv2 L37Q_L50G(SEQ ID NO:128)、hu3D6VLv2L37Q_L50D(SEQ ID NO:129)、hu3D6VLv2 L37Q_L54T(SEQ ID NO:130)、hu3D6VLv2 L37Q_S52G(SEQ IDNO:131), hu3D6VLv2L37Q_L54E (SEQ ID NO:145), hu3D6VLv2 L37Q_L50D_L54G (SEQ ID NO:132), hu3D6VLv2 L37Q_L50D_L54R (SEQ ID NO:133), hu3D6VLv2L37Q_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), hu3D6VLv2L37Q_S52G_L54D_G100Q (SEQ ID NO: 139), hu3D6VLv2L37Q_L50D_L54G_G100Q (SEQ ID NO: 140), hu3D6VLv2L37Q_L50D_L54R_G100Q (SEQ ID NO: 141), hu3D6VLv2L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144).

[0390] Exemplary 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), hu3D6VHv1bA11 L64cV (SEQ ID NO: 150), hu3D6VHv1bA11 L86cV (SEQ ID NO: 151), hu3D6VHv1bA11 L65cV (SEQ ID NO: 153), hu3D6VHv1bA11 L67cV (SEQ ID NO: 154), hu3D6VHv1bA11 L68cV (SEQ ID NO: 155), hu3D6VHv1bA11 L69cV (SEQ ID NO: 157), hu3D6VHv1bA11 L69cV (SEQ ID NO: 158), hu3D6VHv1bA11 L6 NO: 147) and hu3D6VHv1bA11D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148) and any permutation or combination of any one of the humanized 3D6VL light chain variable regions hu3D6VLv1 (SEQ ID NO: 20), hu3D6VLv2 (SEQ ID NO: 21), hu3D6VLv3 (SEQ ID NO: 22) and hu3D6VLv4 (SEQ ID NO: 22). Exemplary antibodies include the 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 L54E (SEQ ID NO: 102), hu3D6VLv2 L54E (SEQ ID NO: 103), hu3D6VLv2 L54E (SEQ ID NO: 104), hu3D6VLv2 L54E (SEQ ID NO: 105), hu3D6VLv2 L54E (SEQ ID NO: 106), hu3D6VLv2 L54E (SEQ ID NO: 107), hu3D6VLv2 L54E 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)、hu3D6VLv2Y49E(SEQ ID NO:107)、hu3D6VLv2 L54V(SEQ ID NO:108)、hu3D6VLv2 L54S(SEQ IDNO: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)、hu3D6VLv2L37Q_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)、hu3D6VLv2L37Q_S52G_L54T(SEQ ID NO:123)、hu3D6VLv2L37Q_S52G_L54D(SEQ ID NO:124)、hu3D6VLv2 L37Q_L54R(SEQ ID NO:125)、hu3D6VLv2L37Q_L54G(SEQ ID NO:126)、hu3D6VLv2 L37Q_L54D(SEQ ID NO:127)、hu3D6VLv2 L37Q_L50G(SEQ ID NO:128)、hu3D6VLv2L37Q_L50D(SEQ ID NO:129)、hu3D6VLv2 L37Q_L54T(SEQ ID NO:130)、hu3D6VLv2 L37Q_S52G(SEQ ID NO:131)、hu3D6VLv2L37Q_L54E(SEQ ID NO:145)、hu3D6VLv2 L37Q_L50D_L54G(SEQ ID NO:132)、hu3D6VLv2 L37Q_L50D_L54R(SEQ ID NO:133)、hu3D6VLv2L37Q_L50E_L54G(SEQ ID NO:134)、hu3D6VLv2L37Q_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), hu3D6VLv2L37Q_S52G_L54D_G100Q (SEQ ID NO:139), hu3D6VLv2L37Q_L50D_L54G_G100Q (SEQ ID NO:140), hu3D6VLv2L37Q_L50D_L54R_G100Q(SEQ ID NO: 141), hu3D6VLv2L37Q_L50V_L54D_G100Q (SEQ ID NO: 142), hu3D6VLv2 L37Q (SEQ ID NO: 143), and hu3D6VLv2 G100Q (SEQ ID NO: 144) and any permutation or combination of any of the following 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), hu3D6VHv1bA11B6H4 (SEQ ID NO: 49), hu3D6VHv1c (SEQ ID NO: 50), hu3D6VHv1d (SEQ ID NO: 51), hu3D6VHv1dA11B6H5 (SEQ ID NO: 52), hu3D6VHv1dA11B6H6 (SEQ ID NO: 53), hu3D6VHv1c NO:47), hu3D6VHv1c (SEQ ID NO:48), hu3D6VHv1d (SEQ ID NO:49), hu3D6VHv1e (SEQ ID NO:50), hu3D6VHv1f (SEQ ID NO:51), hu3D6VHv3 (SEQ ID NO:52), hu3D6VHv3b (SEQ ID NO:53), hu3D6VHv3c (SEQ ID NO:54), hu3D6VHv4 (SEQ ID NO:55), hu3D6VHv4b (SEQ ID NO:56), and hu3D6VHv4c (SEQ ID NO:57).

[0391] The present invention provides an antibody, wherein the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2L37Q_S52G_L54R (L2-DIM4, SEQ ID NO: 122). The present invention provides an antibody, wherein the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54T (L2-DIM5, SEQID NO: 123). The present invention provides an antibody, wherein the humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with the humanized light chain variable region hu3D6VLv2L37Q_S52G_L54R (L2-DIM4, SEQ ID NO: 122). The present invention provides an antibody, wherein the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 L37Q_S52G_L54G (L2-DIM3, SEQ ID NO: 121). The present invention provides an antibody, wherein the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2 S52G (L2-DIM9, SEQ ID NO: 110). The present invention provides an antibody, wherein the humanized heavy chain variable region h3D6VHvb8 (SEQ ID NO: 146) is combined with the humanized light chain variable region hu3D6VLv2 L54G (L2-DIM7, SEQ ID NO: 94). The present invention provides an antibody, wherein the humanized heavy chain variable region hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) is combined with the humanized light chain variable region hu3D6VLv2L50G (L2-DIM22, SEQ ID NO: 103).

[0392] The present invention provides an antibody in which any one of the exemplified humanized heavy chain variable regions is bound to a human heavy chain constant region. An exemplary human heavy chain constant region is provided as SEQ ID NO: 176 (IgG1: allotype G1m17, 1). For example, SEQ ID NO: 178 lists the amino acid sequence of the mature heavy chain of a 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype). For example, SEQ ID NO: 180 lists the amino acid sequence of the heavy chain of a 3D6 humanized variant (hu3D6VHv1bA11 IgG1 G1m17 allotype) having a bovine α-lactalbumin signal peptide at the N-terminus. The present invention provides an antibody in which any one of the exemplified humanized light chain variable regions is bound to a light chain constant region. An exemplary light chain constant region is provided as SEQ ID NO: 177 (κ). For example, SEQ ID NO: 179 lists the amino acid sequence of the mature light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4κ). For example, SEQ ID NO: 181 lists the amino acid sequence of the light chain of the 3D6 humanized variant (hu3D6VLv2 variant L37Q_S52G_L54R, L2-DIM4κ) with a bovine α-lactalbumin signal peptide at the N-terminus.

[0393] The present invention provides variants of the 3D6 humanized antibody, wherein the humanized mature heavy chain variable region is the same as 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), hu3D6VHv1bA11 L82cV (SEQ ID NO: 148), hu3D6VHv1bA11 L82cV (SEQ ID NO: 149), hu3D6VHv1bA11 L82cV (SEQ ID NO: 150), hu3D6VHv1bA11 L82cV (SEQ ID NO: 151), hu3D6VHv1bA11 L82cV (SEQ ID NO: 152), hu3D6VHv1bA11 L82cV (SEQ ID NO: NO: 147) or hu3D6VHv1bA11D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9, SEQ ID NO: 148), and the humanized mature light chain variable region is at least 90%, 95%, 96%, 97%, 98% or 99% identical to 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). NO:96), hu3D6VLv2L50E (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), hu3D6VLv2S52G(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)、hu3D6VLv2L37Q_L50G_L54R(SEQ IDNO:119)、hu3D6VLv2 L37Q_L50G_L54G(SEQ ID NO:120)、hu3D6VLv2 L37Q_S52G_L54G(SEQID NO:121)、hu3D6VLv2 L37Q_S52G_L54R(SEQ ID NO:122)、hu3D6VLv2L37Q_S52G_L54T(SEQ ID NO:123)、hu3D6VLv2 L37Q_S52G_L54D(SEQ ID NO:124)、hu3D6VLv2 L37Q_L54R(SEQ ID NO:125)、hu3D6VLv2L37Q_L54G(SEQ ID NO:126)、hu3D6VLv2 L37Q_L54D(SEQ IDNO:127)、hu3D6VLv2 L37Q_L50G(SEQ ID NO:128)、hu3D6VLv2L37Q_L50D(SEQ ID NO:129)、hu3D6VLv2 L37Q_L54T(SEQ ID NO:130)、hu3D6VLv2 L37Q_S52G(SEQ ID NO:131)、hu3D6VLv2L37Q_L54E(SEQ ID NO:145)、hu3D6VLv2 L37Q_L50D_L54G(SEQ ID NO:132)、hu3D6VLv2 L37Q_L50D_L54R(SEQ ID NO:133)、hu3D6VLv2L37Q_L50E_L54G(SEQ ID NO:134)、hu3D6VLv2 L37Q_L50E_L54R(SEQ ID NO:135)、hu3D6VLv2 L37Q_L50G_L54R_G100Q(SEQ ID NO:136)、hu3D6VLv2L37Q_L50G_L54G_G100Q(SEQ ID NO:137), hu3D6VLv2L37Q_S52G_L54R_G100Q(SEQ ID NO:138), hu3D6VLv2L37Q_S52G_L54D_G100Q(SEQ ID NO:139), hu3D6VLv2L37Q_L50D_L54G_G100Q (SEQ ID NO:140), hu3D6VLv2L37Q_L50D_L54R_G100Q (SEQ ID NO:141), hu3D6VLv2L37Q_L50V_L54D_G100Q (SEQ ID NO:142), hu3D6VLv2 L37Q (SEQ ID NO:143) or hu3D6VLv2 G100Q (SEQ ID NO: 144) shows at least 90%, 95%, 96%, 97%, 98% or 99% identity. In some such antibodies, 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 of SEQ ID NOs:76-80, SEQ ID NOs:90-91, SEQ ID NOs:146-148, SEQ ID NOs:83-85, and SEQ ID NOs:93-145 are retained.

[0394] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the indicated 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.

[0395] In some humanized 3D6 antibodies, position H91 in the VH region is occupied by F.

[0396] 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.

[0397] 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.

[0398] In some humanized 3D6 antibodies, position H58 in the VH region is occupied by I.

[0399] 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, 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.

[0400] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H54 is occupied by D, 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.

[0401] In some humanized 3D6 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H17 is occupied by S or T, H20 is occupied by L or I, H23 is occupied by T or K, H28 is occupied by N or T, H38 is occupied by K or R, H42 is occupied by E or G, H43 is occupied by Q or K, H54 is occupied by N or D, 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.

[0402] In some humanized 3D6 antibodies, positions H91, H93, and H94 in the VH region are occupied by F, S, and T, respectively, as in huVHvb 1. In some humanized 3D6 antibodies, positions H1, H5, H11, H20, H23, H38, H42, H43, H66, H75, H76, H81, H91, H93, H94, H108, and H109 in the VH region are occupied by E, V, V, I, K, R, G, K, R, T, D, E, F, S, T, L, and V, respectively, as in huVHvb2. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H38, H42, H43, H58, H66, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, V, V, T, I, K, R, G, K, I, R, T, D, M, E, R, S, T, L and V, respectively, as in huVHvb3. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, I, R, V, T, D, M, E, R, S, T, L and V, respectively, as in huVHvb4. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H58, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, I, R, V, T, D, M, E, R, S, T, L and V, respectively, as in huVHvb5. In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H91, H93, H94, H108 and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, F, S, T, L and V, respectively, as in huVHvb6.In some humanized 3D6 antibodies, positions H1, H5, H11, H17, H20, H23, H28, H38, H42, H43, H54, H56, H66, H67, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, V, V, T, I, K, T, R, G, K, D, E, R, V, T, D, M, E, R, S, T, L and V, respectively, as in huVHvb7.

[0403] In some humanized 3D6 antibodies, position H60 is occupied by E, such as in hu3D6VHv1bA11D60E (h3D6VHvb8). In some humanized 3D6 antibodies, position H82C is occupied by V, such as in hu3D6VHv1bA11 L82cV. In some humanized 3D6 antibodies, positions H60, H80, H81, H82c and H83 are occupied by E, M, E, V and R, such as in hu3D6VHv1bA11D60E_L80M_Q81E_L82cV_T83R (h3D6VHvb9).

[0404] The heavy chain variable region of any of the antibodies referenced above may be modified to further reduce immunogenicity. For example, in some humanized antibodies, position H80 is occupied by M and / or position H82c is occupied by V.

[0405] 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.

[0406] In some humanized 3D6 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L7 is T or S, L10 is T or S, L15 is I or L, L17 is Q or E, L24 is K or R, L37 is L or Q, L45 is K or R, L83 is L or V, L86 is H or Y, L100 is A or Q, and L106 is L or I.

[0407] In some humanized 3D6 antibodies, positions L7, L10, L15, L83, L86 and L106 in the VL region are occupied by S, S, L, V, Y and I, respectively, as in huVLvb2. In some humanized 3D6 antibodies, positions L7, L10, L15, L17, L24, L37, L45, L83, L86, L100 and L106 in the VL region are occupied by S, S, L, E, R, Q, R, V, Y, Q and I, respectively, as in huVLvb3.

[0408] The light chain variable region of any of the antibodies referenced above may be modified to further reduce immunogenicity. For example, in some humanized antibodies, position L47 is occupied by G, N, D, E, P, T, S, or A; position L48 is occupied by G or D; position L49 is occupied by E; position L50 is occupied by E, D, G, or V; position L52 is occupied by G; and / or position L54 is occupied by D, G, N, E, Q, K, R, T, V, or S. The heavy chain variable region of any of the antibodies referenced above may be modified to further reduce immunogenicity. For example, in some humanized antibodies, position H80 is occupied by M and / or position H82c is occupied by V.

