EA041230B1 - ANTIBODIES RECOGNIZING TAU - Google Patents

Description

Cross-reference to related applications

This application claims priority under 35 USC §119(e) pursuant to U.S. Provisional Application No. 62/330800, filed May 2, 2016, which is hereby incorporated by reference in its entirety for all purposes.

Link to sequence listing

The sequence listing recorded in file 497448SEQLIST.txt is 59 kilobytes, was created on May 2, 2017, and is incorporated herein by reference.

State of the art

Tau is a well-known human protein that can exist in phosphorylated forms (see, for example, Goedert, Proc. Natl. Acad. Sci. USA, 85:4051-4055 (1988); Goedert, EMBO J., 8: 393-399 (1989), Lee, Neuron, 2:1615-1624 (1989), Goedert, Neuron, 3:519-526 (1989), Andreadis, Biochemistry, 31:10626-10633 (1992). plays a role in the stabilization of microtubules, particularly in the central nervous system.Total tau (c-tau, i.e. phosphorylated and unphosphorylated forms) and phospho-tau (p-tau, i.e. phosphorylated tau) is released by the brain in response on neuronal damage and neurodegeneration, and elevated levels of tau have been reported in the CSF (cerebrospinal fluid) of patients suffering from Alzheimer's disease compared to the general population (Jack et al., Lancet Neurol, 9:119-28 (2010) ).

Tau is an essential component of neurofibrillary tangles, which, along with plaques, are a hallmark of Alzheimer's disease. The coils form abnormal fibrils that are 10 nm in diameter, occurring in pairs and interlacing in a helical pattern at regular intervals of 80 nm. Tau in neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated), with phosphate groups attached to specific sites on the molecule. In Alzheimer's disease, a significant presence of neurofibrillary tangles is observed in layer II neurons of the entorhinal cortex, in CA1 regions and areas of the supporting structure of the hippocampus, the amygdala, and in deeper layers (layers III, V and superficial VI) of the neocortex. It has also been reported that hyperphosphorylated tau interferes with microtubule assembly, which can cause neural network disruption.

Tau inclusions are part of the definition of the neuropathology of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and Pick's disease.

Brief description of the claimed invention

In one aspect, the present invention provides an isolated monoclonal antibody that specifically binds to tau. Examples of such antibodies bind to an epitope within amino acid residues 55-78, 60-75 or 61-70 of SEQ ID NO: 3 (corresponding to amino acid residues 113-136, 118-133 or 119-128, respectively, of SEQ ID NO: 1) .

Some of these antibodies compete with the 16G7 antibody for binding to human tau. Some of these antibodies bind to the same epitope on human tau as 16G7.

Some antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody 16G7, wherein 16G7 is a mouse antibody that is characterized by a heavy chain variable region containing an amino acid sequence that contains SEQ ID NO: 7 and a light chain variable region containing an amino acid a sequence that contains SEQ ID NO: 11. In some antibodies, three heavy chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOs: 8, 9 and 10) and three light chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOs: 12, 13 and 14).

For example, the antibody may be 16G7 or a chimeric, veneered, or humanized form of said antibody. In some of these antibodies, the heavy chain variable region has >85% human sequence identity. In some of these antibodies, the light chain variable region has >85% human sequence identity. In some of these antibodies, the heavy chain variable region and the light chain variable region each share >85% human germline sequence identity. In some of these antibodies, the mature heavy chain variable region contains three heavy chain CDRs that are as defined according to the Cabot/Chothia pooled definition (SEQ ID NOS: 8, 9 and 10) and three light chain CDRs that are as determined according to the Cabot/Chothia summary definition (SEQ ID NOs: 12, 13 and 14); with the proviso that position H31 is S or G, position H60 is N or A, and position 64 is K or Q. In some of these antibodies, CDR-H1 contains an amino acid sequence containing SEQ ID NO: 49. In some of these antibodies, CDR-H2 contains an amino acid sequence containing SEQ ID NO: 50. In some of these antibodies, CDR-H2 contains an amino acid sequence containing SEQ ID NO: 51. In some of these antibodies, the antibody is a humanized antibody.

In some of these antibodies, CDR-H1 contains an amino acid sequence containing SEQ ID NO: 49, and CDR-H2 contains an amino acid sequence containing

SEQ ID NO: 50. In some of these antibodies, CDR-H1 contains an amino acid sequence containing SEQ ID NO: 49, and CDR-H2 contains an amino acid sequence containing

SEQ ID NO: 51.

Some antibodies are a humanized or chimeric 16G7 antibody that specifically binds to human tau, where 16G7 is a mouse antibody that is characterized by a mature heavy chain variable region according to SEQ ID NO: 7 and a mature light chain variable region according to SEQ ID NO: 11. Some such antibodies are a humanized antibody containing a humanized heavy chain mature variable region that contains three 16G7 heavy chain CDRs and a humanized light chain mature variable region that contains three 16G7 light chain CDRs. In some of these antibodies, the CDRs correspond to a definition selected from the group consisting of definitions according to Cabot, Chothia, Cabot/Chothia summary definition, AbM, and contact definition.

In some such antibodies, the humanized mature heavy chain variable region contains three 16G7 heavy chain CDRs according to the Cabot/Chothia pooled definition (SEQ ID NO: 8-10) and the humanized mature light chain variable region contains three 16G7 light chain CDRs according to the pooled Cabot/Chothia definition. (SEQ ID NO: 12-14).

In some such antibodies, the humanized mature heavy chain variable region contains three 16G7 heavy chain CDRs according to Cabot (SEQ ID NO: 39, SEQ ID NO: 9, and SEQ ID NO: 10) and the humanized mature light chain variable region contains three 16G7 light chain CDRs. according to Cabot (SEQ ID NO: 12-14).

In some such antibodies, the humanized mature heavy chain variable region contains three 16G7 heavy chain CDRs according to Chothia (SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 10) and the humanized mature light chain variable region contains three 16G7 light chain CDRs. according to Chothia (SEQ ID NO: 12-14).

In some such antibodies, the humanized mature heavy chain variable region contains three 16G7 heavy chain CDRs according to AbM (SEQ ID NO: 8, SEQ ID NO: 42, and SEQ ID NO: 10)) and the humanized mature light chain variable region contains three light chain CDRs. 16G7 according to AbM (SEQ ID NO: 12-14).

In some such antibodies, the humanized mature heavy chain variable region contains three 16G7 heavy chain CDRs as defined by the contact definition (SEQ ID NO: 46-48) and the humanized mature light chain variable region contains three 16G7 light chain CDRs as defined by the contact definition (SEQ ID NO: 43 -45).

In some antibodies, the humanized mature heavy chain variable region contains an amino acid sequence at least 90% identical to any of SEQ ID NOs: 15-27, and the humanized mature light chain variable region contains an amino acid sequence at least 90% identical to any of SEQ ID NO: 28-30.

In certain such antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H5 occupies Q, H7 occupies P, H9 occupies S, H19 occupies V, H11 occupies L, H12 occupies V, H13 occupies R, H20 is L, H40 is R, H48 is I, H66 is K, H67 is A, H69 is L, H71 is V, H73 is I, H75 is S, H80 is V, H82a is T, H82b is S, H83 occupies T and H85 occupies E. In some of these antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83 and H85 in the VH region occupy E, Q, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S, V, T, S , T and E respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H5 occupies Q, H7 occupies P, H9 occupies S, H10 occupies V, H11 occupies L, H12 occupies V, H13 occupies R , H20 occupies L, H48 occupies I, H69 occupies L, H71 occupies V, H82a occupies T, H82b occupies S, H83 occupies T, and H85 occupies E. H12, H13, H20, H48, H69, H71, H82a, H82b, H83 and H85 in the VH region occupy E, Q, P, S, V, L, V, R, L, I, L, V, T, S , T and E, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H5 occupies Q, H11 occupies L, H12 occupies V, H20 occupies L, H48 occupies I, H69 occupies L, H71 occupies V , H82a occupies T, H82b occupies S, H83 occupies T, and H85 occupies E. In some antibodies, H1, H5, H11, H12, H20, H48, H69, H71, H82a, H82b, H83 and H85 in the VH region occupy E, Q, L, V, L, I, L, V, T, S, T, and E, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H12 occupies V, H31 occupies G, H40 occupies R, H48 occupies I, H60 occupies A, H69 occupies L, H73 occupies I , and H83 occupies T. In some antibodies, positions H1, H12, H31, H40, H48, H60, H69, H73, and H83 in the VH region are occupied by E, V, G, R, I, A, L, I, and T, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied

- 2 041230 amino acid, as indicated: H1 is E, H12 is V, H40 is R, H48 is I, H69 is L, H73 is I, and H83 is T. In some antibodies, positions H1, H12, H40, H48, H69 ,

H73 and H83 in the VH region occupy E, V, R, I, L, I, and T, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H12 occupies V, H40 occupies R, H48 occupies I, H66 occupies K, H67 occupies A, H69 occupies L, H71 occupies V , H73 occupies I, and H83 occupies T. In some antibodies, positions H1, H12, H40, H48, H66, H67, H69, H71, H73, and H83 in the VH region are occupied by E, V, R, I, K, A, L , V, I, and T, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H12 occupies V, H40 occupies R, H48 occupies I, H69 occupies L, H73 occupies I, and H83 occupies T.

In some antibodies, the positions H1, H12, H40, H48, H69, H73, and H83 in the VH region are E, V, R, I, L, I, and T, respectively. In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H7 occupies P, H71 occupies V, and H73 occupies I.

In some antibodies, positions H7, H71, and H73 in the VH region are P, V, and I, respectively. In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H31 occupies G, and H60 occupies A.