[0409] In some humanized 3D6 antibodies, position L54 is occupied by D, such as in hu3D6VLv2L54D. In some humanized 3D6 antibodies, position L54 is occupied by G, such as in hu3D6VLv2 L54G. In some humanized 3D6 antibodies, position L54 is occupied by N, such as in hu3D6VLv2 L54N. In some humanized 3D6 antibodies, position L54 is occupied by E, such as in hu3D6VLv2L54E. In some humanized 3D6 antibodies, position L50 is occupied by E, such as in hu3D6VLv2 L50E. In some humanized 3D6 antibodies, position L54 is occupied by Q, such as in hu3D6VLv2 L54Q. In some humanized 3D6 antibodies, position L50 is occupied by D, such as in hu3D6VLv2 L50D. In some humanized 3D6 antibodies, position L54 is occupied by K, such as in hu3D6VLv2L54K. In some humanized 3D6 antibodies, position L54 is occupied by R, such as in hu3D6VLv2 L54R. In some humanized 3D6 antibodies, position L54 is occupied by T, such as in hu3D6VLv2 L54T. In some humanized 3D6 antibodies, position L50 is occupied by G, such as in hu3D6VLv2 L50G. In some humanized 3D6 antibodies, position L48 is occupied by G, such as in hu3D6VLv2I48G. In some humanized 3D6 antibodies, position L48 is occupied by D, such as in hu3D6VLv2 I48D. In some humanized 3D6 antibodies, position L47 is occupied by G, such as in hu3D6VLv2 L47G. In some humanized 3D6 antibodies, position L49 is occupied by E, such as in hu3D6VLv2 Y49E. In some humanized 3D6 antibodies, position L54 is occupied by V, such as in hu3D6VLv2L54V. In some humanized 3D6 antibodies, position L54 is occupied by S, such as in hu3D6VLv2 L54S. In some humanized 3D6 antibodies, position L52 is occupied by G, such as in hu3D6VLv2 S52G. In some humanized 3D6 antibodies, position L47 is occupied by N, such as in hu3D6VLv2 L47N. In some humanized 3D6 antibodies, position L47 is occupied by D, such as in hu3D6VLv2L47D. In some humanized 3D6 antibodies, position L47 is occupied by E, such as in hu3D6VLv2 L47E. In some humanized 3D6 antibodies, position L47 is occupied by P, such as in hu3D6VLv2 L47P. In some humanized 3D6 antibodies, position L47 is occupied by T, such as in hu3D6VLv2 L47T. In some humanized 3D6 antibodies, position L47 is occupied by S, such as in hu3D6VLv2L47S.In some humanized 3D6 antibodies, position L47 is occupied by A, such as in hu3D6VLv2L47A. In some humanized 3D6 antibodies, position L50 is occupied by V, such as in hu3D6VLv2 L50V.

[0410] In some humanized 3D6 antibodies, positions L37, L50 and L54 are occupied by Q, G and R, respectively, such as in hu3D6VLv2L37Q_L50G_L54R. In some humanized 3D6 antibodies, positions L37, L50 and L54 are occupied by Q, G and G, respectively, such as in hu3D6VLv2L37Q_L50G_L54G. In some humanized 3D6 antibodies, positions L37, L52 and L54 are occupied by Q, G and G, respectively, such as in hu3D6VLv2 L37Q_S52G_L54G. In some humanized 3D6 antibodies, positions L37, L52 and L54 are occupied by Q, G and R, respectively, such as in hu3D6VLv2 L37Q_S52G_L54R. In some humanized 3D6 antibodies, positions L37, L52 and L54 are occupied by Q, G and T, respectively, such as in hu3D6VLv2 L37Q_S52G_L54T. In some humanized 3D6 antibodies, positions L37, L52 and L54 are occupied by Q, G and D, respectively, such as in hu3D6VLv2 L37Q_S52G_L54D.

[0411] In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and R, respectively, such as in hu3D6VLv2 L37Q_L54R. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and G, respectively, such as in hu3D6VLv2 L37Q_L54G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and D, respectively, such as in hu3D6VLv2 L37Q_L54D. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and G, respectively, such as in hu3D6VLv2 L37Q_L50G. In some humanized 3D6 antibodies, positions L37 and L50 are occupied by Q and D, respectively, such as in hu3D6VLv2 L37Q_L50D. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and T, respectively, such as in hu3D6VLv2 L37Q_L54T. In some humanized 3D6 antibodies, positions L37 and L52 are occupied by Q and G, respectively, such as in hu3D6VLv2 L37Q_S52G. In some humanized 3D6 antibodies, positions L37 and L54 are occupied by Q and E, respectively, such as in hu3D6VLv2 L37Q_L54E.

[0412] In some humanized 3D6 antibodies, positions L37, L50 and L54 are occupied by Q, D and G, respectively, such as in hu3D6VLv2L37Q_L50D_L54G. In some humanized 3D6 antibodies, positions L37, L50 and L54 are occupied by Q, D and R, respectively, such as in hu3D6VLv2L37Q_L50D_L54R. In some humanized 3D6 antibodies, positions L37, L50 and L54 are occupied by Q, E and G, respectively, such as in hu3D6VLv2 L37Q_L50E_L54G. In some humanized 3D6 antibodies, positions L37, L50 and L54 are occupied by Q, E and R, respectively, such as in hu3D6VLv2 L37Q_L50E_L54R.

[0413] In some humanized 3D6 antibodies, positions L37, L50, L54 and L100 are occupied by Q, G, R and Q, respectively, such as in hu3D6VLv2 L37Q_L50G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54 and L100 are occupied by Q, G, G and Q, respectively, such as in hu3D6VLv2 L37Q_L50G_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54 and L100 are occupied by Q, G, R and Q, respectively, such as in hu3D6VLv2 L37Q_S52G_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L52, L54 and L100 are occupied by Q, G, D and Q, respectively, such as in hu3D6VLv2L37Q_S52G_L54D_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54 and L100 are occupied by Q, D, G and Q, respectively, such as in hu3D6VLv2L37Q_L50D_L54G_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54 and L100 are occupied by Q, D, R and Q, respectively, such as in hu3D6VLv2L37Q_L50D_L54R_G100Q. In some humanized 3D6 antibodies, positions L37, L50, L54 and L100 are occupied by Q, V, D and Q, respectively, such as in hu3D6VLv2L37Q_L50V_L54D_G100Q.

[0414] In some humanized 3D6 antibodies, position L37 is occupied by Q, such as in hu3D6VLv2L37Q. In some humanized 3D6 antibodies, position L100 is occupied by Q, such as in hu3D6VLv2G100Q.

[0415] Some humanized 3D6 antibodies comprise a mature heavy chain variable region comprising CDR H1, CDR H2 and CDR H3 having SEQ ID NOs: 8, 9 and 10, respectively, except that position H28 can be occupied by N or T, H54 can be occupied by N or D, H56 can be occupied by D or E, position H58 can be occupied by V or I, and position H60 can be occupied by D or E, and a mature light chain variable region comprising CDR L1, CDR L2 and CDR L3 having SEQ ID NOs: 12, 13 and 14, respectively. L3, except that position L24 can be occupied by K or R, position L50 can be occupied by L, E, D, G, or V, position L52 can be occupied by S or G, and position L54 can be occupied by L, D, G, N, E, Q, K, R, T, V, or S, wherein at least one of the following positions is occupied by 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, H57 is occupied by E 8 is occupied by I, H60 is occupied by E, H82c is occupied by V, L10 is occupied by T, L17 is occupied by E, L24 is occupied by R, L37 is occupied by Q, L47 is occupied by G, N, D, E, P, T, S or A, L48 is occupied by G or D, L49 is occupied by E, L50 is occupied by E, D, G or V, L52 is occupied by G, L54 is occupied by D, G, N, E, Q, K, R, T, V or S, L83 is occupied by L, L86 is occupied by H, L100 is occupied by Q, and L106 is occupied by L.

[0416] Some humanized 3D6 antibodies comprise three light chain CDRs and three heavy chain CDRs of monoclonal antibody 3D6, wherein 3D6 is a mouse antibody characterized by having a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:11, except that position H27 can be occupied by F or Y, position H28 can be occupied by N or T, position H29 can be occupied by I or F, position H30 can be occupied by K or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, position H60 can be occupied by D, A, or E, position H61 can be occupied by P or E, position H102 can be occupied by F or Y, position L50 can be occupied by L, E, D, G, or V, and position L52 can be occupied by S or G. The amino acids in the formula (a) and (b) are represented by the amino acids in the table below: 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.

[0417] In some humanized 3D6 antibodies, the variable heavy chain has ≥85% identity to a human sequence. In some humanized 3D6 antibodies, the variable light chain has ≥85% identity to a human sequence. In some humanized 3D6 antibodies, each of the variable heavy chain and the variable light chain has ≥85% identity to a human germline sequence. In some humanized 3D6 antibodies, the three heavy chain CDRs are as defined by the Kabat / Chothia complex (SEQ ID NOs: 8, 9, and 10), and the three light chain CDRs are as defined by the Kabat / Chothia complex (SEQ ID NOs: 12, 13, and 14); provided that position H28 is occupied by N or T, position H54 is occupied by N or D, position H56 is occupied by D or E, position H58 is occupied by V or I, position H60 is occupied by D or E, position L24 is occupied by K or R, position L50 is occupied by L, E, D, G, or V, position L52 is occupied by S or G, and position L54 is occupied by L, D, G, N, E, Q, K, R, T, V, or S. In some humanized 3D6 antibodies, the Kabat / Chothia complex 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 NO: 150 to SEQ ID NO: 175.

[0418] The CDR regions of such humanized antibodies may be identical or substantially identical to the CDR regions of 3D6, and the CDR regions may be defined by any conventional definition (e.g., Chothia, or a composite of Chothia and Kabat), but are preferably as defined by Kabat.

[0419] Unless otherwise indicated, variable region framework positions conform to Kabat numbering. Other such variants typically differ from the sequences of the exemplified Hu3D6 heavy and light chains by a small amount (e.g., typically no more than 1, 2, 3, 5, 10, or 15) of substitutions, deletions, or insertions. Such differences are typically in the framework, but may also be present in the CDRs.

[0420] The possibility of other variation of humanized 3D6 variant is other back mutation in variable region framework.Many framework residues that do not contact with CDR in humanized mAb can adapt to the amino acid replacement of the corresponding position from donor mouse mAb or other mice or people's antibody, and even many potential CDR contact residues also allow replacement.Even the amino acid in CDR can also be changed, for example, using the residue found in the corresponding position of the people's acceptor sequence for providing the variable region framework.In addition, alternative people's acceptor sequence can be used, for example, for heavy chain and / or light chain.If different acceptor sequences are used, one or more back mutations recommended above can not be carried out, because corresponding donor and acceptor residues are identical in the absence of back mutation.

[0421] Preferably, the substitutions or backmutations (whether conservative or not) in the humanized 3D6 variant have no substantial effect on the binding affinity or potency of the humanized mAb, ie, its ability to bind tau.

[0422] The humanized 3D6 antibodies were further characterized by their ability to bind both phosphorylated and unphosphorylated tau as well as misfolded / aggregated forms of tau.

[0423] D. Chimeric Antibodies and Faced Antibodies

[0424] The present invention also provides non-human antibodies, specifically chimeric forms and veneered forms of the exemplified 3D6 antibody.

[0425] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of non-human antibodies (e.g., mice) are combined with human light chain constant regions and human heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of mouse antibodies and are about two-thirds human sequences. In one embodiment, the chimeric 3D6 antibody has a heavy chain amino acid sequence of SEQ ID NO: 72 and a light chain amino acid sequence of SEQ ID NO: 73.

[0426] A veneer antibody is a type of humanized antibody that retains some and usually all CDRs and some non-human variable region framework residues of a non-human antibody, but replaces other variable region framework residues that may contribute to B cell epitopes or T cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28: 489, 1991) with residues from the corresponding positions of human antibody sequences. The result is an antibody in which the CDRs are completely or substantially from a non-human antibody, and the variable region framework of the non-human antibody becomes more human-like by substitution. The veneer version of the 3D6 antibody is included in the present invention.

[0427] E. Human Antibodies

[0428] Human antibodies to tau or fragments thereof (e.g., amino acid residues 199-213 and / or 262-276 of SEQ ID NO:3, corresponding to amino acid residues 257-271 and / or 320-334 of SEQ ID NO:1, respectively, or amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1, or any combination of two, three or all four thereof) are provided by a variety of techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter (supra), or otherwise, 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 specificity for a particular epitope by using only fragments of tau, such as tau fragments containing only amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively) or containing only amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1 as target antigens, and / or by screening antibodies against a collection of tau variants, such as tau variants containing various mutations within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively) or within amino acid residues 259-268 or 290-299 or 321-330 or 353-362 of SEQ ID NO:1.

[0429] Methods for producing human antibodies include the three-body hybridoma 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; using transgenic mice that include human immunoglobulin genes (see, e.g., Lonberg et al., WO 93 / 12227 (1993); U.S. Pat. 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; 5,571,836 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996); and Kucherlapati, WO 91 / 10741 (1991)); phage display methods (see, e.g., Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047; U.S. Pat. No. 5,877,218; U.S. Pat. No. 5,871,907; U.S. Pat. No. 5,858,657; U.S. Pat. No. 5,837,242; U.S. Pat. No. 5,733,743; and U.S. Pat. No. 5,565,332); and the 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).

[0430] F. Selection of constant regions

[0431] The heavy and light chain variable regions of chimeric, veneered or humanized antibodies can be linked to at least a portion of a human constant region. The selection of constant regions depends in part on whether antibody-dependent cell-mediated 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 λ or κ. Numbering conventions for constant regions include EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991), IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol., 29, 185-203 (2005)) and IMGT exon numbering (Lefranc, supra).

[0432] One or more amino acids at the amino or carboxyl terminus of the light chain and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in a portion 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, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU numbering is used in this paragraph for constant regions) for increasing the half-life of the antibody. The substitution at any one or all of positions 234, 235, 236 and / or 237 reduces the affinity for Fcγ receptors, especially FcγRI receptors (see, e.g., US 6,624,821). The alanine substitution 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 for reducing effector function. Optionally, positions 234, 236 and / or 237 in human IgG2 are replaced by alanine, and position 235 is replaced by glutamine (see, e.g., US 5,624,821). In some antibodies, mutations at one or more positions 241, 264, 265, 270, 296, 297, 322, 329 and 331 (EU numbering) of human IgG1 are used. In some antibodies, mutations at one or more positions of positions 318, 320 and 322 (EU numbering) of human IgG1 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.

[0433] The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as individual heavy chains, light chains, Fab, Fab', F(ab')2 and Fv, or as a single chain antibody in which the heavy and light chain mature variable domains are connected by a spacer.

[0434] Human constant regions show allotypic variation and homologous allotypic variation between different individuals, i.e., at one or more polymorphic positions, constant regions can be different in different individuals. Homologous allotypes are distinguished from allotypes in that serum that recognizes homologous allotypes binds to non-polymorphic regions of one or more other isotypes. Thus, for example, another heavy chain constant region belongs to IgG1 G1m3 with or without C-terminal lysine. Reference to human constant regions includes constant regions with any arrangement of residues of any natural allotype or position occupying natural allotypes. Exemplary heavy chain constant regions are SEQ ID NO: 176, with or without C-terminal lysine, and exemplary light chain constant regions are SEQ ID NO: 177.

[0435] G. Expression of recombinant antibodies

[0436] Many methods are known for producing chimeric and humanized antibodies using antibody expression cell lines (e.g., hybridomas). For example, well-known methods can be used to clone and sequence the immunoglobulin variable regions of antibodies. In one method, mRNA prepared by hybridoma cells is used to clone the heavy chain variable VH region by RT-PCR. Universal primers are used as 5' primers and g2b constant region specific 3' primers for VH region leader peptides including translation start codons. Exemplary primers are described in U.S. Patent Publication US2005 / 0009150 by Schenk et al. (hereinafter referred to as "Schenk"). Sequences from multiple independently derived clones can be compared to ensure that no changes are introduced during the amplification process. The sequence of the VH region can also be determined or confirmed by sequencing the VH fragments obtained by the 5'RACE RT-PCR method and the 3'g2b specific primers.

[0437] The variable VL region of light chain can be cloned in a similar manner. In one method, a common primer set for the amplification of the VL region is designed using a 5' primer designed to hybridize with the VL region including the translation initiation codon and a 3' primer specific for the Ck region downstream of the VJ junction region. In a second method, a 5' RACE RT-PCR method is used to clone the VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequence is then combined with the sequence encoding the constant region of a human (or other non-human species).

[0438] In one method, the heavy chain variable region and the light chain variable region are reengineered to encode the splicing donor sequence downstream of the corresponding VDJ or VJ junction, and they are cloned into a mammalian expression vector (such as pCMV-h 1 for heavy chain and pCMV-Mcl for light chain). These vectors encode human 1 and C kappa constant regions as exon fragments downstream of the inserted variable region box. After sequence verification, the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned medium is collected 48 hours after transfection, and antibody yield is assessed by Western blot analysis or antigen binding is assessed by ELISA. Chimeric antibodies are humanized as described above.

[0439] Chimeric, veneered, humanized and human antibodies are usually produced by recombinant expression. Recombinant polynucleotide constructs usually include expression control sequences operably linked to the coding sequence of the antibody chain, including naturally associated or heterologous expression control elements, such as promoters. The expression control sequence can be a promoter system in a vector capable of transforming or transfecting a eukaryotic or prokaryotic host cell. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of cross-reactive antibodies.

[0440] These expression vectors are usually replicable in the host organism either as an episome or as an integrated part of the host chromosomal DNA. Typically, the expression vector contains a selection marker, for example, ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence.

[0441] Escherichia coli (E. coli) is a prokaryotic host that can be used to express antibodies, especially antibody fragments. Microorganisms such as yeast can also be used for expression. Saccharomyces is a yeast host with suitable vectors with expression control sequences, replication origins, termination sequences, etc. as needed. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, etc.

[0442] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting complete heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma, including Sp2 / 0 and NS0. The cell can be non-human. The expression vectors of these cells may include expression control sequences, such as replication origins, promoters, enhancers (Queen et al., Immunol. Rev. 89: 49 (1986)) and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Expression control sequences may include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, etc. See Co et al., J. Immunol. 148: 1149 (1992).

[0443] Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequently expressed in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes such as casein or beta lactoglobulin.

[0444] The vector containing the DNA segment of interest can be transferred to the host cell by a method depending on the cell host type. For example, for prokaryotic cells, calcium chloride transfection is usually utilized, while for other cell hosts, calcium phosphate treatment, electroporation, lipofection, gene gun method or virus-based transfection can be used. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection. In order to produce transgenic animals, the transgene can be microinjected into a fertilized oocyte or can be incorporated into the genome of an embryonic stem cell or an induced pluripotent stem cell (iPSC), and the nucleus of such cells is transferred into an enucleated oocyte.

[0445] One or more vectors encoding antibody heavy chain and light chain have been introduced into cell culture, and cell bank can be screened for growth rate and product quality in serum-free medium. The cell bank of optimal yield can then carry out single cell cloning based on FACS to produce monoclonal system. The specific productivity of each cell more than 50pg or 100pg every day can be used, which corresponds to the product titer greater than 7.5g / L culture. The antibody produced by single cell cloning can also carry out turbidity, filtering characteristics, PAGE, IEF, UV scanning, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrum and the test of binding assay (such as ELISA or Biacore). Selected clone can be stored in multiple bottles and frozen storage subsequently, for later use.

[0446] Once expressed, antibodies can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0447] Methods for commercial production of antibodies can be used, including codon optimization, choice of promoter, choice of transcription elements, choice of terminator, blood-free single cell cloning, cell banking, use of selection markers for copy number expansion, CHO terminator, or improving protein titer (see, e.g., US 5,786,464; US 6,114,148; US 6,063,598; US 7,569,339; WO2004 / 050884; WO2008 / 012142; WO2008 / 012142; WO2005 / 019442; WO2008 / 107388; WO2009 / 027471; and US 5,888,809).

[0448] IV. Active Immunogens

[0449] Agents used for active immunization are used to induce the same types of antibodies in a patient as 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 consecutive 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 of SEQ ID NO:1, respectively), such as a peptide comprising 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 a peptide of 3 to 15 or 3 to 12 or 5 to 12 or 5 to 8 consecutive amino acids from 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 comprising SEQ ID NO:1 residues 259-268 or 290-299 or 321-330 or 353-362 peptides. 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 spanning tau). Fragments of tau consisting of, including, or overlapping with the epitope can then be used as immunogens. Such fragments are typically used in a non-phosphorylated form.

[0450] If used, heterologous carriers and adjuvants can be the same as those used to generate monoclonal antibodies, but can also be selected for better pharmaceutical suitability for use in humans. Suitable carriers include serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or toxoids from other pathogenic bacteria such as diphtheria (e.g., CRM197), Escherichia coli, cholera or Helicobacter pylori (H.pylori), or attenuated toxin derivatives. T cell epitopes are also suitable carrier molecules. Some conjugates can be formed by connecting the agent of the present invention to an immunostimulatory polymer molecule (e.g., tripalmitoyl-S-glycerocysteine ​​(Pam3Cys), mannan (mannose polymer) or glucan (β1→2 polymer)), cytokines (e.g., IL-1, IL-1α peptide and β peptide, IL-2, γ-INF, IL-10, GM-CSF) and chemokines (e.g., MIP1-α and β, and RANTES). The immunogen may be connected to a carrier with or without an intervening amino acid (e.g., gly-gly). Additional carriers include virus-like particles. Virus-like particles (VLPs), also referred to as pseudoviruses or virus-derived particles, represent a subunit structure consisting of multiple copies of a viral capsid and / or envelope protein that can self-assemble into a VLP with a limited spherical symmetry in vivo. (Powilleit et al., (2007) PLoS ONE 2(5): e415.) Alternatively, a peptide immunogen may be connected to at least one artificial T cell epitope that can bind to most MHC class II molecules, such as a pan DR epitope ("PADRE"). PADRE is described in US 5,736,142, WO 95 / 07707 and Alexander J et al., Immunity, 1:751-761 (1994). Active immunogens may exist in a polymeric form, wherein multiple copies of an immunogen and / or its carrier exist as a single covalent molecule.

[0451] The fragments are usually administered with a pharmaceutically acceptable adjuvant. The adjuvant increases the titer of the induced antibody and / or the binding affinity of the induced antibody relative to the use of the peptide alone. A variety of adjuvants can be used in combination with immunogenic fragments of tau to induce an immune response. Preferred adjuvants enhance the inherent response to the immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Preferred adjuvants include aluminum salts such as aluminum hydroxide and aluminum phosphate, 3De-O-acylated monophosphoryl lipid A (MPL TM ) (See GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa). Stimulon TMQS-21 is a triterpene glycoside or saponin isolated from the bark of the soap bark tree (Quillaja Saponaria Molina) found in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (Powell & Newman, ed., Plenum Press, NY, 1995); US 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 with saline containing Tween 80. Ribi also contains a purified mycobacterial product and bacterial monophosphoryl lipid A, which act as immunostimulants. Another adjuvant is CpG (WO 98 / 40100). The adjuvant 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.

[0452] Analogs of natural fragments of tau that induce antibodies against tau can also be used. For example, one or more or all of the L-amino acids can be replaced by D amino acids in such peptides. The order of amino acids can also be reversed (retropeptide). Optionally, the peptide includes all D-amino acids in the opposite order (retro-inverso peptide). Peptides and other compounds do not necessarily have significant amino acid sequence similarity with tau peptides, but still act as mimics of tau peptides and induce similar immune responses. Anti-idiotypic antibodies of monoclonal antibodies against tau as described above can also be used. Such anti-Id antibodies simulate antigens and produce immune responses against the antigens (see Essential Immunology, edited by Roit, Blackwell Scientific Publications, Palo Alto, CA 6th edition, page 181).

[0453] Peptide (and optionally the carrier fused with peptide) can also be used in the form of nucleic acid encoding the peptide and expressed in situ in the patient. The nucleic acid segment encoding immunogen is usually connected to a regulatory element, such as a promoter and enhancer, which allow the DNA segment to be expressed in the expected target cells of the patient. In order to express in hemocytes, as desired by the induction of immune response, promoter elements and enhancer elements from light chain immunoglobulin genes or heavy chain immunoglobulin genes or CMV main immediate early promoter and enhancer are suitable for guiding expression. The regulatory element and encoding sequence connected are usually cloned into a vector. Antibodies can also be used in the form of nucleic acids encoding antibody heavy chains and / or light chains. If both heavy and light chains exist, the chain is preferably connected as a single-chain antibody. Antibodies for passive administration can also be prepared, for example, by affinity chromatography from the serum of the patient treated with the peptide immunogen.

[0454] DNA can be delivered in naked form (i.e., without colloid or encapsulating material). Alternatively, many viral vector systems can be used, including retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors {see, e.g., Bett et al., J. Virol. 67, 591 1 (1993)), including retroviral derived vectors, such as MMLV, HIV-1 and ALV; adeno-associated viral vectors {see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), lentiviral vectors such as those based on HIV or FIV gag sequences, viral vectors from the Pox family (including vaccinia virus and fowl pox virus), viral vectors from the alphavirus genus such as derived from Sindbis virus and Semliki Forest virus (Semliki Forest virus). Virus) (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No. 5,643,576), and rhabdoviruses such as vesicular stomatitis virus (see WO 96 / 34625), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al., WO 94 / 12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).

[0455] The DNA encoding the heavy chain and / or light chain of an immunogen or an antibody, or a vector containing the DNA can be packaged into a liposome. Suitable lipids and related analogs are described in US 5,208,036, US 5,264,618, US 5,279,833 and US 5,283,185. The vector and DNA encoding the heavy chain and / or light chain of an immunogen or an antibody can also be adsorbed onto or associated with a particle carrier, the example of which includes a polymethyl methacrylate polymer and polylactic acid and poly (lactide-co-glycolide), (see, e.g., McGee et al., J. Micro Encap. 1996).

[0456] Vectors encoding heavy and / or light chains of antibodies or segments from these vectors can be incorporated into ex vivo cells, for example, into cells transplanted from individual patients (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, which are then reimplanted into the patient, usually after selecting cells that have incorporated the transgene. (See, e.g., WO 2017 / 091512). Exemplary patient-derived cells include induced pluripotent stem cells (IPSCs) derived from patients or other types of stem cells (embryonic stem cells, hematopoietic stem cells, neural stem cells, or mesenchymal stem cells).

[0457] The vector encoding the heavy and / or light chains of the antibody or a segment from the vector can be introduced into any region of interest (such as an albumin gene or other safe harbor gene) in an ex vivo cell. The cells combined with the vector can be implanted with or without prior differentiation. The cells can be implanted into a specific tissue (such as a secretory tissue or a pathological location), or systemically (such as by infusion into the blood). For example, the cells can be implanted into the patient's secretory tissue (such as the liver), which is optionally differentiated into cells present in the tissue, such as hepatocytes in the case of the liver. The expression of the antibody in the liver causes the antibody to be secreted into the blood.

[0458] H. Antibody Screening Assay

[0459] Antibodies can be initially screened for the intended binding specificity as described above. Active immunogens can also be screened for the ability to induce antibodies with such binding specificity. In this case, laboratory animals are immunized with the active immunogen and the resulting sera are tested for the appropriate binding specificity.

[0460] Then the antibody with desired binding specificity can be tested in cells and animal models. Cells for this screening are preferentially neuronal cells. Cell models of tau pathology have been reported, wherein neuroblastoma cells are transfected with four repeat domains of tau optionally with mutations associated with tau pathology (e.g., δK280, see Khlistunova, Current Alzheimer Research 4, 544-546 (2007)). In another model, tau is induced in neuroblastoma N2a cell lines by adding doxycycline. Cell models enable the study of the toxicity of tau to cells in soluble or aggregated states, the appearance of tau aggregates after opening tau gene expression, the dissolution of tau aggregates after closing gene expression again, and the effect of antibodies in inhibiting the formation of tau aggregates or disaggregating them.

[0461] Antibodies or active immunogens can also be screened in transgenic animal models of diseases associated with tau. Such transgenic animals may include a tau transgene (e.g., any of the human isoforms) and optionally, a particularly human APP transgene, such as a kinase that phosphorylates tau, ApoE, presenilin, or alpha synuclein. Such transgenic animals are configured to develop at least one sign or symptom of a disease associated with tau.

[0462] An exemplary transgenic animal is the K3 line of mice (Itner et al., Proc. Natl. Acad. Sci. USA 105(41): 15997-6002 (2008)). These mice have a human tau transgene with a K 369I mutation (which is associated with Pick's disease) and a Thy 1.2 promoter. This model shows a rapid course of neurodegeneration, motor deficits, and degeneration of afferent fibers and cerebellar granule cells. Another exemplary animal is the JNPL3 line of mice. These mice have a human tau transgene with a P301L mutation (which is associated with frontotemporal dementia) and a Thy 1.2 promoter (Taconic, Germantown, NY, Lewis et al., Nat Genet. 25: 402-405 (2000)). These mice have a more gradual course of neurodegeneration. Mice develop neurofibrillary tangles in several brain regions and the spinal cord (which is hereby incorporated by reference in its entirety). This is an excellent model for studying the consequences of tangle development and for screening therapeutics that can inhibit the production of these aggregates. Another advantage of these animals is the relatively early onset of pathology. In homozygous lines, behavioral abnormalities associated with tau pathology can be observed at least as early as 3 months, but the animals remain relatively healthy until at least 8 months of age. In other words, at 8 months, the animals are ambulatory, feed themselves, and can perform behavioral tasks well enough to allow monitoring of treatment effects. Active immunization of these mice with -AI wI KLH-PHF-1 for 6 to 13 months produces titers of approximately 1,000 and shows fewer neurofibrillary tangles, less pSer422, and reduced weight loss relative to untreated control mice.

[0463] The activity of the antibody or active agent can be evaluated by various criteria, including a reduction in the amount of total tau or phosphorylated tau, a reduction in other pathological features (such as amyloid deposits of Aβ), and inhibition or delay of behavioral defects. Active immunogens can also be tested for the induction of antibodies in serum. Both passive and active immunogens can be tested for antibodies that cross the blood-brain barrier and enter the brain of transgenic animals. Antibodies or fragments of induced antibodies can also be tested in non-human primates that develop symptoms of diseases characterized by tau, either naturally or by induction. The test of antibodies or active agents is usually performed in conjunction with controls, wherein parallel experiments are performed, except that there is no antibody or active agent (e.g., replaced by a vehicle). Then the reduction, delay or inhibition of disease signs or symptoms attributable to the antibody or active agent to be tested can be assessed relative to the control.

[0464] V. Patients Suitable for Treatment

[0465] The existence of neurofibrillary tangles has been found in several diseases, including Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, C-type Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant (LBVAD) of Alzheimer's disease, chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), and progressive supranuclear palsy (PSP). The scheme of the present invention can also be used to treat or prevent any of these diseases. Due to the extensive association between neurological diseases and conditions and tau, the protocols of the present invention can be used to treat or prevent any subject that exhibits elevated levels of tau or phosphorylated tau (e.g., in CSF) compared to the mean in individuals without neurological diseases. The protocols of the present invention can also be used to treat or prevent neurological diseases in individuals with mutations in tau associated with neurological diseases. The methods of the present invention are particularly suitable for treating or preventing Alzheimer's disease, especially in patients.