In some antibodies, positions H31 and H60 in the VH region are G and A, respectively. In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies Q or E, H5 occupies V or Q, H7 occupies S or P, H9 occupies A or S, H10 occupies E or V, H11 occupies V or L, H12 occupies K or V, H13 occupies K or R, H20 occupies V or L, H31 occupies G or S, H37 occupies V or A, H 38 occupies R or K, H40 occupies A or R, H48 occupies M or I, H60 occupies A or N, H64 occupies Q or K, H66 occupies R or K, H67 occupies V or A, H69 occupies M or L, H71 occupies R or V, H73 occupies T or I, H75 occupies I, S or A, H80 occupies M or V, H82a occupies S or T, H82b occupies R or S, H83 occupies R or T, and H85 occupies D or E.

In some antibodies, positions H1, H5, H7, H9, H10, H1 1, H12, H13, H20, H37, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83 and H85 in the VH region are E, Q, P, S, V, L, V, R, L, A, K, R, I, K, A, L, V, I, S, V, T, S, T and E, respectively.

In some antibodies, the positions H1, H5, H1 1, H12, H20, H48, H69, H71, H75, H82a, H82b, H83 and H85 in the VH region are E, Q, L, V, L, I, L, V, A, T, S, T, and E, respectively.

In some antibodies, the positions H1, H12, H40, H48, H66, H67, H69, H71, H73, and H83 in the VH region are E, V, R, I, K, A, L, V, I, and T, respectively.

In some antibodies, positions H1, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73 and H83 in the VH region are occupied by E, V, G, R, I, A, K, A, L, V , I and T, respectively.

In some antibodies, the positions H1, H12, H40, H48, H69, H73, and H83 in the VH region are E, V, R, I, L, I, and T, respectively.

In some antibodies, the positions H1, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73 and H83 in the VH region are Q, V, G, R, I, A, K, A, L, V , I and T, respectively.

In some antibodies, the H1, H7, and H73 positions in the VH region are occupied by Q, P, V, and I, respectively. In some antibodies, the positions H1, H12, H31, H40, H48, H60, H64, H69, H73, and H83 in the VH region are E, V, G, R, I, A, Q, L, I, and T, respectively.

In some antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 in the VH region are occupied by E, Q, P, S, V , L, V, R, L, I, L, V, A, T, S, T, and E.

In some antibodies, the positions H1, H5, H1 1, H12, H20, H48, H66, H67, H69, H71, H73, H75, H82a, H82b, H83 and H85 in the VH region are E, Q, L, V, L, I, K, A, L, V, I, S, T, S, T, and E.

In some antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H38, H40, H48, H66, H67, H69, H71, H73I, H75, H80, H82a, H82b, H83 and H85 in the VH regions are occupied by E, Q, P, S, V, L, V, R, L, K, R, I, K, A, L, V, I, S, V, T, S, T, and E, respectively.

In some antibodies, H1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, and H85 positions in the VH region occupy Q, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S, V, T, S, T, and E, respectively.

In some antibodies, positions H7, H71, and H73 in the VH region are P, V, and I, respectively. In some antibodies, the position of H71 in the VH region is occupied by V.

In some antibodies, the positions H1, H7, H71, and H73 in the VH region are occupied by E, P, V, and I.

In some antibodies, at least one of the following positions in the VL region is occupied by an amino acid, as indicated: L4 occupies L, L9 occupies S, L15 occupies V, L22 occupies S, and L43 occupies S. In some antibodies, positions L4, L9, L15, L22 and L43 in the VL region occupy L, S, V, S and S, respectively.

In some antibodies, at least one of the following positions in the VL region is occupied by an amino acid, as indicated: L4 occupies L, L9 occupies S, L15 occupies V, L18 occupies K, L19 occupies V, L21 occupies M, L22 occupies S, and L43 occupies S. In some antibodies L4, L9, L15, L18, L19, L21,

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L22 and L43 in the VL region occupy L, S, V, K, V, M, S, and S, respectively.

In some antibodies, at least one of the following positions in the VL region is occupied by an amino acid, as indicated: L4 occupies M or L, L9 occupies D or S, L15 occupies L or V, L18 occupies R or K, L19 occupies A or V, L21 occupies I or M, L22 occupies N or S, L43 occupies P or S, and L48 occupies I or M. In some antibodies, L4, L9, L15, L18, L19, L21, L22, and L43 occupy L, S, V, K, V, M, S and S, respectively.

In some antibodies, positions L4, L9, L15, L22, and L43 in the VL region are occupied by L, S, V, S, and S, respectively. In some antibodies, positions L4, L9, L15, L18, L19, L21, L22, and L43 in the VL region are occupied by L, S, V, K, V, M, S, and S, respectively.

Some antibodies contain a mature heavy chain variable region containing an amino acid sequence at least 95% identical to any of SEQ ID NOs: 15-27 and a mature light chain variable region containing an amino acid sequence at least 95% identical to any of SEQ ID NO: 28-30. Some antibodies contain a mature heavy chain variable region containing an amino acid sequence at least 98% identical to any of SEQ ID NOs: 15-27 and a mature light chain variable region containing an amino acid sequence at least 98% identical to any of SEQ ID NO: 28-30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of any of SEQ ID NOs: 15-27 and the mature light chain variable region contains the amino acid sequence of any of SEQ ID NOs: 28-30. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 15 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 15 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 15 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 16, and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 16, and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 16, and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 17 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 17 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 17 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 18 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 18 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 19 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 19 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 19 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 20 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 20 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 20 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

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In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 21 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 21 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 21 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 22 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 22 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 22 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 23 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 23 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 23 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 24 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 24 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 24 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 25, and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 25, and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 25, and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 26 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27 and the mature variable region of the heavy chain the light chain region contains the amino acid sequence of SEQ ID NO: 29. In some antibodies, the mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27 and the mature light chain variable region contains the amino acid sequence of SEQ ID NO: 30.

For example, the antibody may be a chimeric antibody.

For example, the antibody may be a veneered antibody.

The antibody can be an intact mouse antibody, a chimeric, veneered or humanized antibody or binding fragment, a single chain Fab antibody fragment, a Fab'2 fragment, or a single chain Fv. Some of the antibodies are of the human IgG1 isotype, while others may be of the human IgG2 or IgG4 isotype. Some antibodies contain 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 the natural human heavy chain constant region that has reduced binding to the Fcy receptor compared to the natural human heavy chain constant region.

Some antibodies may contain at least one mutation in the constant region, such as a mutation that reduces the fixation or activation of complement by 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 contain alanine at positions 318, 320 and 322. Some antibodies may be at least 95% w/w. clean. The antibody may be conjugated to a therapeutic, cytotoxic, cytostatic, neurotrophic, or neuroprotective agent.

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

According to another aspect, the present invention provides a nucleic acid encoding the heavy chain and/or light chain of any of the antibodies disclosed herein, a recombinant expression vector containing the nucleic acid, and a host cell transformed with the recombinant expression vector.

According to another aspect, the present invention provides methods for humanizing any non-human antibody described herein, for example, mouse 16G7 antibodies, wherein 16G7 is characterized by a mature heavy chain variable region according to SEQ ID NO: 7 and a mature light chain variable region according to SEQ ID NO: 11. Such methods may include selecting one or more acceptor antibodies, synthesizing a nucleic acid encoding a humanized heavy chain containing a mouse heavy chain CDR and a nucleic acid encoding a humanized light chain containing a mouse antibody light chain CDR, and expressing nucleic acids in a host cell to produce a humanized antibody.

Also provided are methods for producing antibodies, such as a humanized, chimeric, or veneered antibody, such as a humanized, chimeric, or veneered form of 16G7. In such methods, cells transformed with nucleic acids encoding the heavy and light chains of an antibody are cultured so that the cells secrete the antibody. The antibody can then be purified from the cell culture medium.

Cell lines producing any of the antibodies disclosed herein can be obtained by introducing into cells a vector encoding the heavy and light chains of the antibody and a selectable marker, expanding cells under conditions to select cells containing an increased number of copies of the vector, isolating single cells from selected cells; and creating a bank of cells cloned from a single cell selected based on the output of the antibody.

Some cells can be expanded under selective conditions and screened for cell lines naturally expressing and secreting at least 100 mg/L/10 ⁶ cells/24 hours. Single cells can be isolated from the selected cells. The cells cloned from the single cell can then be used to create a cell bank. Single cells can be selected depending on desired properties such as antibody yield. Illustrative cell lines are cell lines expressing 16G7.

The present invention also provides methods for inhibiting or reducing tau aggregation in a subject who suffers from tau-mediated amyloidosis or is at risk of developing tau-mediated amyloidosis, said methods comprising administering to the subject an effective antibody regimen disclosed herein, and thereby inhibiting or reducing aggregation tau in the subject. Illustrative antibodies include humanized versions of 16G7.

Also provided are methods of treating or preventing a tau-induced disease in a subject, said methods comprising administering an effective antibody regimen disclosed herein and thereby treating or preventing the disease. Examples of such a disease are Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration , argyrophilic granular disease, glial globular tauopathy, amyotrophic lateral sclerosis/parkinsonism-dementia Guam, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer disease (LBVAD), or progressive supranuclear palsy (PNP). In some methods, the tau-induced disease is Alzheimer's disease. In some methods, the patient is an ApoE4 carrier.

Methods for reducing impaired tau transmission are also provided, said methods comprising administering an effective antibody regimen disclosed herein and thereby reducing tau transmission.

Methods for inducing tau phagocytosis are also provided, said methods comprising administering an effective antibody regimen disclosed herein and thereby inducing tau phagocytosis.

Also provided are methods for inhibiting tau aggregation or accumulation, said methods comprising administering an effective antibody regimen disclosed herein and thereby inhibiting tau aggregation or accumulation.

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Methods for inhibiting tau tangle formation are also provided, said methods comprising the use of an effective antibody regimen disclosed herein.

The present invention also provides a method for detecting tau protein deposits in a subject suffering from or at risk of a tau aggregation or accumulation disease, said methods comprising administering to the subject an antibody disclosed herein and detecting an antibody bound to tau in subject. Examples of such a disease are Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration , argyrophilic granular disease, globular glial tauopathy, amyotrophic lateral sclerosis/Parkinsonism-Guam dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD), or progressive supranuclear palsy (PNP). In some embodiments, the antibody is administered to the subject via intravenous injection. In some embodiments, the antibody is administered directly to the subject's brain via intracranial injection or by drilling a hole in 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).

The present invention also provides a method for measuring the effectiveness of a treatment in a subject who is receiving treatment for a disease associated with tau aggregation or accumulation, said method comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering to the subject an antibody disclosed herein, and detecting a first amount of tau-bound antibody in the subject, administering to the treatment subject, measuring a second level of tau deposition in the subject after treatment by administering the antibody to the subject, and detecting tau-bound antibody in the subject, wherein a decrease in the level of tau deposition is indicative of about a positive response to treatment.

The present invention also provides a method for measuring the effectiveness of a treatment in a subject who is receiving treatment for a disease associated with tau aggregation or accumulation, said method comprising measuring a first level of tau protein deposits in the subject prior to treatment by administering to the subject an antibody disclosed herein, and detecting a first amount of tau-bound antibody in the subject, administering to the treatment subject, measuring a second level of tau protein deposits in the subject after treatment by administering the antibody to the subject, and detecting a second amount of tau-bound antibody in the subject, with no change in the level of tau deposits -protein or a slight increase in deposits of tau protein indicates a positive response to treatment.

Brief description of the drawings

In FIG. 1 shows the results of experiments designed to map the epitope or epitopes to which the mouse monoclonal antibody 16G7 binds.

In FIG. 2 shows an alignment of the heavy chain variable regions of mouse antibody 16G7 and humanized versions of antibody 16G7. CDRs according to the Cabot/Chothia consolidated definition are shown in bold. Positions in the heavy chain variable regions of the mouse 16G7 antibody and in humanized versions of the 16G7 antibody where amino acid residues differ from the Majority sequence are boxed.

In FIG. 3 shows an alignment of the light chain variable regions of mouse antibody 16G7 and humanized versions of antibody 16G7. CDRs as defined by Cabot are shown in bold. Positions in the light chain variable regions of the mouse 16G7 antibody and in humanized versions of the 16G7 antibody at which amino acid residues differ from the Majority sequence are boxed.

In FIG. 4A and 4B are experimental results demonstrating that 16G7 immune-capture tau from human Alzheimer's tissue.

In FIG. 5 shows the results of disaggregation assays for selected anti-tau mouse monoclonal antibodies.

In FIG. 6 shows the affinity of the mouse 16G7 antibody for tau.

In FIG. 7 shows the binding kinetics of the 16G7 chimeric antibody and the humanized 16G7 VHV2a, VHV2b, and VHV6a H7L2 antibodies for tau.

In FIG. 8 shows the avidity of mouse 16G7 Fab and intact mouse 16G7 antibody for aggregated tau.

Brief description of the sequences

SEQ ID NO: 1 shows the amino acid sequence of the human tau isoform (SwissProt P10636-8).

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

SEQ ID NO: 3 shows the amino acid sequence of the human tau isoform (SwissProt P10636-6) (human tau 4R0N).

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

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

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

SEQ ID NO: 7 shows the amino acid sequence of the heavy chain variable region of the mouse antibody 16G7.

SEQ ID NO: 8 shows the amino acid sequence of the mouse 16G7 antibody CDR-H1 according to the Cabot/Chothia pooled definition.

SEQ ID NO: 9 shows the amino acid sequence of the CDR-H2 mouse antibody 16G7 according to Cabot.

SEQ ID NO: 10 shows the amino acid sequence of the CDR-H3 mouse antibody 16G7 according to Cabot.

SEQ ID NO: 11 shows the amino acid sequence of the light chain variable region of the mouse antibody 16G7.

SEQ ID NO: 12 shows the amino acid sequence of the CDR-L1 mouse antibody 16G7 according to Cabot.

SEQ ID NO: 13 shows the amino acid sequence of the CDR-L2 mouse antibody 16G7 according to Cabot.

SEQ ID NO: 14 shows the amino acid sequence of the CDR-L3 mouse antibody 16G7 according to Cabot.

SEQ ID NO: 15 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv1 antibody.

SEQ ID NO: 16 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv2 antibody.

SEQ ID NO: 17 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv2a antibody.

SEQ ID NO: 18 shows the amino acid sequence of the heavy chain variable region of the humanized antibody 16G7 hu16G7VHv2aB7.

SEQ ID NO: 19 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv2b antibody.

SEQ ID NO: 20 shows the amino acid sequence of the heavy chain variable region of the humanized antibody 16G7 hu16G7VHv2bH7.

SEQ ID NO: 21 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv3 antibody.

SEQ ID NO: 22 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv4 antibody.

SEQ ID NO: 23 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv5V antibody.

SEQ ID NO: 24 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv5VR antibody.

SEQ ID NO: 25 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv6a antibody.

SEQ ID NO: 26 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv6b antibody.

SEQ ID NO: 27 shows the amino acid sequence of the heavy chain variable region of the humanized 16G7 hu16G7VHv7 antibody.

SEQ ID NO: 28 shows the amino acid sequence of the light chain variable region of the humanized 16G7 hu16G7VLv1 antibody.

SEQ ID NO: 29 shows the amino acid sequence of the light chain variable region of the humanized 16G7 hu16G7VLv2 antibody.

SEQ ID NO: 30 shows the amino acid sequence of the light chain variable region of the humanized 16G7 hu16G7VLv3 antibody.

SEQ ID NO: 31 shows the amino acid sequence of the heavy chain variable region of the acceptor. No. AAA18265.1.

SEQ ID NO: 32 shows the amino acid sequence of the heavy chain variable region of the acceptor. No. ADW08092.1.

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SEQ ID NO: 33 shows the amino acid sequence of the heavy chain variable region of the acceptor. No. IMGT#IGHV1-2*02.

SEQ ID NO: 34 shows the amino acid sequence of the acceptor light chain variable region with account. No. AAB24404.1.

SEQ ID NO: 35 shows the amino acid sequence of the acceptor light chain variable region with account. No. AEK69364.1.

SEQ ID NO: 36 shows the amino acid sequence of the acceptor light chain variable region with account. No. IMGT#IGKV4-1*01.

SEQ ID NO: 37 shows the nucleic acid sequence encoding the heavy chain variable region of the mouse antibody 16G7.

SEQ ID NO: 38 shows the nucleic acid sequence encoding the light chain variable region of the mouse antibody 16G7.

SEQ ID NO: 39 shows the amino acid sequence of the CDR-H1 mouse antibody 16G7 according to Cabot.

SEQ ID NO: 40 shows the amino acid sequence of the CDR-H1 mouse antibody 16G7 according to Chothia.

SEQ ID NO: 41 shows the amino acid sequence of the CDR-H2 mouse antibody 16G7 according to Chothia.

SEQ ID NO: 42 shows the amino acid sequence of the CDR-H2 mouse antibody 16G7 according to AbM.

SEQ ID NO: 43 shows the amino acid sequence of the mouse 16G7 antibody CDR-L1 according to the contact definition.

SEQ ID NO: 44 shows the amino acid sequence of the CDR-L2 mouse antibody 16G7 according to the contact definition.

SEQ ID NO: 45 shows the amino acid sequence of the mouse 16G7 antibody CDR-L3 according to the contact definition.

SEQ ID NO: 46 shows the amino acid sequence of the mouse 16G7 antibody CDR-H1 according to the contact definition.

SEQ ID NO: 47 shows the amino acid sequence of the CDR-H2 mouse antibody 16G7 according to the contact definition.

SEQ ID NO: 48 shows the amino acid sequence of the CDR-H3 mouse antibody 16G7 according to the contact definition.

SEQ ID NO: 49 shows the amino acid sequence of an alternative CDR-H1 humanized antibody 16G7 according to the Cabot-Chothia summary definition.

SEQ ID NO: 50 shows the amino acid sequence of an alternative CDR-H2 humanized 16G7 antibody according to Cabot.

SEQ ID NO: 51 shows the amino acid sequence of an alternative CDR-H2 humanized 16G7 antibody according to Cabot.

SEQ ID NO: 52 shows the consensus amino acid sequence of the heavy chain variable regions of the mouse 16G7 antibody and the humanized 16G7 antibodies (Majority is marked in FIG. 2).

SEQ ID NO: 53 shows the consensus amino acid sequence between the light chain variable regions of the mouse 16G7 antibody and the humanized 16G7 antibodies (Majority is marked in FIG. 3).

SEQ ID NO: 54 shows the amino acid sequence of the heavy chain of the 16G7 chimeric antibody.

SEQ ID NO: 55 shows the light chain amino acid sequence of the 16G7 chimeric antibody.

Definitions.

Monoclonal antibodies or other biological molecules are typically presented in isolated form. This means that the antibody or other biological molecule, as a rule, are at least 50% wt./wt. free from interfering proteins and other contaminants generated during their preparation or purification, but does not exclude the possibility that the monoclonal antibody is combined with an excess of a pharmaceutically acceptable carrier or carriers or other excipient intended to facilitate the use of the antibody. Sometimes monoclonal antibodies are at least 60, 70, 80, 90, 95 or 99% wt./wt. free from interfering proteins and contaminants generated during the production or purification process. Often, an isolated monoclonal antibody or other biological molecule is the dominant macromolecular component remaining after purification.

Specific binding of an antibody to its target antigen means an affinity and/or avidity of at least 106, 10 ⁷ , 10 ⁸ , 109, 10 ¹⁰ , 10 ¹¹ , or 10 ¹² M ^-1 . Specific binding is detectably higher in magnitude and distinguishable from non-specific binding,

- 9 041230 arising from at least one unrelated target. Specific binding may result from the formation of bonds between specific functional groups or a particular spatial arrangement (eg, lock and key), while non-specific binding is usually due to van der Waals forces. However, specific binding does not necessarily mean that the antibody binds to one and only one target.