[0466] Patients suitable for treatment include individuals with a risk of illness but no symptoms, and patients currently showing symptoms. Patients with a risk of illness include those with a known genetic risk of the disease. Such individuals include those individuals with relatives who have experienced the disease, and those individuals whose risk is determined by genetic or biochemical marker analysis. The genetic markers of risk include mutations in tau, such as those discussed above, and mutations in other genes associated with neurological diseases. For example, the ApoE4 allele in heterozygous and even especially homozygous forms is associated with the risk of Alzheimer's disease. Other markers of Alzheimer's disease risk include mutations in the APP gene, specifically mutations at position 717 and position 670 and 671 (respectively referred to as Hardy and Swedish mutations), mutations in presenilin genes PS1 and PS2, family history of AD, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be identified by PET imaging, by the presence of characteristic dementia and risk factors as described above. In addition, many diagnostic tests can be used to identify individuals suffering from AD. These include measurement of CSF tau or phosphorylated-tau and Aβ42 levels. Elevated tau or phosphorylated-tau and reduced Aβ42 levels mean the presence of AD. Some mutations are associated with Parkinson's disease. Mutations in Ala30Pro or Ala53 or other genes associated with Parkinson's disease such as leucine-rich repeat kinase PARK8. According to the criteria of DSM IV TR, individuals can also be diagnosed with any of the neurological diseases mentioned above.

[0467] In asymptomatic patients, treatment can start at any age (e.g., 10 years old, 20 years old, 30 years old). However, generally, treatment does not have to be started before the patient reaches 40, 50, 60 or 70 years old. It is usually necessary to carry out multiple doses of treatment over a period of time. Treatment can be monitored by measuring the antibody level over time. If the response decreases, a booster dose is required. In the case of potential Down syndrome patients, treatment can be started before birth or shortly after birth by administering a therapeutic agent to the mother.

[0468] I. Nucleic Acids

[0469] The present invention also provides nucleic acids encoding any of the above-mentioned heavy and light chains (e.g., SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:76-80, SEQ ID NO:90-91, SEQ ID NO:146-148, SEQ ID NO:83-85, SEQ ID NO:93-145, and SEQ ID NO:178-181). An exemplary nucleic acid encoding a heavy chain of the present invention is SEQ ID NO:182, and an exemplary nucleic acid encoding a light chain of the present invention is SEQ ID NO:183. Optionally, such nucleic acids further encode a signal peptide and can be expressed with a signal peptide connected to a variable region. The coding sequence of the nucleic acid can be operably linked to a regulatory sequence to ensure expression of the coding sequence, such as a promoter, an enhancer, a ribosome binding site, a transcription termination signal, and the like. The regulatory sequence can include a promoter, for example, a prokaryotic promoter or a eukaryotic promoter. The nucleic acid encoding the heavy or light chain can be codon optimized for expression in a host cell. The nucleic acid encoding the heavy and light chains can encode a selective gene. Nucleic acids encoding heavy and light chains can exist in isolated form or can be cloned into one or more vectors. Nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding heavy and light chains can be joined as a continuous nucleic acid in, for example, an expression vector, or can be separated, for example, cloned into their own expression vectors respectively.

[0470] J. Conjugated Antibodies

[0471] Conjugated antibodies that specifically bind to an antigen such as tau can be used to detect the presence of tau; monitor and evaluate the efficacy of therapeutic agents for treating patients diagnosed with the following diseases: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP); inhibit or reduce Aggregation of tau;Inhibit or reduce tau fibril formation;Reduce or remove tau deposits;Stabilize the non-toxic conformation of tau;Or treat the following diseases in patients or achieve the prevention of the following diseases: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitis Parkinson's disease, post-traumatic dementia or pugilistic dementia, 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 dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). For example, such antibodies can be conjugated with other therapeutic moieties, other proteins, other antibodies and / or detectable labels. See WO 03 / 057838; US 8,455,622. Such therapeutic moieties may be any agent that can be used to treat, combat, alleviate, prevent or ameliorate an unwanted condition or disease in a patient, such as Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0472] The therapeutic part of conjugation may include cytotoxic agents, cell growth inhibitors, neurotrophic agents, neuroprotective agents, radiotherapeutic agents, immunomodulators, or any bioactive agent that promotes or enhances the activity of antibodies. Cytotoxic agents may be any agents that are toxic to cells. Cytostatic agents may be any agents that inhibit cell proliferation. Neurotrophic agents may be any agents that promote neuronal maintenance, growth or differentiation, including chemical agents or protein agents. Neuroprotective agents may be agents that protect neurons from acute damage or degenerative processes, including chemical agents or protein agents. Immunomodulators may be any agents that stimulate or inhibit the development or maintenance of immune responses. Radiotherapeutic agents may be any molecules or compounds that emit radiation. If such therapeutic parts are coupled to tau-specific antibodies, such as antibodies described herein, the coupled therapeutic parts will have specific affinity for tau-related disease-affecting cells relative to normal cells. Therefore, the administration of conjugated antibodies directly targets cancer cells, while having minimal damage to surrounding normal healthy tissues. This may be particularly useful for therapeutic parts that are too toxic to be administered alone. In addition, a smaller amount of therapeutic parts may be used.

[0473] Some such antibodies can be modified to act as immunotoxins. See, for example, U.S. Pat. No. 5,194,594. For example, the plant-derived cytotoxin ricin can be coupled to an antibody using the bifunctional reagent S-acetylmercaptosuccinic anhydride for antibodies and 3-(2-pyridyldithio) propionic acid succinimidyl ester for ricin. See Pietersz et al., Cancer Res. 48(16): 4469-4476 (1998). The coupling results in the loss of the B chain binding activity of ricin, while not compromising the toxic potential of the A chain of ricin, nor the activity of the antibody. Similarly, saporin, an inhibitor of ribosome assembly, can be coupled to an antibody via a disulfide bond between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18: 1215-1222 (2004).

[0474] Some of these antibodies may be linked to a radioactive isotope. Examples of radioactive isotopes include, for example, yttrium 90 (90Y), Indium 111 (111In), 131 I. 99 mTc, radioactive silver-111, radioactive silver-199, and bismuth 213. The attachment of radioisotopes to antibodies can be performed using conventional bifunctional chelates. For radiosilver-111 and radiosilver-199 attachment, a thiol linker can be used. See Hazra et al., Cell Biophys. 24-25: 1-7 (1994). Attachment of silver radioisotopes can involve reduction of immunoglobulins with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1: S91-S95 (2001).

[0475] Some such antibodies can be linked to other therapeutic moieties. Such therapeutic moieties can have, for example, cytotoxicity, cell growth inhibition, neurotrophicity or neuroprotective properties. For example, antibodies can be conjugated to toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binders (e.g., auristatin), or minor groove binders such as calicheamicin. Other representative therapeutic moieties include agents known to be useful for the treatment, management, or amelioration of Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0476] Antibodies can also be coupled with other proteins. For example, antibodies can be coupled with Fynomers. Fynomers are small binding proteins (e.g., 7 kDa) derived from human Fyn SH3 domains. They can be stable and soluble, and they can lack cysteine ​​residues and disulfide bonds. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. They are suitable for producing multi-specific fusion proteins based on antibodies. For example, Fynomers can be fused to the N-terminus and / or C-terminus of antibodies to produce bispecific and trispecific FynomAbs with different architectures. Fynomer libraries can be used, and Fynomers can be selected by screening techniques using FACS, Biacore, and cell-based assays that allow effective selection of Fynomers with optimal properties. Examples of fynomers 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(Pt 6):1124-1137 (2013); and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

[0477] The antibodies disclosed herein may also be coupled or conjugated to one or more other antibodies (e.g., to form an antibody heteroconjugate). Such other antibodies may bind to different epitopes within tau or may bind to different target antigens.

[0478] The antibody can also be coupled to a detectable label. Such antibodies can be used, for example, to diagnose Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, 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 dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), and / or to evaluate the efficacy of treatment. Such antibodies are particularly useful in subjects suffering from or susceptible to Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, amyotrophic lateral sclerosis / Parkinson's dementia complex of Guam, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP), or in performing such assays in appropriate biological samples obtained from such patients. Representative detectable labels that can be coupled or linked to the antibody include various enzymes, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radiosilver-111, radiosilver-199, bismuth-199, fluorescein ... 213 ,iodine( 131 I. 125 I. 123 I. 121 I), carbon ( 14 C) Sulfur ( 5 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In, 111 In), Technetium ( 99 Tc), thallium ( 201 Ti、Ga( 68 Ga, 67 Ga), Palladium ( 103 Pd), Mo (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 Such as 68 Ge, 57 Co. 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn and 117 Tin; positron-emitting metals, using various positron emission tomography techniques; non-radioactive paramagnetic metal ions; and molecules that are radiolabeled or conjugated to specific radioisotopes.

[0479] The connection of the radioisotope to the antibody can be carried out with conventional bifunctional chelates. For radioactive silver-111 and radioactive silver-199 connection, a thiol linker can be used. See Hazra et al., Cell Biophys. 24-25: 1-7 (1994). The connection of the silver radioisotope can involve reducing the immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl. 1: S91-S95 (2001).

[0480] Therapeutic moieties, other proteins, other antibodies, and / or detectable labels can be coupled or conjugated to the antibodies of the invention directly or indirectly through an intermediate (eg, a linker). See, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol. Rev., 62: 119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers that release the coupled therapeutic moiety, protein, antibody, and / or detectable label under acidic or reducing conditions, upon exposure to a specific protease, or under other defined conditions can be used.

[0481] VI. Pharmaceutical Compositions and Methods of Use

[0482] In preventive applications, antibodies or agents for inducing antibodies or pharmaceutical compositions of the antibodies or agents are administered to patients susceptible to the disease or at risk of the disease in a regimen (dose, frequency and route of administration) that can effectively reduce the risk of the disease (e.g., Alzheimer's disease), reduce the severity of the disease, or delay the onset of at least one sign or symptom of the disease. Specifically, the regimen preferably effectively inhibits or delays tau or phosphorylated-tau and paired filaments formed therefrom in the brain, and / or inhibits or delays its toxic effects and / or inhibits / or delays the development of behavioral defects. In therapeutic applications, antibodies or agents for inducing antibodies are administered to patients suspected of having or already having the disease in a regimen (dose, frequency and route of administration) that can effectively improve or at least inhibit further deterioration of at least one sign or symptom of the disease (e.g., Alzheimer's disease). Specifically, the regimen preferably effectively reduces or at least inhibits further increases in the levels of tau, phosphorylated tau or paired filaments formed therefrom associated with toxicity and / or behavioral defects.

[0483] A regimen is considered therapeutically or prophylactically effective if the outcome achieved by an individual treated patient is more favorable than the average outcome in a control population of comparable patients not treated by the methods of the invention, or if the outcome of the treated patient is demonstrated to be more favorable relative to control patients in a controlled clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial) at a level of p<0.05 or 0.01 or even 0.001.

[0484] The effective dose will vary depending on many different factors, such as the mode of administration, the target site, the physiological state of the patient, whether the patient is an ApoE carrier, whether the patient is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic.

[0485] An exemplary dosage range of the antibody is about 0.01 to 60 mg / kg, or about 0.1 to 3 mg / kg, or 0.15-2 mg / kg, or 0.15-1.5 mg / kg of patient body weight. The antibody can be administered daily, every other day, weekly, every two weeks, monthly, quarterly, or according to any other schedule determined by empirical analysis. Exemplary treatment requires long-term administration of multiple doses, such as at least six months. Other exemplary treatment regimens require administration once every two weeks, or once a month, or once every 3 to 6 months.

[0486] For human administration, the amount of the agent for active administration varies in the range of 0.1 μg to 500 μg per patient and more generally in the range of 1 μg to 100 μg or 1 μg to 10 μg per injection. The schedule of injection can vary significantly, from once a day, to once a year, to once a decade. A typical regimen consists of immunization, followed by booster injections at intervals such as 6 weeks or two months. Another regimen consists of immunization, followed by booster injections after 1 month, 2 months, and 12 months. Another regimen requires a lifelong injection once every two months. Alternatively, booster injections can be performed irregularly, as indicated by monitoring immune responses.

[0487] The antibody or the agent for inducing the antibody is preferably administered via an external route (i.e., the route by which the administered or induced antibody crosses the blood-brain barrier to reach the intended location in the brain). The route of administration includes topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, or intramuscular. The preferred route for antibody administration is intravenous and subcutaneous. The preferred route for active immunization is subcutaneous and intramuscular. This type of injection is most commonly performed in the arm or leg muscles. In some methods, the agent is injected directly into a specific tissue where deposits accumulate, such as an intracranial injection.

[0488] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form (i.e., a dose for a single administration). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulating agents, such as suspending agents, stabilizers and / or dispersants. Alternatively, the antibody can be in lyophilized form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.

[0489] The regimen of the invention may be administered in combination with another agent that is effective in treating or preventing the disease being treated. For example, in the case of Alzheimer's disease, the regimen of the invention may be combined with immunotherapy for Aβ (WO / 2000 / 072880), cholinesterase inhibitors or memantine, or in the case of Parkinson's disease, the regimen of the invention may be combined with immunotherapy for alpha synuclein WO / 2008 / 103472, levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, amantadine or anticholinergic agents.

[0490] The antibody is administered with an effective regimen, which means a dosage, route of administration and frequency of administration that delays onset, reduces severity, inhibits further deterioration and / or improves at least one sign or symptom of the disorder being treated. If the patient already suffers from the disorder, the regimen can be referred to as a therapeutically effective regimen. If the patient is at a high risk of suffering from the disorder relative to the general population but has not yet developed symptoms, the regimen is referred to as a preventive effective regimen. In some cases, treatment or preventive efficacy can be observed in individual patients relative to historical controls or past experiences of the same patient. In other cases, treatment or preventive efficacy can be demonstrated in preclinical or clinical trials of a population of treated patients relative to a control population of untreated patients.

[0491] An exemplary dosage of the antibody is 0.1-60 mg / kg (e.g., 0.5, 3, 10, 30, or 60 mg / kg), or 0.5-5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4, or 5 mg / kg) or 10-4000 mg or 10-1500 mg as a fixed dose. The dosage depends on the patient's condition and response to previous treatment (if any), whether the treatment is preventive or therapeutic, and whether the condition is acute or chronic, and other factors.

[0492] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Some antibodies can be administered to the systemic circulation by intravenous or subcutaneous administration. Intravenous administration can, for example, be by infusion over a period of time such as 30-90 min.

[0493] The frequency of administration depends on the half-life of the antibody in the circulation, the patient's condition and the route of administration and other factors. The frequency can be daily, weekly, monthly, quarterly or irregularly in response to changes in the patient's condition or the progression of the illness being treated. An exemplary frequency of intravenous administration is between weekly and quarterly within a continuous treatment process, but more frequent or less frequent administration is also possible. For subcutaneous administration, an exemplary administration frequency is daily to monthly, but more frequent or less frequent administration is also possible.

[0494] The number of doses used depends on whether the disease is acute or chronic and the response of the disease to treatment. For acute or chronic conditions, 1 to 10 doses are usually sufficient. Sometimes a single bolus dose, optionally in a separate form, is sufficient for acute or chronic conditions. Treatment can be repeated for acute or chronic conditions. For chronic conditions, antibodies can be administered regularly, for example, weekly, biweekly, monthly, quarterly, or every six months for at least 1 year, 5 years, or 10 years, or for the patient's lifetime.