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

Light chains are classified as kappa or lambda. Heavy chains, which are classified as gamma, mu, alpha, delta, or epsilon, define the isotype of the antibody as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a J region of 12 or more amino acids, while the heavy chain also contains a D region of approximately 10 or more amino acids. (See, in general terms, Fundamental Immunology Manual, Paul, W., ed., 2nd ed. Raven Press, NY., 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).)

The variable region of an immunoglobulin light or heavy chain (which is 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 (framework, FR) that is punctuated by three complementarity-determining regions. or CDR (complementarity determining regions). The framework regions serve to align the CDRs for specific binding to an antigen epitope. CDRs contain antibody amino acid residues primarily responsible for antigen binding. From amino to carboxy, 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.

The distribution of amino acids to each of the VL and VH domains follows any generally accepted CDR definition. Common definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), Chothia definition (Chothia & Lesk, J. Mol. Biol., 196:901-917, 1987 ; Chothia et al., Nature, 342:878-883, 1989); Chothia and Cabot's CDR definition in which CDR-H1 is Chothia and Cabot's summary CDR definition; AbM definition used in the Oxford University Antibody Molecular Modeling Software university, and the contact definition according to Martin et al. different light chains are assigned the same number When an antibody is said to contain a CDR according to a specific CDR definition (e.g., Cabot), this definition indicates the minimum amount of residue in the CDRs present in the antibody (i.e. CDR according to Cabot). This does not exclude that other residues are also present that fall under another generally accepted definition of CDR, but outside the specified definition. For example, an antibody containing Cabot-defined CDRs includes, among other possibilities, an antibody in which the CDRs contain Cabot CDR residues and no other CDR residues, and an antibody in which the H1 CDR is the Chothia-Cabot CDR H1, and other CDRs contain CDR residues according to Cabot and do not contain additional CDR residues based on other definitions.

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Table 1

Common definitions of CDRs using Cabot numbering

A loop Cabot Chothia Consolidated definition of Chothia and Cabot ALM contact definition L1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36 L2 L50-L56 L50-L56 L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96 H1 H31-H35V H26-H32..H34* H26-H35V* H26-H35V H30-H35V H2 H50-H65 H52-H56 H50-H65 H50-H58 H47-H58 NZ H95-H102 H95-H102 H95-H102 H95-H102 H93-H101

*CDR-H1 according to Chothia may end in H32, H33 or H34 (depending on the buttonhole length). This is because Cabot's numbering scheme places insertions of additional residues at 35A and 35B, while Chothia's numbering places them at 31A and 31B. If neither H35A nor H35B (Cabot numbering) are present, Chothia's CDR-H1 loop ends at H32. If only H35A is present, this loop ends at H33. If both H35A and H35B are present, this loop ends at H34.

The term antibody includes intact antibodies and their binding fragments. Typically, fragments compete with the intact antibody from which they were derived for specific target binding, including individual heavy chains, Fab light chains, Fab', F(ab') ₂ , F(ab)c, Dab, nanobodies, and Fv. Fragments can be obtained by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term antibody also includes a bispecific antibody and/or a humanized antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody that contains two different heavy/light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990) ; Kostelny et al., J. Immunol., 148:1547-53 (1992)). In some bispecific antibodies, the two different heavy/light chain pairs comprise a humanized 16G7 heavy chain/light chain pair and a heavy chain/light chain pair specific for a different epitope on tau than that to which 16G7 binds.

In some bispecific antibodies, one heavy chain/light chain pair is a humanized 16G7 antibody, as discussed in more detail below, and the other heavy chain/light chain pair is derived from an antibody that binds to a receptor that is expressed at the blood-brain barrier, such as the insulin receptor. , insulin-like growth factor (IGF) receptor, leptin receptor, or lipoprotein receptor, or transferrin receptor (Friden et al., Proc. Natl. Acad. Sci., USA, 88:4771-4775, 1991; Friden et al., Science , 259:373-377, 1993). Such a bispecific antibody can be transported across the blood-brain barrier by receptor-mediated transcytosis. The uptake of the bispecific antibody into the brain can be further enhanced by designing the bispecific antibody to reduce its affinity for the blood-brain barrier receptor. Reduced affinity for the receptor resulted in a wider distribution in the brain (see, for example, Atwal et al., Sci. Trans. Med., 3, 84ra43, 2011; Yu et al., Sci. Trans. Med., 3 , 84ra44, 2011).

Exemplary bispecific antibodies can also be (1) a dual-variable-domain antibody (DVD-Ig) in which each light chain and heavy chain contain two variable domains in tandem via a short peptide bond (Wu et al ., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVDIg™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010));

(2) Tandab (tandem antibody), which is the fusion of two single chain diabodies to produce a tetravalent bispecific antibody containing two binding sites for each of the target antigens;

- 11 041230 (3) a flexibody antibody, which is a combination of scFv with a diabody to form a multivalent molecule;

(4) a so-called dock and lock molecule based on the dimerization and docking domain in protein kinase A, which, when applied with a Fab, produces a trivalent bispecific binding protein consisting of two identical Fab fragments attached to great Fab fragment; or (5) a so-called Scorpion molecule containing, for example, two scFvs fused to both ends of the human Fc region.

Examples of platforms suitable for generating bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab arm replacement, Genmab).

The term epitope refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or non-contiguous amino acids that have come together as a result of tertiary folding of one or more proteins. Epitopes formed by adjacent amino acids (also known as linear epitopes) are generally resistant to denaturing solvents, while epitopes formed by tertiary folding (also known as conformational epitopes) are usually destroyed by treatment with denaturing solvents. An epitope typically contains at least 3 and more often at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, vol. 66, Glenn E. Morris, ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be identified using a conventional immunoassay that detects the ability of one antibody to compete with the binding of another antibody to the target antigen. The epitope of an antibody can also be determined by X-ray crystallography of an antibody bound to its antigen to identify contact residues. Alternatively, two antibodies target the same epitope if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. Two antibodies target overlapping epitopes if certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody.

Antibody competition is determined by an assay in which the antibody of interest inhibits the specific binding of a reference antibody to a known antigen (see, for example, Junghans et al., Cancer Res., 50:1495, 1990). The test antibody competes with the reference antibody if an excess of test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50% as measured in the analysis of competitive binding. Some test antibodies inhibit reference antibody binding by at least 75%, 90%, or 99%. Antibodies identified by competitive assay (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope that the reference antibody binds to cause steric barrier.

The term pharmaceutically acceptable means that the carrier, diluent, excipient or excipient is compatible with the other ingredients of the formulation and is not substantially harmful to its recipient.

The term patient includes a human or other mammalian subject receiving prophylactic or therapeutic treatment.

An individual is at increased risk of developing a disease if the subject has at least one known risk factor (eg, genetic, biochemical, family history, and exposure to situational factors) that places individuals with that risk factor at a statistically significant greater risk of developing the disease compared to individuals without risk factor.

The term biological sample means a sample of biological material in a biological source or derived from a biological source, such as a human or mammalian subject. Such samples can be organs, organelles, tissues, tissue sections, body fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations obtained from these samples. The term biological sample may also cover any material resulting from the processing of a sample. The resulting material may include cells or their progeny. Processing of a biological sample may include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering substances, and the like.

The term control sample means a biological sample that is known or

- 12 041230 is suspected to not contain areas affected by tau disease, or which is at least known or suspected to not contain areas of this type of disease affected. Control samples can be obtained from individuals not suffering from tau-induced disease. Alternatively, control samples can be obtained from patients suffering from tau-induced disease. Such samples may be obtained simultaneously with a biological sample believed to contain tau-induced disease, or at another time. A biological sample and a control sample can be obtained from the same tissue. Preferably, control samples consist of essentially or completely normal, healthy areas and can be used in comparison with a biological sample that is considered to contain areas affected by tau-induced disease. Preferably, the tissue in the control sample is of the same type as the tissue in the biological sample. Preferably, the tau-affected cells believed to be in the biological sample originate from the same cell type (eg, neurons or glia) as the cell type in the control sample.

The term disease means any abnormal condition that impairs physiological function. The term is used in a broad sense to cover any disorder, ailment, anomaly, pathology, disease, condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

The term symptom refers to the subjective manifestation of the disease, such as an altered gait, as perceived by the subject. A symptom refers to the objective manifestation of a disease observed by a physician.

The term positive response to treatment means a more favorable response in an individual patient or an average response in a patient population compared to an average response in an untreated control population.

For the purpose of classifying amino acid substitutions as conservative or non-conservative amino acids are grouped as follows:

Group I (hydrophobic side chains): met, ala, val, leu, ile;

Group II (neutral hydrophilic side chains): cys, ser, thr;

Group III (acid side chains): asp, glu;

Group IV (main side chains): asn, gin, 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 are substitutions of amino acids of one class for amino acids of another class.

Percent sequence identity is determined by antibody sequences aligned as closely as possible using the Cabot numbering system. After alignment, if a region of an antibody of interest (e.g., a full mature heavy or light chain variable region) is compared to the same region of a reference antibody, the percent sequence identity between the regions of interest and the reference antibody is the number of positions occupied by the same amino acids in the regions. of both the antibody of interest and the reference antibody divided by the total number of aligned positions of the two regions, without counting breaks, multiplied by 100 to convert to a percentage.

Compositions or methods containing or including one or more of these elements may contain or include other elements not specifically indicated. For example, a composition that contains or includes an antibody may contain the antibody by itself or in combination with other components.

Specifying a range of values includes all integer values within or within the specified range and all subranges defined by integer values within the range

Unless it is clear from the context to the contrary, the term roughly covers insignificant variations, such as values within a standard error (e.g., SEM - standard error of the mean) of a specified value.