[0495] A. Diagnostic and Monitoring Methods

[0496] In vivo imaging, diagnostic methods, and optimizing immunotherapy

[0497] The present invention provides methods for imaging tau protein deposits (e.g., neurofibrillary tangles and tau inclusions) in patients. The methods are performed by administering to the patient an agent such as an antibody that binds to tau (e.g., a mouse, humanized, chimeric, or veneered 3D6 antibody) and then detecting the agent after it binds. Antibodies that bind to tau epitopes within amino acid residues 199-213 or 262-276 of SEQ ID NO:3 (corresponding to amino acid residues 257-271 or 320-334 of SEQ ID NO:1, respectively) or within amino acid residues 259-268 or 290-299 or 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. By using antibody fragments lacking full-length constant regions, such as Fab, the clearance response to the administered antibody can be avoided or reduced. In some methods, the same antibody can be used as both a therapeutic and a diagnostic agent.

[0498] The diagnostic reagent can be administered to the patient by intravenous injection, or directly into 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 the treatment method. Typically, the reagent is labeled, but in some methods, the main reagent with affinity for tau is not labeled and a second labeling agent is used to bind the main reagent. The choice of label depends on the detection means. For example, fluorescent labels are suitable for optical detection. The use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Positron emission tomography (PET) or single photon emission computed tomography (SPECT) can also be used to detect radioactive labels.

[0499] The in vivo imaging method of tau protein deposits can be used to diagnose tau diseases (such as Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy and Pick's disease) or confirm its diagnosis or susceptibility to this disease. For example, the method can be used for patients showing symptoms of dementia. If the patient has abnormal neurofibrillary tangles, the patient may suffer from Alzheimer's disease. Alternatively, if the patient has abnormal tau inclusions, the patient may suffer from frontotemporal lobar degeneration depending on the position of the inclusions. The method can also be used for asymptomatic patients. The presence of abnormal tau protein deposits indicates susceptibility to future symptomatic diseases. The method can also be used to monitor disease progression and / or the response to treatment in patients previously diagnosed with tau related diseases.

[0500] Diagnosis can be performed by comparing the number, size and / or intensity of the marker locus with the corresponding baseline value. The baseline value can represent the average level of a population of non-diseased individuals. The baseline value can also represent a previous level determined in the same patient. For example, a baseline value can be determined in a patient before starting tau immunotherapy treatment, and then the measured value is compared with the baseline value. A decrease relative to the baseline value conveys a signal of a positive response to treatment.

[0501] In some patients, the diagnosis of tau disease can be assisted by performing PET scanning. PET scanning can be performed using, for example, conventional PET imagers and auxiliary equipment. Scanning generally includes one or more brain regions commonly known to be associated with tau protein deposits and one or more regions in which there is usually little (if any) deposit to serve as a control.

[0502] The signal detected in the PET scan can be represented as a multidimensional image. The multidimensional image can be the two-dimensional representation of the cross section through the brain, the three-dimensional representation of the three-dimensional brain, or the four-dimensional representation of the change of the three-dimensional brain over time. Color scales with different colors can be used to indicate different labeling amounts and speculatively indicate the detected tau protein deposits. The scan result can also be presented digitally using the numbers related to the amount of labeling amount detected and therefore the amount of tau protein deposits. The mark present in the brain region associated with the deposit of a known specific tau disease (for example, Alzheimer's disease) can be compared with the mark present in the region known to be unrelated to the deposit, to provide a ratio indicating the deposit degree in the previous region. For the same radiolabeled ligand, this type of ratio provides a comparable measure of tau protein deposits and their changes between different patients.

[0503] In some methods, a PET scan is performed simultaneously with an MRI or CAT scan or during the same patient visit. An MRI or CAT scan provides more detail of the brain's anatomy than a PET scan. However, images from a PET scan can be superimposed on the MRI or CAT scan images, thereby more accurately indicating the location of the PET ligand and, by extension, the location of tau deposits relative to the anatomy of the brain. Some machines can perform both a PET scan and an MRI or CAT scan without requiring the patient to change position between scans, thereby facilitating superposition of the images.

[0504] Suitable PET ligands include radiolabeled antibodies of the invention (e.g., mouse, humanized, chimeric, or veneered 3D6 antibodies). The radioisotope used can be, for example, C 11 、N 13 , O 15 、F 18 , or I 123 The interval between administration of the PET ligand and performance of the scan may depend on the PET ligand, and in particular its uptake and clearance rate in the brain, and the half-life of its radiolabel.

[0505] PET scans can also be performed as a preventive measure in asymptomatic patients or in patients who have symptoms of mild cognitive impairment but have not yet been diagnosed with tauopathy but are at high risk of developing tauopathy. For asymptomatic patients, the scan is particularly useful for individuals who are considered to be at high risk of tauopathy due to family history, genetic or biochemical risk factors or middle-aged and elderly people. For example, preventive scans can be started at a patient age between 45 and 75 years old. In some patients, the first scan is performed at the age of 50.

[0506] Preventive scan can be carried out with, for example, between six months and ten years, preferably between 1 to 5 years interval.In some patients, preventive scan is carried out once a year.If the PET scan carried out as a preventive measure indicates abnormally high tau protein deposits level, then immunotherapy can be started and PET scan is carried out subsequently, just as in the patient diagnosed with tau protein disease.If the PET scan carried out as a preventive measure indicates that the level of tau protein deposits is within normal levels, then other PET scans can be carried out in response to the appearance of signs or symptoms of tau protein disease or mild cognitive impairment as before with between six months and 10 years and preferably 1 to 5 years.If and when detecting tau protein deposits higher than normal levels, by combining preventive scan with the administration of tau directed immunotherapy, the level of tau protein deposits can be reduced to or close to normal levels, or at least suppress further increase, and compared with not receiving preventive scan and tau directed immunotherapy, the patient can keep not suffering from tau protein disease (for example, at least 5 years, 10 years, 15 years or 20 years, or the rest of the patient's life) for a longer time.

[0507] The normal level of tau protein deposits can be determined by the amount of brain neurofibrillary tangles or tau inclusions of a representative sample (e.g., a representative sample of disease-free individuals under 50 years old) of an individual in a general population that is not diagnosed with a specific tau disease (e.g., Alzheimer's disease) and is not considered to be in the high risk of developing this disease. Alternatively, if the PET signal according to this method in the brain region in which it is known that tau protein deposits have developed is different from (within the accuracy of measurement) the signal from the brain region in which such deposits are known not to develop usually, then normal levels can be identified in individual patients. By comparing with normal levels (e.g., the variance of external mean and standard deviation), or only by the signal of the rising that exceeds the experimental error in the brain region associated with tau protein deposits compared with the region that is not known to be associated with deposits, the rising level in the individual can be identified. In order to compare the level of tau protein deposits in individual and colony, tau protein deposits should preferably be determined in one or more identical regions of the brain, and these regions include at least one region of tau protein deposits known to be formed and associated with specific tau protein disease (e.g., Alzheimer's disease). Patients with elevated levels of tau deposits are candidates for starting immunotherapy.

[0508] After starting immunotherapy, the reduction in the level of tau protein deposits can first be considered as an indication that the treatment has a desired effect. The observed reduction can be, for example, in the range of 1-100%, 1-50%, or 1-25% of the baseline value. Such effects can be measured in one or more regions of the brain where deposits are known to form, or can be measured by the average value of such regions. The total effect of the treatment can be approximately calculated by adding the increase in tau protein deposits that may exist in the average untreated patient to the percentage reduction relative to the baseline.

[0509] The maintenance of tau protein deposits at approximately constant levels or even a small increase in tau protein deposits can also indicate a response to treatment, although a non-optimal response. Such responses can be compared with the time course of tau protein deposit levels in patients with specific tau diseases (e.g., Alzheimer's disease) who are not receiving treatment to determine whether immunotherapy has the effect of inhibiting further increases in tau protein deposits.

[0510] Monitoring of changes in tau protein deposits allows immunotherapy or other treatment regimens to be adjusted in response to treatment. PET monitoring provides an indication of the nature and extent of the response to treatment. Then, it can be determined whether to adjust the treatment, and if necessary, the treatment can be adjusted in response to PET monitoring. Therefore, PET monitoring allows tau-directed immunotherapy or other treatment regimens to be adjusted before other biomarkers, MRI or cognitive measurements have been detectably responded. Significant changes mean that the comparison of parameter values ​​after treatment relative to baseline provides some evidence that treatment has or has not yet produced a beneficial effect. In some cases, the change in parameter values ​​in the patient himself provides evidence that treatment has or has not yet produced a beneficial effect. In other cases, the change in the value in the patient (if any) is compared with the change in the value in the representative control group of the patient who has not undergone immunotherapy (if any). The difference (e.g., the variance of the mean value plus the standard deviation) of the response of a specific patient and the normal response of the control patient can also provide evidence that the immunotherapy regimen has or has not achieved a beneficial effect in the patient.

[0511] In some patients, monitoring indicates a detectable decrease in tau deposits, but the level of tau deposits remains above normal. In such patients, if there are no unacceptable side effects, the treatment regimen can be continued as is, or even the frequency of administration and / or dosage can be increased (if the maximum recommended dose has not yet been reached).

[0512] If monitoring indicates that the level of tau protein deposits in the patient has been reduced to a normal or near-normal level of tau protein deposits, the immunotherapy regimen can be adjusted from an induction regimen (i.e., reducing the level of tau protein deposits) to a maintenance regimen (i.e., maintaining tau protein deposits at a substantially constant level). This regimen can be achieved by reducing the dose and / or frequency of administering the immunotherapy.

[0513] In other patients, monitoring can indicate that immunotherapy has some beneficial effects but non-optimal effect.Optimal effect can be defined as the percentage reduction (measured or calculated in its representative region formed therein in the whole brain or known tau protein deposits) of the change of tau protein deposits experienced by the representative sample of the tau disease patient of immunotherapy at a given time point after starting therapy.Experience less reduced patients, or its tau protein deposits remain constant or even increase but the degree is less than the degree expected in the absence of immunotherapy (for example, as inferred from the control group of the patient of immunotherapy) can be classified as experiencing positive but non-optimal response.Such patients can optionally carry out scheme adjustment, wherein increased the dosage of agent and or the frequency of administration.

[0514] In some patients, tau deposits may increase in a manner similar to or greater than tau deposits in patients not receiving immunotherapy. If such an increase persists for a period of time, such as 18 months or 2 years, even after any increase in the frequency or dosage of the agent, then immunotherapy may be discontinued as needed in favor of other treatments.

[0515] The above description of diagnosis, monitoring and adjustment of the treatment of tau disease focuses primarily on the use of PET scanning. However, any other technique for visualizing and / or measuring tau protein deposits suitable for the use of the tau antibodies of the present invention (e.g., mouse, humanized, chimeric or veneered 3D6 antibodies) may be used instead of PET scanning to perform such methods.

[0516] Also provided are methods for detecting an immune response to tau in a patient suffering from or susceptible to a disease associated with tau. The methods can be used to monitor the course of therapeutic and prophylactic treatments using agents provided herein. The antibody spectrum after passive immunization typically shows an immediate peak in antibody concentration followed by an exponential decay. In the absence of re-administration, the decay approaches pre-treatment levels over a period of days to months depending on the half-life of the administered antibody. For example, the half-life of some human antibodies is about 20 days.

[0517] In some methods, a baseline measurement of antibodies against tau in a subject is performed before administration, a second measurement is performed shortly thereafter to determine the peak antibody level, and one or more further measurements are performed at intervals to monitor the decay of antibody levels. When the antibody level has dropped to a predetermined percentage (e.g., 50%, 25%, or 10%) of the peak less than the baseline or the baseline, another dose of the antibody is administered. In some methods, the peak or subsequently determined level below the background is compared with a previously determined reference level to construct a beneficial preventive or therapeutic treatment regimen in other subjects. If the measured antibody level is significantly less than the reference level (e.g., less than the mean of the reference value in the subject population benefiting from the treatment minus one, or preferably, two standard deviations), an additional dose of the antibody is indicated.

[0518] Also provided are methods 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 methods can be used to diagnose or confirm the diagnosis of a disease associated with tau or its susceptibility. The methods can also be used for asymptomatic subjects. The presence of tau indicates susceptibility to future symptomatic diseases. These methods can also be used to monitor 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, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0519] Biological samples obtained from subjects suffering from, suspected of suffering from, or having a risk of suffering from the following diseases can be contacted with antibodies disclosed herein to assess the presence of tau: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, Niemann-Pick type C, supranuclear palsy, frontotemporal dementia, frontotemporal degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). For example, the tau levels in such subjects can be compared with those levels present in healthy subjects. Alternatively, the tau levels in such subjects receiving treatment for the disease can be compared with those tau levels in subjects not treated for the following diseases: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, C-type Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant (LBVAD) of Alzheimer's disease, chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). Some such tests relate to tissue biopsies obtained from such subjects. ELISA determination can also be a useful method, for example, for assessing tau in a fluid sample.

[0520] VII.Medicine Box

[0521] The invention also provides a kit (e.g., container) comprising an antibody disclosed herein and related materials such as instructions for use (e.g., package inserts). The instructions for use may include, for example, instructions for administering the antibody and optionally one or more additional agents. The container of the antibody may be a unit dose, bulk (e.g., multi-dose packaging), or a subunit dose.

[0522] Package insert refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products.

[0523] The kit may also include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0524] VIII. Other Applications

[0525] Antibodies can be used to detect tau or its fragments in the case of clinical diagnosis or treatment or research. For example, antibodies can be used to detect the presence of tau in a biological sample as an indication that the biological sample includes tau deposits. The binding of the antibody to the biological sample and the binding of the antibody to the control sample can be compared. The control sample and the biological sample may include cells of the same tissue origin. The control sample and the biological sample may be from the same individual or different individuals and may be obtained on the same occasion or on different occasions. If necessary, multiple biological samples and multiple control samples are evaluated on multiple occasions to counteract random changes that are unrelated to the differences between the samples. Direct comparisons can then be made between one or more biological samples and one or more control samples to determine whether the binding of the antibody to the one or more control samples (i.e., the presence of tau) increases, decreases, or is the same. Relative to one or more control samples, the increase in the binding of the antibody to the one or more biological samples indicates the presence of tau in the one or more biological samples. In some cases, the increased antibody binding is statistically significant. Optionally, binding of the antibody 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 binding of the antibody to a control sample.

[0526] In addition, these antibodies can be used to detect the presence of tau in a biological sample to monitor and evaluate the efficacy of therapeutic agents used to treat patients diagnosed with the following diseases: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). To be diagnosed as suffering from Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitis Parkinson's disease, post-traumatic dementia or pugilistic dementia, Pick's disease, C-type Niemann-Pick disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or biological samples of patients with progressive supranuclear palsy (PSP) are evaluated to establish a baseline (i.e., a baseline of tau presence in the sample) of the binding of the antibody to the sample before starting therapy with a therapeutic agent. In some cases, multiple biological samples from patients are evaluated on multiple occasions to establish a baseline and a measure of random variation that is unrelated to treatment. The therapeutic agent is then administered in a regimen. The regimen may include multiple administrations over a period of time. Optionally, the binding of the antibody (i.e., the presence of tau) is evaluated in multiple biological samples from the patient on multiple occasions to establish a measure of stochastic variation and to show a trend in response to immunotherapy. Various assessments of the binding of the antibody to the biological sample are then compared. If only two assessments are performed, a direct comparison can be made between the two assessments to determine whether the antibody binding (i.e., the presence of tau) increases, decreases, or remains the same between the two assessments. If more than two measurements are performed, the measurements can be analyzed as a time course that begins before treatment with the therapeutic agent and progresses throughout the course of treatment.In patients in whom the binding of the antibody to the biological sample (i.e., the presence of tau) has been reduced, it can be inferred that the therapeutic agent is effective in treating the following diseases in the patient: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or pugilistic dementia, 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 dementia complex, cortical basal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP). The reduction in antibody binding can be statistically significant. Optionally, 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 antibody binding may be combined with assessment of other signs and symptoms of the following diseases: Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, ALS / Parkinson's dementia complex of Guam, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

[0527] Antibodies can also be used as research reagents for laboratory studies to detect tau or its fragments. In such uses, the antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in the form of a kit with all necessary reagents to perform the detection assay. Antibodies can also be used to purify tau, or a binding partner of tau, for example, by affinity chromatography.

[0528] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference as a whole for all purposes, to the extent that each individual item is specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, then the version associated with the accession number on the effective application date of the present application is meaningful. The effective application date means the application date (if applicable) earlier than the actual application date or the priority application related to the accession number. Similarly, if different versions of publications, websites, etc. are announced at different times, then the version published most recently on the effective application date of the present application is meaningful, unless otherwise specified. Unless otherwise specifically specified, any feature, step, element, embodiment or aspect of the present invention may be used in combination with any other. Although the present invention has been described in considerable detail by way of illustration and example for the purpose of clarity and understanding, it is apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0529] Example

[0530] Example 1. Identification of tau monoclonal antibodies

[0531] Monoclonal antibodies against tau were generated as follows. Immunization was performed with recombinant 383a.a. human tau with an N-terminal His tag containing the P301S mutation (4RON) [immunogen A] or recombinant 383a.a. human tau containing the P301S mutation and lacking an N-terminal His tag (4RON) [immunogen B]. The immunogens were emulsified in RIBI adjuvant.

[0532] 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. On days 43 and 98, mice were bled and titered against immunogen A; on day 101, animals with the highest titers were boosted with a terminal immunization of 50 μg of immunogen B. 1 / 2Intraperitoneal and 1 / 2 intravenous delivery. The fused hybridomas were screened for both immunogens via ELISA, and the positive hybridomas with the highest signal were subjected to epitope mapping (see Example 2). 3D6 reacts with a peptide 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, this corresponds to amino acid residues 257-271 of SEQ ID NO: 1 and amino acid residues 320-334 of SEQ ID NO: 1).

[0533] Example 2. Epitope mapping of antibody 3D6

[0534] A series of overlapping biotinylated peptides spanning the entire 383aa 4RON human tau protein were used to map the murine 3D6 antibody. Additional peptides were used to model potential post-translational modifications at the C- and N-termini of the protein.

[0535] Biotinylated peptides are bound to individual wells of streptavidin-coated ELISA plates. The plates are blocked and treated with mouse 3D6 and then incubated with horseradish peroxidase-conjugated anti-mouse antibodies. After thorough washing, OPD is applied to the plates and developed. The absorbance of the plates at 450 nm is read. Background subtraction is performed using the absorbance values ​​of the wells without the primary antibody, and the threshold value for positive binding is set to 0.2 absorbance units.

[0536] 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 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 amino acid residues 320-334 (of SEQ ID NO: 1). Figure 1 )

[0537] Example 3. Design of humanized 3D6 antibody

[0538] The starting point or donor antibody for humanization was the mouse antibody 3D6. The heavy chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:7. The light chain variable amino acid sequence of mature m3D6 is provided as SEQ ID NO:11. The heavy chain Kabat / Chothia complex 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.

[0539] The variable kappa (Vk) of 3D6 belongs to mouse Kabat subgroup 2, which corresponds to human Kabat subgroup 2, and the variable heavy chain (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]. In Vk, the 16-residue Chothia CDR-L1 belongs to canonical class 4, the 7-residue Chothia CDR-L2 belongs to class 1, and the 9-residue Chothia CDR-L3 belongs to class 1 [Martin AC and Thornton JM (1996) J. Mol. Biol. 263: 800-15. [Martin & Thornton, 1996]. The 10-residue Chothia CDR-H1 belongs to class 1, and the 17-residue Chothia CDR-H2 belongs to class 2 [Martin & Thornton, 1996]]. CDR-H3 is not classified in the canonical category. The protein sequences in the PDB database were searched [Deshpande N et al., (2005) Nucleic Acids Res. 33: D233-7.] to find structures that would provide a rough structural model of 3D6. To build the Fv model of 3D6, the structure of the mouse anti-pyroglutamate-Aβ antibody Fab c#24 (pdb code 5MYX) was used [Piechotta, A. et al., 2017, J Biol Chem. 292: 12713-12724] with a resolution of 1.4A. It retains the same ring canonical structure as 3D6.

[0540] The framework of 3D6 VH has a high 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 length on the CDR-H1 and H2 loops. Similarly, the framework of 3D6 VL has a high sequence similarity with the corresponding region of the human antibody ARX71335 VL cloned by Dafferner, AJ et al. (2017; Direct submission). The variable light domains of 3D6 and ARX71335 antibodies also share the same length on the CDR-L1, L2 and L3 loops. Therefore, the framework regions of 48G7VH (2RCS-VH) and ARX71335 VL were selected as the acceptor sequences of the CDRs of 3D6. Models of the 3D6 CDRs grafted onto the corresponding human frameworks of VH and VL were constructed and used as a guide for further backmutations.

[0541] The heavy and light chain variant sequences generated by the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the humanness of the heavy and light chains, as outlined by the WHO INN Committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (review) (Internet) 2014. Available from: http: / / www.who.int / medicines / services / inn / BioRev2014.pdf) Where possible, residues were changed to align with the corresponding human germline sequence to enhance humanness and reduce potential immunogenicity. For the humanized VLvb2 variants and VLvb3 variants, mutations were introduced to make the sequence more similar to the human germline gene IGKV2-30*02 (SEQ ID NO: 27) For the humanized VHvb2, VHvb3, VHvb4, VHvb5, VHvb6 and VHvb6 variants, mutations were introduced to make the sequence more similar to the human germline gene IGHV1-69-2*01 (SEQ ID NO: 25)

[0542] Versions of hu3D6-VH and hu3D6-VL were designed to allow for evaluation of the contribution of various framework residues to antigen binding, thermal stability, and immunogenicity, as well as for optimization of glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface exposed charged patches, surface exposed charge patches, deamination, and protease sensitivity. Positions considered for mutation included the following:

[0543] - Define canonical CDR conformations (summarized in Martin, ACR (2010) Protein sequence and structure analysis of antibody variable domains. In: Kontermann R and Dübel S (eds.). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.),

[0544] - within the fine-tuning region (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224 (2): 487-99.),

[0545] - located at the VH / VL domain interface (summarized in Léger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanisation by CDR grafting. In: Shepherd P and Dean C (eds.), Monoclonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.),

[0546] - susceptible to post-translational modifications, such as glycosylation or pyroglutamination,

[0547] - occupied by residues predicted to clash with CDRs based on the 3D6 CDR models grafted onto the VH and VL frameworks, or

[0548] - Occupancy of residues that are rare in sequenced human antibodies, where the parental mouse 3D6 residue or some other residue is more prevalent in the human antibody repertoire.

[0549] The light chain variable regions of the murine 3D6 antibody and various humanized antibodies are shown (Tables 4 and Figure 3 ) and heavy chain variable region (Table 3 and Figure 2 ) comparison.

[0550] Seven humanized heavy chain variable region variants and three humanized light chain variable region variants containing different substitution arrangements were constructed: hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6Hvb5, 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). Exemplary humanized Vk and Vh designs with back mutations based on selected human frameworks and other mutations are shown in Tables 3 and 4, respectively. The bold regions in Tables 3 and 4 represent CDRs defined by the Kabat / Chothia complex. SEQ ID NOs: 76 to 80 and SEQ ID NOs: 90 to 91 contain back mutations and other mutations as shown in Table 5. The amino acids at the positions in hu3D6VHvb1, hu3D6VHvb2, hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7 are listed in Table 6. The amino acids at the positions in hu3D6VLvb1, hu3D6VLvb2, and hu3D6VLvb3 are listed in Table 7.

[0551] Table 3

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558] Table 4

[0559]

[0560]

[0561]

[0562]

[0563]

[0564] Table 5: V of humanized 3D6H 、V L Back mutations and other mutations

[0565]

[0566]

[0567]

[0568] Table 6: Kabat numbering of backmutations and other mutated framework (or CDR) residues (based on Kabat / Chothia complex CDRs) in the heavy chain of the humanized 3D6 antibody

[0569]

[0570]

[0571] Table 7: Kabat numbering of backmutations and other mutated framework residues (based on Kabat / Chothia complex CDRs) in the light chain of the humanized 3D6 antibody

[0572]

[0573]

[0574] Positions where canonical, fine-tuning, or interface residues differ between the mouse and human receptor sequences are candidates for substitution. Examples of canonical / CDR interaction residues include Kabat residues H54 and H94 in Table 3. Examples of fine-tuning residues include Kabat residues H28, H67, H93, and H94 in Table 3. Examples of interface / stacking (VH+VL) residues include Kabat residues H91 and H93 in Table 3.

[0575] The reasons for selecting the positions shown in Table 3 in the heavy chain variable region as substitution candidates are as follows.

[0576] Heavy chain variable region

[0577] hu3D6VHvb1

[0578] - consists of the CDR-H1, H2 and H3 loops of 3D6-VH grafted onto the framework of 48G7-VH (RCS-VH) with back mutations at positions H91 (Y91F), H93 (A93S) and H94 (S94T).

[0579] hu3D6VHvb2

[0580] - All framework substitutions were restored at the following positions: these positions are key positions for defining the Chothia canonical class, are part of the fine-tuning region, or are located at the VH / VL domain interface or contribute to structural stability. 3D6-VH_vb2 incorporates back mutations or substitutions Q1E, Q5V, L11V, L20I, T23K, K38R, E42G, Q43K, K66R, S75T, N76D, Q81E, Y91F, A93S, S94T T108L and L109V to enable assessment of the contribution of these positions to antigen binding affinity and immunogenicity.

[0581] hu3D6VHvb3, hu3D6VHvb4, hu3D6VHvb5, hu3D6VHvb6, and hu3D6VHvb7

[0582] Consists of additional substitutions and increases antibody stability and / or serves to optimize glycosylation, aggregation, N-terminal heterogeneity, thermal stability, surface exposed charged patches, surface exposed charge patches, deamination, and protease sensitivity.

[0583] Q1E: is a stability enhancing mutation used to reduce the possibility of pyroglutamate formation (Liu, supra)

[0584] Q5V: is a frequency-based germline aligned mutation. Val is most common at this position in human sequences. Val is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0585] L11V: is a germline aligned mutation. Val is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0586] S17T: is a germline aligned mutation. Thr is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0587] L20I: is a germline alignment mutation. Ile is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0588] T23K: is a frequency-based germline aligned mutation. Lys is more frequently at this position. Lys is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0589] N28T: This is a Thr residue substitution in CDR-H1.

[0590] K38R: is a frequency-based back mutation. Arg is most frequently present at this position. It is predicted that the Arg at this position forms two H bonds with Glu 46 in addition to one H bond each with Asp86 and Tyr 90 in the heavy chain; therefore, Arg substitution may enhance the stability of the Lys at this position.

[0591] E42G: is a frequency-based germline aligned mutation. Gly is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25). Gly is most frequently at this position. Gly substitution is not predicted to affect stability.

[0592] Q43K: In addition to the main chain H-bonds with Gln 39 and Arg 40, the Lys side chain at this position is predicted to form an H-bond with G42, thus Lys substitution may enhance the stability of Q at this position.

[0593] N54D and D56E are substitutions of CDR residues that are predicted to be non-antigen contacting positions based on homology modeling. It is predicted that the N54D substitution and the D56E substitution will stabilize the antibody structure.

[0594] V58I: is a substitution of a CDR-H2 residue. The germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25) has Ile at this position. This residue is predicted not to contact the antigen.

[0595] K66R: The Arg at this position is predicted to form H bonds and salt bridges with Asp 86 as well as with Ser 82a and Thr 83.

[0596] A67V: is a substitution of a residue in the fine-tuning region. The germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25) has Val at this position.

[0597] S75T: Ser at this position is predicted to form H bonds with Asp 72 and Tyr 76. Thr at this position is also predicted to make these contacts, but the surface exposed Thr residue may enhance antibody stability.

[0598] N76D:Asp is a germline aligned mutation. Asp is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0599] L80M:Met is a germline aligned mutation. Met is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0600] Q81E: Glu is predicted to form an H-bond plus salt bridge with K19, so Glu at this position may enhance antibody stability.

[0601] T83R is used to enhance thermal stability and to increase human origin. Arg is a germline aligned mutation. Arg is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25). Arg is most frequently at this position.

[0602] F91Y: is a mutation of an interface residue and is a frequency-based back mutation. Tyr is usually at this position and can enhance antibody stability.

[0603] A93S: is a back mutation of the fine-tuning region residues and the interface region residues.

[0604] S94T: is a back mutation of the canonical structural residues and fine-tuning residues defined by Chothia.

[0605] T108L:Leu is a germline aligned mutation. Leu is at this position in the human germline gene IMGT#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 the stability of the antibody.

[0606] L109V: is a frequency based mutation. Val is most frequently at this position.

[0607] The reasons for selecting the positions shown in Table 4 in the light chain variable region as substitution candidates are as follows.

[0608] Kappa light chain variable region

[0609] hu3D6VLvb1

[0610] - Consists of the CDR-L1, L2 and L3 loops of 3D6-VL grafted onto the ARX71335 VL framework.

[0611] hu3D6VLvb2 and hu3D6VLvb3

[0612] -Restore all framework substitutions at the following positions: These positions are key positions for defining Chothia canonical classes, are part of fine-tuning regions, or are located at the VH / VL domain interface. Hu3D6-VLvb2 and Hu3D6-VLvb3 also include substitutions that contribute to structural stability hu3D6-VL_vb2 incorporates back mutations T7S, I15L, L83V, H86Y and L106I to enable evaluation of the contribution of these positions to antigen binding affinity and immunogenicity.

[0613] Hu3D6-VL_vb3 all substitutions mentioned for vb2, with additional changes at Q17E, K24R, L37Q, K45R and L106I

[0614] T7S: is a germline aligned mutation. Ser is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0615] T10S: is a frequency-based germline aligned mutation. Ser is frequently at this position. Ser is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0616] I15L: is a germline aligned mutation. Leu is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0617] Q17E: Glu at this position is predicted to form an H-bond with T14 and a salt bridge with Lys 107, two light chain residues, and serve to improve antibody stability.

[0618] K24R: is a mutation of a CDR residue. Both Lys and Arg are predicted to form H bonds and salt bridges with Asp 70 in the light chain. Arg is predicted to be more suitable in this conformation. Arg is also a germline alignment mutation. Arg is at this position in the human germline gene IGKV2-30*02 (SEQ (IDNO: 27).

[0619] L37Q: This is predicted to be a deeply buried residue, Leu is predicted not to interact with surrounding residues, however, Gln is predicted to form H bonds with Q38 and Asp 82 in the light chain. Gln is also a germline aligned mutation. Gln is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0620] K45R: However, Lys is predicted to form H bonds with S56 and Gly 57; Arg is predicted to have a much wider range of interactions with adjacent residues, 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 at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0621] L83V: This is a frequency-based mutation of a residue predicted to be surface exposed. Val is also a germline aligned mutation. Val is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0622] H86Y: Murine 3D6 VL has Tyr at this position. Tyr is also the most frequently occurring residue at this position.