Statistically significant means p<0.05.

Singular forms include plural references to objects unless the context clearly dictates otherwise. For example, the term compound or at least one compound may include a plurality of compounds, including mixtures thereof.

Detailed description of the invention

I. General part.

The present invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within residues 55-78 of SEQ ID NO: 3 (corresponding to residues 113-136 of SEQ ID NO: 1), such as, for example, an epitope within residues 60-75 (corresponding to residues 118-133 of SEQ ID NO: 1) or within residues 61-70 (corresponding to residues 119-128 of SEQ ID NO: 1). Some of these antibodies can specifically bind to two peptides, such as the first peptide containing residues 55-69 of SEQ ID NO: 3 (corresponding to residues 113-127 of SEQ ID NO: 1), as well as

- 13 041230 the second peptide containing residues 64-78 (corresponding to residues 122-136 of SEQ ID NO: 1). Some antibodies bind to tau regardless of phosphorylation status. Certain antibodies of the present invention act to inhibit or delay tau-related pathologies and associated symptom aggravations. Although an understanding of the mechanism is not necessary for the implementation of the present invention in practice, an understanding of the mechanism is not necessary, a decrease in toxicity may result from antibody-induced tau phagocytosis, inhibition of inter- or intramolecular aggregation of tau, or binding to other molecules through stabilization of a non-toxic conformation, through inhibition of intercellular or intracellular transmission pathogenic forms of tau, by blocking tau phosphorylation, by preventing tau from binding to cells, or by inducing proteolytic cleavage of tau, among other mechanisms. The antibodies of the present invention, or agents that induce such antibodies, can be used in methods of treating or preventing Alzheimer's disease and other tau-related diseases.

II. target molecules.

Unless it is clear from the context to the contrary, reference to tau refers to the natural form of human tau, including all isoforms, regardless of whether post-translational modification (eg, phosphorylation, glycation, or acetylation) is present. There are six main isoforms (splice variants) of tau produced in the human brain. The longest of these variants contains 441 amino acids and cleaves off the initial met residue. Residues are numbered according to isoform 441. Thus, for example, mention of phosphorylation at position 404 means position 404 of isoform 441, or the corresponding position of any other isoform when aligned to the maximum isoform 441. Isoform amino acid sequences and Swiss-Prot database numbers are shown below.

P10636-8 (SEQ ID NO:1)

10 20 30 40 50 60 MAEPRQEFEV 70 MEDHAGTYGL 80 90 GDRKDQGGYT 100 1 MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 10 12 0 SETSDAKSTP 130 TAEDVTAPLV 140 150 DEGAPGKQAA 160 AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 170 180 HVTQARMVSK 19 0 SKDGTGSDDK 200 210 KAKGADGKTK 220 IATPRGAAPP GQKGQANATR IPAKTPPAPK 230 240 TPPSSGEPPK 25 0 SGDRSGYSSP 260 270 GSPGTPGSRS 280 RTPPSLPTPPT REPKKVAWR TPPKSPSSAK 290 300 SRLQTAPVPM 310 PDLKNVKSKI 320 330 GSTENLKHQP 34 0 GGGKVQIINK KLDLSNVQSK CGSKDNIKHV 350 360 PGGGSVQIVY 370 KPVDLSKVTS 380 390 KCGSLGNIHH 400 KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 410 420 THVPGGGNKK 43 0 DSPQLATLAD IETHKLTFRE 44 0 EVSASLAKQG NAKAKTDHGA L EIVYKSPWS GDTSPRHLSN VSSTGSIDMV

P10636-7 (SEQ ID NO:2)

- 14 041230

20 30 40 50 60

MAEPRQEFEV 70 MEDHAGTYGL 80 90 GDRKDQGGYT MHQDQEGDTD 100 110 120 AGLKESPLQT PTEDGSEEPG SETSDAKSTP 130 TAEAEEAGIG 140 150 DTPSLEDEAA GHVTQARMVS 16_0 170 180 KSKDGTGSDD KKAKGADGKT KIATPRGAAP 19 0 PGQKGQANAT 200 210 RIPAKTPPAP KTPPSSGEPP 22_0 230 240 KSGDRSGYSS PGSPTGTPGSR SRTPSLPTPP 25 0 TREPKKVAW 260 270 RTPPKSPSSA KSRLQTAPVP 2 8_0 2 90 300 MPDLKNVKSK IGSTENLKHQ PGGGKVQIIN 310 KKLDLSNVQS 320 330 KCGSKDNIKH VPGGGSVQIV 34_0 350 360 YKPVDLSKVT SKCGSLGNIH HKPGGGQVEV 370 KSEKLDFKDR 380 390 VQSKIGSLDN ITHVPGGGNK 4 0 _0 410 KIETHKLTFR ENAKAKTDHG AEIVYKSPW SGDTSPRHLS NVSSTGSIDM VDSPQLATLA P10636-6 (human tau 4PONY) (SEQ ID NO:3) 10 20 30 40 50 60 DEVSASLAKQ GL MAEPRQEFEV 70 MEDHAGTYGL 80 90 GDRKDQGGYT MHQDQEGDTD 100 110 120 AGLKAEEAGI GDTPSLEDEA AGHVTQARMV 130 SKSKDGTGSD 140 150 DKKAKGADGK TKIATPRGAA 16-0 170 180 PPGQKGQANA TRIPAKTPPA PKTPPSSGEP 19 0 PKSGDRSGYS 200 210 SPGSPGTPGS RSRTPSLPTP 22_0 230 240 PTREPKKVAV VRTPPKSPSS AKSRLQTAPV 25 0 PMPDLKNVKS 260 270 KIGSTENLKH QPGGGKVQII 28_0 290 300 NKKLDLSNVQ SKCGSKDNIK HVPGGGSVQI 310 VYKPVDLSKV 320 330 TSKCGSLGNI HHKPGGGQVE 34_0 350 360 VKSEKLDFKD RVQSKIGSLLD NITHVPGGGN KKIETHKLTF 370 380 MVDSPQLATL ADEVSASLAK P10636-5 (SEQ ID NO :4) 10 20 30 RENAKAKTDH GAEIVYKSPV QGL 40 50 60 VSGDTSPRHL SNVSSTGSID MAEPRQEFEV 70 MEDHAGTYGL 80 90 GDRKDQGGYT MHQDQEGDTD 100 110 120 AGLKESPLQT PTEDGSEEPG SETSDAKSTP 130 TAEDVTAPLV 140 150 DEGAPGKQAA AQPHTEIPEG 16_0 170 180 TTAEEAGIGD TPSLEDEAAG HVTQARMVSK 19 0 SKDGTGSDDK 200 210 KAKGADGKTK IATPRGAAPP 22_0 230 240 GQKGQANATR IPAKTPPAPK TPPSSGEPPK 25 0 SGDRSGYSSP 260 270 GSPGTPGSRS RTPSLPTPPT 28_0 290 300 REPKKVAWR TPPKSPSSAK

- 15 041230

SRLQTAPVPM 310 PDLKNVKSKI 320 330 GSTENLKHQP 34 0 GGGKVQIVYK 350 360 PVDLSKVTSK CGSLGNIHHK PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKI ETHKLTFREN AKAKTDHGAE 370 380 390 400 410 IVYKSPWSG DTSPRHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGL P10636-4 (SEQ ID NO:5) 10 20 30 40 50 60 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 100 1 10 12 0 SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGET 130 140 150 160 170 180 KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPTGTPGSR 19 0 200 210 220 230 240 SRTPSLPTPP TREPKKVAW RTPPKSPSSA KSRLQTAPVP MPDLKNVKSK IGSTENLKHQ 25 0 260 270 280 290 300 PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 310 320 330 34 0 350 360 THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPWS GDTSPRHLSN VSSTGSIDMV 370 38 0 DSPQLATLAD EVSASLAKQG L

P10636-2 (SEQ ID NO:6) 10 20 MAEPRQEFEV 30 40 50 60 MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 70 AGHVTQARMV 80 90 SKSKDGTGSD 100 110 120 DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 130 PKTPPSSGEP 140 150 PKSGDRSGYS 160 170 180 SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 190 AKSRLQTAPV 200 210 PMPDLKNVKS 220 230 240 KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH 250 HKPGGGQVEV 260 270 KSEKLDFKDR 280 290 300 VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG 310 320 330 340 350

AEIVYKSPW SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

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

Tau may be phosphorylated at one or more amino acid residues, including tyrosine at amino acid positions 18, 29, 97, 310 and 394, serine at amino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293, 324, 356, 396, 400, 404, 409, 412, 413 and 422; and threonine at amino acid positions 175, 181, 205, 212, 217, 231, and 403. Unless otherwise apparent from the context, reference to tau or fragments thereof includes natural human amino acid sequences, including their isoforms, mutants, and allelic variants.

IV. Antibodies.

A. Binding specificity and functional properties.

The present invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within residues 55-78 of SEQ ID NO: 3 (corresponding to residues 113-136 of SEQ ID NO: 1), such as, for example, an epitope within residues 60-75 of SEQ ID NO: 3 (corresponding to residues 118-133 of SEQ ID NO: 1) or within residues 61-70 of SEQ ID NO: 3 (corresponding to residues 119-128 of SEQ ID NO: 1). Some antibodies bind to tau regardless of phosphorylation status. These antibodies can be obtained by immunization with a tau polypeptide purified from a natural source or recombinantly expressed. Antibodies can be screened for binding to tau in an unphosphorylated form, as well as in a form in which one or more phosphorylation residues are phosphorylated. Such antibodies preferably bind with indistinguishable affinities, or at least within 1.5-, 2-, or 3-fold affinity for phosphorylated tau compared to non-phosphorylated tau (ie, are pan-specific). 16G7 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, for example, the 16G7 epitope. Also included are antibodies that compete for tau binding with any of the aforementioned antibodies, such as, for example, those that compete with 16G7.