[0623] 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 most frequently at this position and is also a germline aligned mutation. Gln is at this position in the human germline gene IGKV2-30*02 (SEQ (IDNO: 27). Gln is predicted to form an H bond with Ser 7, thereby stabilizing the interior of the chain.

[0624] L106I: is a frequency-based germline aligned mutation. Ile is most frequently at this position. Ile is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0625] The designs based on these people's frameworks are:

[0626] Heavy chain variable region

[0627] >hu3D6VHvb1 (SEQ ID NO: 76)

[0628]

[0629] >hu3D6VHvb2 (SEQ ID NO: 77)

[0630]

[0631] >hu3D6VHvb3 (SEQ ID NO: 78)

[0632]

[0633] hu3D6VHvb4 (SEQ ID NO: 79)

[0634]

[0635] >hu3D6VHvb5 (SEQ ID NO: 80)

[0636]

[0637] >hu3D6VHvb6 (SEQ ID NO: 90)

[0638]

[0639] >hu3D6VHvb7 (SEQ ID NO: 91)

[0640]

[0641] Kappa light chain variable region

[0642] hu3D6VLvb1 (SEQ ID NO: 83)

[0643]

[0644] >hu3D6VLvb2 (SEQ ID NO: 84)

[0645]

[0646] >hu3D6VLvb3 (SEQ ID NO: 85)

[0647]

[0648] The humanized sequence was generated using a two-stage PCR protocol that allowed the introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, BA (1999) BioTechniques 26:680-682).

[0649] Example 4. Mouse monoclonal antibodies bind to tau in ELISA assays

[0650] Method: Indirect ELISA: 96-well polystyrene plates were plated with capture antibody anti-6xHis ( Figure 4A ) or polyclonal anti-tau (Dako #A0024, Figure 4B ) for 2 hr at room temperature or 16 hr at 4°C. The coating was removed and the plates were blocked with 1% BSA in 1xPBS for 1 hr and then incubated with human recombinant tau with ( Figure 4A ) or does not have ( Figure 4B ) polyhistidine tag. After washing, the plate was incubated with the indicated antibody, washed, and incubated with HRP-conjugated goat anti-mouse secondary antibody. The plate was developed with TMB and A was measured with a plate reader. 450 .

[0651] Sandwich ELISA: 96-well polystyrene plates were coated with anti-mouse antibodies in 1xPBS for 2 hr at room temperature or 16 hr at 4°C. The coating was removed and the plates were blocked with 1% BSA in 1xPBS for 1 hr. The plates were then incubated with the same concentrations of the indicated antibodies diluted in 0.1% BSA in 1xPBS. The plates were 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 washes between each step. Streptavidin-HRP was added, the plates were developed with TMB, and A was measured with a plate reader. 450 . See Figure 4C .

[0652] Results: Binding of antibodies produced by a panel of hybridomas to tau was determined by a number of different ELISA formats. Detection of tau was confirmed using an indirect format using tau protein immobilized via a polyhistidine tag fused to its N-terminus ( Figure 4A Binding to native untagged protein was also confirmed ( Figure 4B To evaluate the solution affinity of various antibodies, a sandwich ELISA format was used, using the hybridoma antibody being tested as the capture reagent ( Figure 4C ).

[0653] Example 5. Affinity of mouse monoclonal antibodies to tau

[0654] Methods: SPR analysis was performed using Biacore T200 to determine the binding kinetics of murine antibodies to recombinant human tau. To prepare the sensor surface, anti-mouse antibodies (GE Life Sciences) were immobilized on a sensor chip CM5 by amine coupling, and the antibodies were captured at a level to ensure maximum binding of 50RU. Recombinant tau at various concentrations ranging from 10-0.14nM was passed over the capture ligand at a flow rate of 50μL / min in running buffer (HBS+0.05% P-20, 1mg / mL BSA) for 180sec association and 900sec dissociation. The data were double-referenced to an unrelated sensor without antibody ligand and 0nM analyte concentration to account for the dissociation of the ligand from the capture portion. The data were then analyzed using a global 1:1 fit.

[0655] Results: Multiple murine antibodies were selected based on their performance in a battery of ELISA assays and their binding affinities were assessed by SPR. The antibodies were tested in parallel groups and their binding association and dissociation rates were compared to select the highest binders to recombinant human tau. The highest binding affinity was observed with antibody clone 3D6. The binding affinity was Figure 5Shown in.

[0656] Example 6. Mouse monoclonal antibodies prevent human tau from binding to the surface of immortalized neuronal cells

[0657] Methods: Inhibition of Tau binding to B103 neuroblastoma cells using anti-Tau monoclonal antibodies

[0658] 1. B103 cells were cultured at 5 x 10 5 Resuspend in PBS. Plate 50 μL of cell suspension per well in MSD High Bind plates. This gives 25K cells / well. Cover the plate and allow cells to attach for 2 hr at 37°C, 5% CO2.

[0659] 2. After cells attach, remove PBS from wells by inverting the plate and gently tapping to remove excess buffer. Add 50 μL of 3% MSD Blocker A in PBS or other suitable blocking buffer to each well and incubate the plate at room temperature for 1 hr with shaking.

[0660] 3. During the plate blocking step, co-incubate Tau and anti-Tau antibodies as follows:

[0661] a. Start with 2 mg / mL of anti-Tau antibody and serially dilute 1 :2 in PBS to make 7 additional dilutions.

[0662] b. Dilute Tau to 20 nM in PBS. The concentration of Tau in each well will remain constant.

[0663] c. Tau and anti-Tau antibody were mixed 1:1 to obtain a final Tau concentration of 10 nM and an anti-Tau starting concentration of 1 mg / mL.

[0664] d. Incubate the mixture at room temperature with shaking (600 rpm) for approximately 1 h.

[0665] 4. After the plate is blocked (step 2), remove the blocking buffer from the wells by inverting the plate and tapping gently, and wash the plate twice with PBS using a multichannel pipette. Make sure that excess buffer is completely removed. Cool the seeded cells to 4°C before adding the Tau: anti-Tau complex.

[0666] 5. Add 50 μL of the cooled complex (step 3) to the seeded cells and incubate on ice for 30 minutes.

[0667] 6. Wash plates twice with ice-cold PBS as previously described.

[0668] 7. Add 50 μL of 16B5.SULFO-TAG to each well to detect Tau bound to the cell surface. Incubate on ice for 30 minutes.

[0669] 8. Wash the plates twice again with ice-cold PBS as before.

[0670] 9. Add 150 μL of 1X Read Buffer T Without Surfactant (diluted in H2O) to each well and immediately place in MSD SECTOR TM Read on the instrument at 600. Avoid introducing bubbles when adding read buffer.

[0671] 10. Report the relationship between the MSD signal and the concentration of anti-Tau.

[0672] The antibodies tested were anti-tau antibodies 3D6, 16G7, 3H9, 4C5 and 5G8, and an isotype control.

[0673] result:

[0674] The gradual reduction of the SulfoTag anti-tau signal with increasing amounts of the tested antibodies indicated functional blockade of tau binding to the neuronal cell surface. No blocking was observed in isotype controls, 16G7, or 3H9. Increasing amounts of functional blocking activity were observed in 4C5, 5G8, and 3D6. 3D6 exhibited the deepest blocking activity of the antibodies tested. See Figure 6 .

[0675] Example 7. Depolymerization activity

[0676] Methods: Aggregation of recombinant tau - Purified recombinant tau with an N-terminal 6xHis tag was mixed with an equimolar amount of low molecular weight heparin in 1xPBS (pH 7.4) and incubated on a nutator at 37°C for 96 hr. Aggregation of the samples was confirmed by binding to thioflavin T.

[0677] Incubation with Antibodies - Antibodies were incubated with aggregated recombinant tau at the indicated molar ratios for 96 hr at 37°C without rotation or nutation. Aggregation was measured at the end of the experiment by incubating samples with 25 mM Thioflavin T and measuring the emitted fluorescence (450 / 482 ex / em). Signals were background subtracted to buffer samples.

[0678] Results: Figure 7As shown, 3D6 preferentially decomposes intact tau fibrils. Different molar ratios of 3D6 (triangles), isotype controls (circles) and 16G7 (squares) were incubated with amyloid-containing tau fibrils for 96 hours. At the end of this period, the degree of aggregation was assessed by binding to thioflavin T. Compared with isotype control antibodies and 16G7 (anti-tau antibodies bound to different regions of tau), 3D6 preferentially reduced the thioflavin T signal present in the sample.

[0679] Example 8. Immunocapture of tau from human disease tissue by 3D6 and 5G8.

[0680] Method: The high salt soluble protein fraction was prepared into 1 mg / ml. For each immunoprecipitation, 200 μg of sample was used. 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 hr. Protein G magnetic beads were then added to the mixture and incubated for another hour to capture the antibody / antigen complex. The sample was thoroughly washed with 1xPBS, and the beads were boiled in a reduction / denaturation sample buffer to release the captured protein. The resulting samples were separated by SDS-PAGE and Western blotted using a polyclonal anti-tau antibody (Dako, #A0024).

[0681] Results: Figure 8 As shown, 3D6 and 5G8 immunoprecipitated tau from Alzheimer's disease tissue. The high salt soluble fraction was immunoprecipitated with the indicated antibodies and detected from the binding sites of 3D6 and tau antibody A using polyclonal anti-tau antibodies directed against separate regions of the tau molecule. 3D6 robustly captured tau from this fraction. The input (high salt soluble sample) is shown on the right.

[0682] Example 9. Immunohistochemical immunoreactivity of 3D6

[0683] Frontotemporal cortex was obtained from patients without neurodegenerative disease or with Alzheimer's disease, which was confirmed at the time of postmortem assessment. Immunohistochemistry was performed on frozen sections mounted on 10um slides fixed with mild acetone. All staining steps were performed using Leica BOND Rx automatic stainer using Leica consumables. The mouse or human form of 3D6 was incubated with tissue sections, and then the species-appropriate secondary antibody conjugated to HRP polymer was added. When humanized antibodies were used on human tissues, in order to prevent nonspecific binding of endogenous immunoglobulins, the antibodies were non-covalently labeled with biotin-conjugated anti-human monovalent Fab fragments in vitro and then incubated on the tissues. Tissues labeled with primary antibody-biotin Fab fragment complexes were further amplified using the avidin-biotin amplification system (Vector Laboratories, Burlingame, CA). DAB chromogen was used to visualize staining, producing brown deposits. Negative controls consisted of performing the entire immunohistochemistry procedure on adjacent sections with an IgG isotype control antibody.

[0684] The antibodies tested were murine CD6, chimeric 3D6 (which contains VH and VL from a murine antibody with human constant regions, heavy chain SEQ ID NO:72 and light chain SEQ ID NO:73), and the humanized variant hu3D6VHv5 / hu3D6VLv2.

[0685] The staining performed with the mouse, chimeric and humanized forms of 3D6 was qualitatively compared and evaluated for strength and intensity of staining, as well as the localization of immunoreactivity. For the chimeric and humanized forms of 3D6, the staining intensity was similar and showed a similar localization pattern compared to the mouse form of the antibody. Tau was detected in neurofibrillary tangles, protofibrils, neuropil threads and degenerating axons. Significant neuronal somatic staining was also detected.

[0686] Example 10. Affinity of humanized variants for tau

[0687] Methods; Indirect ELISA 96-well polystyrene plates were coated with human recombinant tau suspended in 1xPBS for 2 h at room temperature or 16 h at 4°C. The coating was removed and the plates were blocked with 1% BSA in 1xPBS for 1 h. 1 μg / mL of humanized variant antibodies in 0.1% BSA in 1xPBS was added to the plates for 1 h, then washed and HRP-conjugated goat anti-human antibodies were added. The plates were developed with TMB and A was measured with a plate reader. 450 .

[0688] Sandwich ELISA 96-well polystyrene plates were coated with anti-human antibodies in 1xPBS for 2 hours at room temperature or 16 hours at 4°C. The coating was removed and the plates were blocked with 1% BSA in 1xPBS for 1 hour. Various concentrations of humanized variant antibodies diluted in 0.1% BSA in 1xPBS were added to the plates for 1 hour, then washed and biotinylated recombinant human tau diluted in 0.1% BSA in 1xPBS was added. After washing, streptavidin-HRP was added, the plates were developed with TMB, and A was measured with a plate reader. 450 .

[0689] SPR analysis was performed using Biacore T200 to determine the binding kinetics of h3D6-VHv5-L2 to recombinant human tau. In order to prepare the sensor surface, anti-human antibodies (GE Life Sciences) were fixed on the sensor chip CM5 via amine coupling, and humanized variant antibodies were captured at a certain level to ensure the maximum binding of 50RU. In running buffer (HBS+0.05% P-20, 1mg / mL BSA), recombinant tau at various concentrations within the range of 10nM to 0.14nM was passed through the capture ligand at a flow rate of 50 μL / min, and continued for 180s association and 900s dissociation. The data were double referenced to unrelated sensors and 0nM analyte concentrations without antibody ligands to take into account the dissociation of ligands from the capture portion. The data were then analyzed using a global 1:1 fit.

[0690] Example 11 Immunogenicity of the light chain variable region of hu3D6VLv2

[0691] 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 can be selected for deimmunization of hu3D6VLv2, i.e., regions that indicate further substitutions can be made to reduce potential immunogenicity.

[0692] Table 9. Results of immunogenicity analysis to identify potential peptides for deimmunization

[0693]

[0694] Based on the analysis results shown in Table 9, variants of the hu3D6VLv2 light chain variable region were designed that target the amino acid residues in bold in Table 10. Each variant incorporates one of the following amino acid substitutions, as shown in Table 11.

[0695] Table 10. Amino acid residues targeted for substitution in hu3D6VLv2.

[0696]

[0697] Table 11. Amino acid substitutions in hu3D6VLv2 designed to reduce immunogenicity

[0698]

[0699]

[0700] Additional variants of the hu3D6VLv2 light chain variable region were designed, as shown in Table 12, incorporating substitutions at one or both of the amino acid residues highlighted in Table 10. Some of the variants in Table 12 also incorporated substitution L37Q (SEQ ID NOs: 119-135 and 145), or both substitution L37Q and substitution G100Q (SEQ ID NOs: 136-142).

[0701] The rationale for selecting positions L37Q and G100Q in the light chain variable region as indicated in Tables 12 and 13 as substitution candidates is as follows.

[0702] L37Q is a mutation used to increase the humanization of the sequence. Gln is a germline alignment mutation. Gln is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0703] G100Q is a mutation used to increase the humanization of the sequence. Gln is a germline alignment mutation. Gln is at this position in the human germline gene IGKV2-30*02 (SEQ (ID NO: 27).

[0704] Table 12: Amino Acid Substitutions in hu3D6VLv2 Designed to Reduce Immunogenicity

[0705]

[0706]

[0707] Additional variants of the hu3D6VLv2 light chain variable region were designed to bind L37Q (SEQ ID NO: 143) or to bind G100Q (SEQ ID NO: 144), as shown in Table 13.