The above antibodies can be obtained de novo by immunization with a peptide containing residues 55-78, 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to residues 113-136, 113-127 or 122-136, respectively, of SEQ ID NO: 1 ), or by immunization with a full length tau polypeptide or fragment containing such residues and screening for specific binding to a peptide containing such residues. Such peptides are preferably attached to a heterologous conjugated molecule that contributes to inducing an antibody response to the peptide. The attachment may be direct or indirect by 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. A polyglycine linker (eg 2-6 glycines) with or without a cysteine residue between the glycines and the peptide can also be used. The carrier molecule acts to provide a T cell epitope that contributes to inducing an antibody response to the peptide. Some carriers are commonly used, in particular fisurella hemocyanin (keyhole limpet hemocyanin, KLH), ovalbumin and bovine serum albumin (BSA). Peptide spacers can be added to the peptide immunogen during solid phase peptide synthesis. Carriers are typically added via chemical crosslinking. Some examples of chemical crosslinkers that can be used include cross-N-maleimido-6-aminocaproyl ester or t-maleimidobenzoyl-N-hydroxysuccinimide (MBS) ester (see, for example, Harlow, E. et al. ., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Siniaglia et al., Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47 (1993); Hammer et al., Cell, 74:197-203 (1993); Falk, K. et al., Immunogenetics, 39:230-242 (1994); WO 98/23635; and Southwood et al., J. Immunology, 160:3363-3373 (1998)). The carrier and spacer, if present, may be attached to either end of the immunogen.

The peptide with an optional spacer and carrier can be used to immunize laboratory animals or B cells, as described in more detail below. The ability of hybridoma supernatants to bind to one or more peptides containing residues 55-78, 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to residues 113-136, 113-127 or 122-136, respectively, of SEQ ID NO: : 1), and/or with phosphorylated and non-phosphorylated forms of tau, such as, for example, the full length isoform of tau at position 404 in phosphorylated form. The peptide may be attached to a carrier or other label to facilitate screening analysis. In this case, the carrier or label is preferably different from the combination of the spacer and carrier molecule that was used for immunization to eliminate antibodies specific for the spacer or carrier instead of the tau peptide. Any of the tau isoforms may be used.

The present invention provides monoclonal antibodies that bind to epitopes in

- 17 041230 within Tau. The antibody designated 16G7 is one such exemplary mouse antibody. Unless the context makes it clear to the contrary, reference to 16G7 should be understood to mean any of the mouse, chimeric, veneered, and humanized forms of the antibody. The antibody was deposited as [DEPOSITION NUMBER]. This antibody specifically binds within amino acid residues 55-78, 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to residues 113-136, 113-127 or 122-136, respectively, of SEQ ID NO: 1). This antibody is also characterized by the ability to bind both phosphorylated and unphosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded/aggregated forms of tau.

Some antibodies of the present invention bind to the same or overlapping epitope as the antibody designated 16G7. The sequences of the mature variable regions of the heavy and light chains of this antibody are designated by SEQ ID NO: 7 and SEQ ID NO: 11, respectively. Other antibodies with such binding specificity can be generated by immunizing mice with tau or a portion thereof containing the desired epitope and screening the resulting antibodies for binding to tau, optionally in competition with an antibody containing mouse 16G7 variable regions (IgG1 kappa) . Tau fragments containing the desired epitope may be attached to a carrier that promotes an antibody response to the fragment and/or can be combined with an adjuvant that promotes such a response. Such antibodies can be screened for different binding to tau or a fragment thereof compared to mutants of the indicated residues. Screening, compared with such mutants, more accurately determines binding specificity to allow identification of antibodies whose binding is inhibited by mutagenesis of specific residues and which are likely to have the same functional properties as other exemplary antibodies. Mutations can be systematic substitutions by alanine (or serine, if alanine is already present) for one residue at a time, or over longer periods of time, throughout the target, or throughout the portion of the target where the epitope is known to be located. If the same set of mutations greatly reduces the binding of two antibodies, the two antibodies bind to the same epitope.

Antibodies that have the binding specificity of a selected mouse antibody (eg, 16G7) can also be generated using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for the production of human antibodies. In this method, the heavy or light chain variable region of a selected mouse antibody is used as starting material. If, for example, a light chain variable region is chosen as a starting material, a phage library is constructed in which members exhibit the same light chain variable region (ie, mouse starting material) and different heavy chain variable regions. Heavy chain variable regions can, for example, be obtained from a library of rearranged human heavy chain variable regions. A phage is selected that exhibits strong specific binding to tau or a fragment thereof (eg, at least 10 and preferably at least 109 M ^-1 ). The heavy chain variable regions from that phage are then used as starting material to construct the next phage library. In this library, each phage exhibits the same heavy chain variable region (ie, the region identified from the first display library) and different light chain variable regions. Light chain variable regions can be obtained, for example, from a library of rearranged human light chain variable regions. Again, a phage showing strong tau-specific binding is selected. The resulting antibodies typically have the same or similar epitope specificity as the original mouse material.

The 16G7 heavy chain CDRs according to the Cabot/Chothia summary definition are designated SEQ ID NOs: 8, 9 and 10, respectively, and the 16G7 light chain CDRs according to Cabot are designated SEQ ID NOs: 12, 13 and 14, respectively.

- 18 041230

table 2

CDR 16G7 as defined by Cabot, Chothia, combined definition of Chothia and Cabot, AbM and contact definition

A loop Cabot Chothia Consolidated definition of Chothia and Cabot AbM Contact Definition L1 L24-L34 SEQIDNO:12 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L30-L36 SEQ ID NO: 43 L2 L50-L56 SEQ ID NO: 13 L50-L56 SEQ ID NO: 13 L50-L56 SEQ ID NO: 13 L50-L56 SEQ ID NO: 13 L46-L55 SEQ ID NO: 44 L3 L89-L97 SEQ ID NO: 14 L89-L97 SEQIDNO:14v L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L96 SEQ ID NO:45 H1 H31-H35B SEQ ID NO:39 H26-H32 SEQ ID NO:40 H26-H35B SEQ ID NO:8 H26-H35B SEQ ID NO:8 H30-H35B SEQ ID NO:46 H2 H50-H65 SEQ ID NO: 9 H52-H56 SEQIDNO:41 H50-H65 SEQ ID NO: 9 H50-H58 SEQ ID NO :42 H47-H58 SEQ ID NO:47 NZ H95-H102 SEQ ID NO: 10 H95-H102 SEQ ID NO: 10 H95-H102 SEQ ID NO: 10 H95-H102 SEQ ID NO: 10 H93-H101 SEQ ID NO:48

Other antibodies can be generated by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as 16G7. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to 16G7 in the amino acid sequence of mature heavy and/or light chain variable regions and retain their functional properties and/or that differ from the corresponding antibodies with a small number of functionally non-essential amino acid substitutions (eg conservative substitutions), deletions or insertions are also included in the present invention. Also included are monoclonal antibodies containing at least one or all six CDRs according to any conventional definition, but preferably according to Cabot, which are 90%, 95%, 99%, or 100% identical to the corresponding 16G7 CDRs.

The present invention also provides antibodies containing some or all (eg, 3, 4, 5 and 6) CDRs derived entirely or substantially from 16G7. Such antibodies may comprise a heavy chain variable region that contains at least two, but typically all three CDRs derived entirely or substantially from a 16G7 heavy chain variable region, and/or a light chain variable region that contains at least two, but typically all three CDRs derived wholly or substantially from the 16G7 light chain variable region. Antibodies can contain both heavy and light chains. A CDR is essentially derived from a corresponding 16G7 CDR when it contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that a CDR-H2 (as defined by Cabot) may contain no more than 6, 5, 4, 3, 2 or 1 substitutions, insertions or deletions. Such antibodies may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with 16G7 of the amino acid sequence of mature heavy and/or light chain variable regions and retain their functional properties and/or may differ from 16G7 by a small amount. the number of functionally non-essential amino acid substitutions (eg conservative substitutions), deletions or insertions.

Some antibodies identified in these assays may bind to monomeric, misfolded, aggregated, phosphorylated or non-phosphorylated forms of tau or other forms. Similarly, some antibodies are immunologically reactive for non-pathological and pathological forms and conformations of tau.

B. Antibodies other than human antibodies.

Generation of antibodies other than human, e.g., mouse, guinea pig, primate, rabbit, or rat, against tau or a fragment thereof (e.g., amino acid residues 55-78, 60-75, 61-70, 55-69, or 64-78 SEQ ID NO: 3 corresponding to amino acid residues 113-136, 118-133, 119-128, 113-127 or 122-136, respectively, SEQ ID NO: 1) can be done by, for example, immunizing the animal with tau or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source by peptide synthesis or by recombinant expression. Optionally, the immunogen may be administered fused to a carrier protein or otherwise

- 19 041230 complexed with a carrier protein. Optionally, the immunogen may be administered with an adjuvant. Several types of adjuvants can be used, as described below. For immunization of laboratory animals, Freund's complete adjuvant followed by the use of an incomplete adjuvant is preferred. To obtain polyclonal antibodies, as a rule, rabbits or guinea pigs are used. To obtain monoclonal antibodies, as a rule, mice are used. Antibodies are screened for specific binding to tau or to an epitope within tau (e.g., an epitope containing one or more of amino acid residues 55-78, 60-75, or 61-70 of SEQ ID NO: 3 (corresponding to amino acid residues 113-136 , 118-133 or 119-128, respectively, SEQ ID NO: 1) Such a screen can be performed by determining the binding of an antibody to a population of tau variants, such as tau variants containing amino acid residues 55-78, 60-75, 61-70 , 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to amino acid residues 113-136, 118-133, 119-128, 113-127 or 122-136, respectively, of SEQ ID NO: 1), or mutations within these residues, and determining which tau variants bind to the antibody Binding can be assessed, for example, by Western blotting, FACS, or ELISA.