[0708] Table 13: Additional amino acid substitutions in hu3D6VLv2

[0709]

[0710] Fig. 10A , Fig. 10B , Fig. 10C and Fig. 10DAlignment of the light chain variable regions of the following humanized forms of the 3D6 antibody is shown: hu3D6VLv2 (SEQ ID NO:21), hu3D6VLv2 L37Q (SEQ ID NO:143), hu3D6VLv2 L50G (SEQID NO:103), hu3D6VLv2 S52G (SEQ ID NO:110), hu3D6VLv2 L54G (SEQ ID NO:94), hu3D6VLv2 L54D (SEQ ID NO:93), hu3D6VLv2L54K (SEQ ID NO:100), hu3D6VLv2 L54R (SEQID NO:101), hu3D6VLv2 L54T (SEQ ID NO:102), hu3D6VLv2 L37Q_L50G (SEQ ID NO:128), hu3D6VLv2 L37Q_L50D (SEQ ID NO:129), hu3D6VLv2L37Q_S52G (SEQ ID NO:131), hu3D6VLv2 L37Q_L54G (SEQ ID NO:126), hu3D6VLv2 L37Q_L54R (SEQ ID NO:125), hu3D6VLv2L37Q_L54T (SEQ ID NO:130), hu3D6VLv2 L37Q_L54D (SEQ ID NO:127), hu3D6VLv2 L37Q_L54E (SEQ ID NO:145), hu3D6VLv2L37Q_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), hu3D6VLv2L37Q_S52G_L54D(SEQ IDNO:124), hu3D6VLv2 L37Q_S52G_L54G (SEQ ID NO:121), hu3D6VLv2 L37Q_S52G_L54T(SEQID NO:123), hu3D6VLv2 L37Q_S52G_L54R (SEQ ID NO:122), hu3D6VLv2L37Q_L50D_L54G_G100Q (SEQ ID NO:140), hu3D6VLv2L37Q_L50D_L54R_G100Q (SEQ IDNO:141), hu3D6VLv2L37Q_L50G_L54R_G100Q (SEQ ID NO:136), hu3D6VLv2L37Q_L50G_L54G_G100Q (SEQ ID NO:137), hu3D6VLv2L37Q_L50V_L54D_G100Q (SEQ ID NO:142), hu3D6VLv2L37Q_S52G_L54D_G100Q (SEQ ID NO:139) and hu3D6VLv2L37Q_S52G_L54R_G100Q (SEQ ID NO:138).

[0711] Example 12 Immunogenicity of hu3D6VHv1bA11 (heavy chain variable region)

[0712] Additional heavy chain variable region variants of hu3D6VHv1bA11 (also known as h3D6Hu5) (SEQ ID NO: 18) were designed as shown in Table 14.

[0713] Fig. 9A and Fig. 9B Shown is an alignment of the heavy chain variable region of murine 3D6 (SEQ ID NO:7) and 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 acceptor 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.

[0714] Table 14: Additional amino acid substitutions in hu3D6VHv1bA11 (also known as h3D6Hu5)

[0715]

[0716] The reasons for selecting the positions shown in Table 14 in the heavy chain variable region as substitution candidates are as follows.

[0717] D60E: used to reduce potential proteolysis. Asp-Pro motif is known to be a potential proteolytic cleavage site. There is an Asp-Pro motif at Kabat position 60-61 in hu3D6VHv1bA11 (h3D6Hu5) VH. Based on homology modeling, the Glu substitution at position 60 is considered to be conformationally conservative. Therefore, the D60E substitution is performed to reduce potential proteolysis.

[0718] Q81E is used to enhance thermal stability and to increase human origin. Glu is predicted to form an H-bond plus salt bridge with K19, so Glu at this position can enhance antibody stability. Glu is a germline alignment mutation. Glu is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0719] L82cV is used to reduce immunogenicity. Computer analysis of humanized hu3D6VHv1bA11 (h3D6Hu5) immunogenicity using the IEDB (Immune Epitope Database) immunogenicity analysis tool predicted potential immunogenic peptides including L82c. The L82cV substitution was designed to reduce immunogenicity. Valine substitution at this position maintains loop conformation.

[0720] L80M is used to reduce immunogenicity; and to increase human origin. Met is a germline aligned mutation. Met is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25).

[0721] T83R is used to enhance thermal stability and to increase human origin. Arg is a germline aligned mutation. Arg is at this position in the human germline gene IMGT#IGHV1-69-2*01 (SEQ (ID NO: 25). Arg is most frequently at this position.

[0722] Example 13 Analysis of humanized 3D6 variants

[0723] Several features of humanized 3D6 variants with predicted deimmunizing substitutions were analyzed, including target binding affinity, activity in cell-based assays, thermal stability, expression characteristics, and number of substitutions. In all cases, the results were compared to the parental sequence hu3D6VHv1bA11 / hu3D6VLv2 to determine if any loss of activity or stability was recorded.

[0724] Target binding analysis was performed using Biacore T200 to compare the binding affinity of humanized 3D6 variants and recombinant human 4R0N tau. Anti-human Fc antibodies were fixed on sensor chip CM3 via amine coupling, and humanized 3D6 variants were captured to equal levels. In running buffer (HBS+0.05% P-20, 1mg / mL BSA), various concentrations of recombinant 4R0N human tau (in the range of 0.02nM to 12.5nM) were passed through the capture ligand at a speed of 50 μL / min, and continued to associate / dissociate for 180 seconds / 420 seconds (as a single cycle). For both unrelated sensors and 0nM analyte concentrations without antibodies, blank values ​​were subtracted from the data. Using Biacore evaluation software, global 1:1 fitting was used for analysis.

[0725] Affinity assays revealed a number of deimmunized variants that retained the affinity of the parental hu3D6VHv1bA11 / hu3D6VLv2 by dividing the K D Compared with the parental hu3D6VHv1bA11 / hu3D6VLv2 D Determined by comparison. Determined to have a comparable K within 4-fold of hu3D6VHv1bA11 / hu3D6VLv2 (Table 15) D Antibodies include hu3D6VHv1bA11 / L2-DIM4, hu3D6VHv1bA11 / L2-DIM9, hu3D6VHv1bA11 / L2-DIM5, hu3D6VHv1bA11 / L2-DIM3, h3D6VHvb8 / L2-DIM4, hu3D6VHv 1bA11 / L2-DIM7, h3D6VHvb8 / L2-DIM7, h3D6VHvb8 / L2-DIM8, hu3D6VHv1bA11 / L2-DIM12, hu3D6VHv1bA11 / L2-DIM21 and hu3D6VHv1bA11 / L2-DIM22. hu3D6VHv1bA11 / L2-DIM4 exhibited improved affinity compared to the parental hu3D6VHv1bA11 / hu3D6VLv2 as demonstrated by association rates, dissociation rates, and Kd numbers.

[0726] Table 15. Affinity of 3D6 humanized variants

[0727]

[0728]

[0729] In addition, as a secondary feature, the thermal stability and titer of all deimmunized variants were analyzed. Based on affinity measurements, the thermal stability levels and titer levels of antibodies comparable to hu3D6VHv1bA11 / hu3D6VLv2 were compared, and the antibodies in Table 16 are listed in order based on the difference in Tm with hu3D6VHv1bA11 / hu3D6VLv2.

[0730] Thermal stability values ​​were determined using differential scanning calorimetry (DSC). All DSC scans were performed using a VP-Capillary DSC system (Malvern). All samples were prepared at 0.5 mg / mL in 1xPBS and were based on 1xPBS. Approximately 0.5 mL of protein solution and buffer were introduced into the sample and reference cells. Calorimetric scans were performed from 25°C to 110°C at a scan rate of 60°C / h under constant pressure. Origin software was used for analysis. The reported values ​​are the temperatures at which the maximum heat capacity of the Fab peak was recorded.

[0731] Titer was determined as follows. After expression in 293 suspension cells, the antibody was purified using protein A chromatography using standard methods. After purification, the antibody was exchanged into 1xPBS and the protein concentration was determined by absorbance at 280nm. Titer was calculated by dividing the final yield of purified protein by the starting volume of the expression culture and reported in mg / L. hu3D6VHv1bA11 / L2-DIM4 exhibited higher thermal stability and titer than parent hu3D6VHv1bA11 / hu3D6VLv2. Both thermal stability level and transient expression level (titer) are important considerations for drug development.

[0732] Table 16. Thermal stability and titer of 3D6 humanized variants

[0733]

[0734]

[0735] Based on analysis of the findings listed above, t...

Claims

1. An isolated antibody or antigen-binding antibody fragment thereof that specifically binds to human tau, comprising: a mature heavy chain variable region comprising a CDR-H1 having SEQ ID NO: 8, a CDR-H2 having SEQ ID NO: 9 or SEQ ID NO: 149, and a CDR-H3 having SEQ ID NO: 10, respectively, wherein the heavy chain variable region is at least 90% identical to SEQ ID NO: 18; and A mature light chain variable region comprising a CDR L1 having SEQ ID NO: 12, a CDR L2 having SEQ ID NO: 150, 151, 153, 156, 158, 159, 160, 163, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 174, and a CDR L3 having SEQ ID NO: 14, wherein the light chain variable region is at least 90% identical to SEQ ID NO:

122.

2. The antibody or antigen-binding antibody fragment of claim 1 , wherein at least one of Kabat positions H12, H13, H17, H24, H40, H43, H48, H66, H67, H76, H80, H81 and H91 can be occupied by V, K, T, A, R, Q, I, R, A, D, L, Q and F, respectively, and at least one of Kabat positions L2, L12, L15, L37, L39, L45, L60 and L100 can be occupied by V, P, L, Q, R, R, D and Q, respectively.

3. The antibody or antigen-binding antibody fragment of claim 1, wherein CDR L2 comprises SEQ ID NO: 150, 151, 163, 167, 168 or 169.

4. The antibody or antigen-binding antibody fragment of claim 3, wherein 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.

5. The antibody or antigen-binding antibody fragment of claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 146, and the light chain variable region comprises SEQ ID NO: 94 or 122.

6. The antibody or antigen-binding antibody fragment of claim 3, wherein the heavy chain variable region comprises SEQ ID NO: 18 or 146, and the light chain variable region comprises SEQ ID NO:

122.

7. The antibody or antigen-binding antibody fragment of any one of claims 1-6, wherein the antibody or antigen-binding antibody fragment 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.

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

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

10. The antibody or antigen-binding antibody fragment of claim 9, wherein the antigen-binding antibody fragment is a single-chain antibody, Fab, or Fab'2 fragment.

11. The antibody or antigen-binding antibody fragment of claim 9, wherein the antigen-binding antibody fragment is a Fab fragment or a single-chain Fv.

12. The antibody or antigen-binding antibody fragment of claim 8, wherein the antibody has a human IgG1 isotype.

13. The antibody or antigen-binding antibody fragment of any one of claims 1-7 and 9-11, wherein the mature light chain variable region is fused to a light chain constant region, and the mature heavy chain variable region is fused to a heavy chain constant region.

14. The antibody or antigen-binding antibody fragment of claim 13, wherein the heavy chain constant region is a mutant form of a native human heavy chain constant region, and the mutant form of the native human heavy chain constant region has reduced binding to an Fcγ receptor relative to the native human heavy chain constant region.

15. The antibody or antigen-binding antibody fragment of claim 13 or 14, wherein the heavy chain constant region is of the IgG1 isotype.

16. The antibody or antigen-binding antibody fragment of claim 15, wherein the heavy chain constant region has the amino acid sequence of SEQ ID NO:

176.

17. The antibody or antigen-binding antibody fragment of claim 13, wherein the mature heavy chain variable region fused to the heavy chain constant region has the amino acid sequence of SEQ ID NO:

178.

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

180.

19. The antibody or antigen-binding antibody fragment of claim 13, wherein the light chain constant region has the amino acid sequence of SEQ ID NO:

177.

20. The antibody or antigen-binding antibody fragment of claim 13, wherein the mature light chain variable region fused to the light chain constant region has the amino acid sequence of SEQ ID NO:

179.

21. The antibody or antigen-binding antibody fragment of claim 20, wherein the light chain has the amino acid sequence of SEQ ID NO:

181.

22. The antibody or antigen-binding antibody fragment of claim 17, wherein 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.

23. The antibody or antigen-binding antibody fragment of claim 18, wherein 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.

24. The antibody or antigen-binding antibody fragment of claim 13 or 14, having at least one mutation in the heavy chain constant region.

25. The antibody or antigen-binding antibody fragment of claim 24, wherein the at least one mutation reduces complement fixation or activation of the heavy chain constant region.

26. The antibody or antigen-binding antibody fragment of claim 25, which has a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 of the heavy chain constant region according to EU numbering.

27. The antibody or antigen-binding antibody fragment of claim 26, which has alanine at positions 318, 320, and 322 according to EU numbering.

28. The antibody or antigen-binding antibody fragment of claim 8, wherein the antibody has a human IgG2 or human IgG4 isotype.

29. The antibody or antigen-binding antibody fragment of any one of claims 1-28, wherein the antibody or antigen-binding antibody fragment is conjugated to a therapeutic agent, a cytotoxic agent, a cytostatic agent, a neurotrophic agent, or a neuroprotective agent.

30. A pharmaceutical composition comprising an antibody or antigen-binding antibody fragment as defined in any one of claims 1 to 29 and a pharmaceutically acceptable carrier.

31. A nucleic acid encoding the heavy chain and light chain of the antibody of any one of claims 8, 12 and 28.

32. A method for producing an antibody, the method comprising: (a) culturing cells transformed with the nucleic acid of claim 31 in a cell culture medium so that the cells secrete the antibody; as well as (b) purifying the antibody from the cell culture medium.

33. A method for preparing a cell line that produces an antibody, the method comprising: (a) introducing a vector comprising the nucleic acid of claim 31 and a selection marker into a cell; (b) propagating the cells under conditions that select for cells having an increased copy number of the vector; (c) isolating single cells from the selected cells; as well as (d) Cell banks are constructed from single cell clones selected based on antibody production.

34. A method for producing a humanized, chimeric or veneered antibody as claimed in claim 7, the method comprising: (a) culturing cells transformed with nucleic acids encoding the heavy and light chains of the humanized, chimeric or veneered antibody in a cell culture medium so that the cells secrete the humanized, chimeric or veneered antibody; and (b) purifying the humanized, chimeric or veneered antibody from the cell culture medium.

35. A method for preparing a cell line producing the humanized, chimeric or veneered antibody of claim 7, the method comprising: (a) introducing a vector encoding the heavy chain and light chain of the humanized, chimeric or veneered antibody and a selection marker into a cell; (b) propagating the cells under conditions that select for cells having an increased copy number of the vector; (c) isolating single cells from the selected cells; as well as (d) banking cells from single cell clones selected based on the production of the humanized, chimeric or veneered antibody.

36. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for inhibiting or reducing tau aggregation in a subject having or at risk of developing tau-mediated amyloidosis.

37. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for treating a tau-related disease or achieving prevention of a tau-related disease in a subject.

38. The use of claim 37, wherein the tau-related disease is Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

39. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for reducing the abnormal propagation of tau in a subject.

40. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for inducing phagocytosis of tau in a subject.

41. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for inhibiting tau aggregation or deposition in a subject.

42. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for inhibiting the formation of tau tangles in a subject.

43. Use of the antibody or antigen-binding antibody fragment of any one of claims 1-29 in the manufacture of a medicament for detecting tau protein deposits in a subject suffering from or at risk of suffering from a disease associated with tau aggregation or deposition.

44. The use of claim 43, wherein the disease associated with tau aggregation or deposition is Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic Parkinson's disease, post-traumatic dementia or dementia pugilistica, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic grain disease, globular glial tauopathy, Guam amyotrophic lateral sclerosis / Parkinson's dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), or progressive supranuclear palsy (PSP).

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