C. Humanized antibodies.

A humanized antibody is a genetically engineered antibody in which CDRs from a non-human donor antibody are transferred into human antibody acceptor sequences (see, for example, Queen, US 5530101 and 5585089; Winter, US 5225539; Carter, US 6407213; Adair , US 5859205; and Foote, US 6881557). Acceptor antibody sequences can be, for example, a mature human antibody sequence, a combination of such sequences, a human antibody consensus sequence, or a germline sequence. Thus, a humanized antibody is an antibody containing at least three, four, five, or all of the CDRs derived wholly or substantially from the donor antibody, and variable region framework sequences and constant regions, if present, derived wholly or substantially from human antibody sequences. Similarly, a humanized heavy chain contains at least one, two, and usually all three CDRs derived entirely or substantially from the donor antibody's heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, derived essentially from the sequences framework region of the variable region and the constant region of the human heavy chain. Similarly, a humanized light chain contains at least one, two, and usually all three CDRs derived entirely or essentially from the light chain of the donor antibody, and a light chain variable region framework sequence and a light chain constant region, if present, derived essentially from the sequences framework region of the variable region and the constant region of the human light chain. Unlike nanobodies and dAbs, a humanized antibody contains a humanized heavy chain and a humanized light chain. The CDRs in a humanized antibody are derived essentially from the corresponding CDRs in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (by any conventional definition, but preferably defined by Cabot) are identical between the corresponding CDRs. An antibody variable region framework region or an antibody chain constant region sequence is derived substantially from a human variable region framework region or human constant region sequence, respectively, when at least 85%, 90%, 95%, or 100% of the respective residues, as determined by Cabot, are identical. To be classified as humanized according to the 2014 World Health Organization (WHO) International Nonproprietary Names (INN) definition of humanized antibodies, an antibody must share at least 85% identity with human germline (i.e., presomatic) antibody sequences. hypermutations). Mixed antibodies are antibodies in which one antibody chain (eg, heavy chain) meets the cut-off, but the other chain (eg, light chain) does not meet the cut-off. An antibody is classified as chimeric if neither chain meets the cut-off, even if the variable region frameworks for both chains were derived essentially from a human with some mouse backmutations. See Jones et al. (2016), The INNs and outs of antibody nonproprietary names, mAbs 8:1, 1-9, DOI: 10.1080/19420862.2015.1114320. See also WHOINN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: http://www.who.int/medicines/services/inn/BioRev2014.pdf) , incorporated herein by reference. For the avoidance of doubt, the term humanized in this document is not limited to the definition of humanized antibodies according to the 2014 WHO INN. herein, have less than 85% sequence identity to human germline sequences. Some of the humanized antibody heavy chains provided herein are

- 20 041230 are characterized by about 60 to 100% sequence identity with human germline sequences, such as, for example, in the range of about 60 to 69%, 70 to 79%, 80 to 84%, or 85% to 89% . Some heavy chains fall short of the 2014 WHO INN definition and are characterized, for example, by sequence identity of approximately 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78 , 79, 80, 81, 82, 83, or 84% with human germline sequences, while other heavy chains meet the definition according to the 2014 WHO INN and have approximately 85, 86, 87, 88, 89% or greater sequence identity with human germline sequences. Some of the humanized antibody light chains provided herein have about 60% to 100% sequence identity with human germline sequences, such as, for example, about 80% to 84% or 85% to 89%. Some light chains fall short of the 2014 WHO INN definition and have, for example, approximately 81, 82, 83, or 84% sequence identity with human germline sequences, while other light chains meet the 2014 WHO INN definition and are characterized approximately 85%, 86%, 87%, 88%, 89% or greater sequence identity with human germline sequences. Certain humanized antibodies provided herein that are chimeric as defined by the 2014 WHO INN contain heavy chains with less than 85% identity with human germline sequences paired with light chains that have less than 85% identity with sequences human germ line. Some humanized antibodies provided herein are mixed as defined by the 2014 WHO INN, e.g., contain a heavy chain with at least 85% sequence identity with human germline sequences paired with a light chain that has sequence identity less than 85% with human germline sequences or vice versa. Certain humanized antibodies provided herein meet the definition of a humanized antibody according to the 2014 WHO INN and contain a heavy chain with at least 85% sequence identity with human germline sequences paired with a light chain that has a sequence identity of at least 85 % with human germline sequences. Illustrative antibodies that meet the definition of humanized antibodies according to the 2014 WHO INN include antibodies containing a mature light chain with the amino acid sequence of SEQ ID NO: 29 paired with a mature heavy chain that contains the amino acid sequence of SEQ ID NO: 20, SEQ ID NO : 25, SEQ ID NO: 26 or SEQ ID NO: 27. Additional humanized antibodies of the present invention include antibodies containing a mature heavy chain that contains the amino acid sequence of any of SEQ ID NO: 15-27 paired with a mature light chain that contains the amino acid sequence of any of SEQ ID NO: 28-30.

Although humanized antibodies often contain all six CDRs (defined according to any conventional definition, but preferably according to Cabot's definition) from a mouse antibody, they may also not be made with all CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouse antibody (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).

In some antibodies, only a portion of the CDRs, namely the subset of CDR residues required for binding, referred to as SDRs, are required to maintain binding in a humanized antibody. Non-antigen-contacting, non-SDR CDR residues can be identified based on prior studies (e.g., H60-H65 residues in CDR H2 are often unnecessary), from Cabot's CDR regions outside of Chothia's hypervariable loops. , J., Mol. Biol., 196:901, 1987), by molecular modeling and/or empirically or as described in Gonzales et al., Mol. Immunol., 41:863, 2004. In such humanized antibodies, at positions where one or more donor CDR residues are missing or where the entire donor CDR is missing, the amino acid occupying the position may be the amino acid occupying the corresponding position (according to Cabot numbering). ) in the sequence of the acceptor antibody. The number of such donor amino acid substitutions in the CDR for inclusion reflects the balance of competing considerations. Such substitutions are potentially beneficial in reducing the number of mouse amino acids in a humanized antibody, and thereby reducing potential immunogenicity, and/or in meeting the WHO INN definition of a humanized antibody. However, substitutions can also cause affinity changes, and a significant decrease in affinity is preferably avoided.

Substitution positions within the CDR and amino acid substitution can also be empirically selected.

Human acceptor antibody sequences can optionally be selected from a variety of known human antibody sequences to obtain a high degree of identity.

- 21 041230 sequences (eg, 65-85% identity) between the human acceptor sequence variable region framework regions and the corresponding donor antibody chain variable region framework regions.

An example of a heavy chain acceptor sequence is the mature human heavy chain variable region with accession code NCBI AAA18265.1 (SEQ ID NO: 31). An example of a heavy chain acceptor sequence is the mature human heavy chain variable region with NCBI accession code ADW08092.1 (SEQ ID NO: 32). An example of a heavy chain acceptor sequence is the mature human heavy chain variable region with NCBI accession code IMGT# IGHV1-2*02 (SEQ ID NO: 33). IMGT# IGHV1-2*02 contains CDR-H1 and CDR-H2, which share the same canonical form as the mouse 16G7 heavy chain and is a member of Cabot's human heavy chain subgroup 1. An example of a light chain acceptor sequence is a mature human light chain variable region with NCBI accession code AAB24404.1 (SEQ ID NO: 34). An example of a light chain acceptor sequence is a mature human light chain variable region with NCBI accession code AEK69364.1 (SEQ ID NO: 35). An example of a light chain acceptor sequence is a mature human light chain variable region with NCBI accession code IMGT#IGKV4-1*01 (SEQ ID NO: 36). IMGT#IGKV4-1*01 has the same canonical classes for CDR-L1, CDR-L2 and L3 as 16G7 mice and belongs to human kappa subgroup 1.

If more than one human antibody acceptor sequence is selected, a combination or hybrid of these acceptors can be used, and the amino acids used at various positions in the humanized light chain and heavy chain variable regions can be from any of the human acceptor antibody sequences used.

Certain amino acids from the human variable region framework residues can be selected for substitution based on their possible effect on CDR conformation and/or antigen binding. The study of such possible influences is carried out through modeling, studying the characteristics of amino acids at specific locations, or empirical observation of the effects of substitution or mutagenesis of specific amino acids.

For example, when an amino acid differs between a mouse variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be replaced with an equivalent framework amino acid from a mouse antibody when amino acid (1) is reasonably expected to non-covalently directly bind to an antigen;

(2) adjacent to or within the CDR as defined by Chothia but not by Cabot;

(3) otherwise interacts with the CDR region (eg, within about 6 A of the CDR region) (eg, as identified by modeling a light or heavy chain on a decoded structure of a homologous known immunoglobulin chain); or (4) is a residue participating in the VL-VH interaction surface.

The present invention provides humanized forms of the mouse 16G7 antibody, including 13 exemplary humanized mature heavy chain variable regions:

hul6G7VHvl (SEQ ID NO: 15), hul6G7VHv2) (SEQ ID NO: 16), hul6G7VHv2a (SEQ ID NO: 17), 16G7VHv2aB7 (SEQ ID NO: 18), hul6G7VHv2b (SEQ ID NO: 19), hul6G7VHv2bH7 (SEQ ID NO:20), hul6G7VHv3 (SEQ ID NO:21), hul6G7VHv4 (SEQ ID NO:22), hul6G7VHv5V (SEQ ID NO:23), hul6G7VHv5VR (SEQ ID NO:24), hul6G7VHv6a (SEQ ID NO:25), hul6G7VHv6b (SEQ ID NO:26 hul6G7VHv7 (SEQ ID NO:27), and 3 exemplary humanized mature light chain variable regions: hul6G7VLvl (SEQ ID NO:28), hu!6G7VLv2 (SEQ ID NO:29) and hu!6G7VLv3 ( SEQ ID NO:30).

In an embodiment, humanized sequences are generated using a two-step PCR protocol that allows multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W. and Malcolm, B.A. (1999) BioTechniques 26:680-682)].

Skeleton remains from classes (1) to (3) as defined by Queen, US 5530101 are sometimes referred to alternatively as canonical and vernier remains. Framework residues that help define 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 maintain the conformation of the antigen-binding loop and play a role in fine-tuning antibody-antigen match are sometimes referred to as vernier residues (Foote & Winter, J. Mol. Biol, 224:487-499 (1992)).

Other framework residues that are candidates for replacement are those that form a potential glycosylation site. Other candidates for replacement are

- 22 041230 acceptor framework human amino acids that are unusual for human immunoglobulin at this position. These amino acids can be replaced with amino acids from the equivalent position of the mouse donor antibody or from the equivalent position of more typical human immunoglobulins.

Other framework residues that are candidates for replacement are N-terminal glutamine residues (Q), which can be replaced with glutamic acid (E) to minimize the possibility of pyroglutamate conversion [Y. Diana Liu et al., 2011, J. Biol. Chem., 286:11211-11217]. The conversion of glutamic acid (E) to pyroglutamate (pE) is slower than that of glutamine (Q). Due to the loss of the primary amine during the conversion of glutamine to pE, the antibodies become more acidic. Incomplete conversion results in heterogeneity in the antibody, which can be observed as multiple peaks using charge-based analytical methods. Differences in heterogeneity may indicate a lack of process control. Exemplary humanized antibodies with N-terminal glutamine to glutamate substitutions are

SEQ ID NO: 15 (hul6G7VHvl), SEQ ID NO: 16 (hul6G7VHv2), SEQ ID NO: 17 (hul6G7VHv2a), SEQ ID NO: 18 (16G7VHv2aB7), SEQ ID NO: 19 (hul6G7VHv2b), SEQ ID NO: 20 (hul6G7VHv2bH7), SEQ ID NO:21 (hul6G7VHv3), SEQ ID NO:22 (hul6G7VHv4), SEQ ID NO:23 (hul6G7VHv5V), SEQ ID NO:24 (hul6G7VHv5VR) and SEQ ID NO:27 (hul6G7VHv7).

Exemplary humanized antibodies are humanized forms of mouse 16G7, designated Hu16G7.

The mouse 16G7 antibody contains mature heavy and light chain variable regions containing amino acid sequences that contain SEQ ID NO: 7 and SEQ ID NO: 11, respectively. The present invention provides 13 exemplary humanized mature heavy chain variable regions:

hul6G7VHvl, hul6G7VHv2), hul6G7VHv2a, 16G7VHv2aB7, hul6G7VHv2b, hul6G7VHv2bH7, hul6G7VHv3, hul6G7VHv4, hul6G7VHv5V, hul6G7VHv5VR, hul6Gv7VHv6a, hul6G7VH7, and hul6G7VH7

The present invention also provides 3 exemplary mature human light chain variable regions: hu16G7VLv1, hu16G7VLv2 and hu16G7VLv3. In FIG. 2 and 3 show alignments of the heavy chain variable region and the light chain variable region, respectively, of mouse 16G7 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/or antigen binding, mediation of interaction between heavy and light chains, interaction with a constant region, presenting a site for desired or undesired post-translational modification, presenting an unusual residue for its position in the variable sequence human region and therefore potentially immunogenic, acquisition of aggregation potential, and for other reasons, the following 33 variable region framework positions were considered candidates for substitutions in 3 exemplary mature human light chain variable regions and 13 exemplary mature human heavy chain variable regions, further exemplified:

L4 (M4L), L9 (D9S), L15 (L15V), L18 (R18K), L19 (A19V), L21 (I21M), L22 (N22S), L43 (P43S), L48 (I48M), Hl (Q1E), H5 (V5Q),

H7 (S7P), H9 (A9S), HH (E10V), NI (VI IL), H12 (K12V), H13 (K13R), H20 (V20L), H37 (V37A), H38 (R38K), H40 (A40R) , H48 (M48I), H66 (R66K), H67 (V67K), H69 (M69L), H71 (R71V), H73 (T73I), H75 (I75S or I75A), H80 (M80V), H82a (S82a-T), H82b (R82b-S), H83 (R83T), H85 (D85E).

The following 3 variable region CDR positions were considered candidates for substitutions in 13 exemplary mature human heavy chain variable regions, further cited in the examples: H31 (S31G), H60 (N60A) and H64 (K64Q).

Here, as elsewhere, the first-mentioned residue is the residue of a humanized antibody formed by transferring the CDR according to Cabot or the CDR according to the Chothia-Cabot summary definition in the case of CDR-H1 into the human acceptor framework, and the second-mentioned residue is the residue considered to replace such a remainder. Thus, within the framework regions of the variable region, the first-mentioned residue is a human residue, and within the CDR, the first-mentioned residue is a mouse residue.

Exemplary antibodies include any permutations or combinations of the exemplary mature heavy and light chain variable regions, e.g.,

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VHvl/VLvl, VHvl/VLv2, VHvl/VLv3, VHv2/VLvl, VHv2/VLv2, VHv2/VLv3,

VHv2a/VLvl, VHv2a/VLv2, VHv2a/VLv3, VHv2aB7/VLvl, VHv2aB7/VLv2,

VHv2aB7/VLv3 VHv2b/VLvl VHv2b/VLv2 VHv2b/VLv3 VHv2bH7/VLvl VHv2bH7/VLv2 VLv3, VHv5/VLvl, VHv5/VLv2, VHv5/VLv3, VHv5VR/VLvl, VHv5VR/VLv2, VHv5VR/VLv3, VHv6a/VLvl, VHv6a/VLv2, VHv6a/VLv3, VHv6b/VLvl, VHv6b/VLv2, VHv3, VHv7/VLvl, VHv7/VLv2 or VHv7/VLv3.

В настоящем изобретении предложены варианты гуманизированного антитела 16G7, в которых гуманизированная зрелая вариабельная область тяжелой цепи демонстрирует по меньшей мере 90, 95, 96, 97, 98 или 99% идентичности с hul6G7VHvl, hul6G7VHv2, hul6G7VHv2a, 16G7VHv2aB7, hul6G7VHv2b, hul6G7VHv2bH7, hul6G7VHv3, hul6G7VHv4 , hul6G7VHv5V, hul6G7VHv5VR, hul6G7VHv6a, hul6G7VHv6b and hul6G7VHv7 (SEQ ID NOs: 15-27), and the humanized mature light chain variable region shows at least 90%, 95%, 96%, 97%, 98% or 99% identity with hu16G7VLv1. hu16G7VLv2 and hu16G7VLv3 (SEQ ID NOs: 28-30). Some of these antibodies retain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or all 36 of the backmutations or other mutations found in SEQ ID NOs: 15-27 and SEQ ID NOs : 28-30.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H5 occupies Q, H7 occupies P, H9 occupies S, H10 occupies V, H11 occupies L, H12 occupies V, H13 occupies R, H20 occupies L, H40 occupies R, H48 occupies I, H66 occupies K, H67 occupies A, H69 occupies L, H71 occupies V, H73 occupies I, H75 occupies S, H80 occupies V, H82a occupies T, H82b occupies S , H83 occupies T, and H85 occupies E. In some humanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83 and H85 occupy E, Q, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S, V, T in the VH region , S, T, and E, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H5 occupies Q, H7 occupies P, H9 occupies S, H10 occupies V, H11 occupies L, H12 occupies V, H13 occupies R, H20 occupies L, H48 occupies I, H69 occupies L, H71 occupies V, H82a occupies T, H82b occupies S, H83 occupies T, and H85 occupies E. In some humanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H48, H69, H71, H82a, H82b, H83 and H85 occupy E, Q, P, S, V, L, V, R, L, I, L, V in the VH region , T, S, T, and E, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H5 occupies Q, H11 occupies L, H12 occupies V, H20 occupies L, H48 occupies I, H69 occupies L, H71 occupies V, H82a occupies T, H82b occupies S, H83 occupies T, and H85 occupies E. the VH regions are occupied by E, Q, L, V, L I, L, V, T, S, T, and E, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H12 occupies V, H31 occupies G, H40 occupies R, H48 occupies I, H60 occupies A, H69 occupies L, H73 occupies I, and H83 occupies T. In some humanized 16G7 antibodies, positions H1, H12, H31, H40, H48, H60, H69, H73, and H83 in the VH region are occupied by E, V, G, R, I, A, L, I and T, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid as indicated: H1 occupies E, H12 occupies V, H40 occupies R, H48 occupies I, H69 occupies L, H73 occupies I, and H83 occupies T. In some humanized 16G7 antibodies, the positions H1, H12, H40, H48, H69, H73, and H83 in the VH region are E, V, R, I, L, I, and T, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies E, H12 occupies V, H40 occupies R, H48 occupies I, H66 occupies K, H67 occupies A, H69 occupies L, H71 occupies V, H73 occupies I, and H83 occupies T. In some humanized 16G7 antibodies, positions H1, H12, H40, H48, H66, H67, H69, H71, H73, and H83 in the VH region are occupied by E, V, R, I, K , A, L, V, I, and T, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid as indicated: H1 occupies E, H12 occupies V, H40 occupies R, H48 occupies I, H69 occupies L, H73 occupies I, and H83 occupies T. In some humanized 16G7 antibodies, the positions H1, H12, H40, H48, H69, H73, and H83 in the VH region are E, V, R, I, L, I, and T, respectively.

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In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H7 occupies a P, H71 occupies a V, and H73 occupies an I. In some humanized 16G7 antibodies, the H7, H71, and H73 positions in the VH region occupy a P , V and I, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H31 occupies a G and H60 occupies an A. In some humanized 16G7 antibodies, the H31 and H60 positions in the VH region occupy G and A, respectively.

In some humanized 16G7 antibodies, the positions H1, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73, and H83 in the VH region are Q, V, G, R, I, A, K, A, L , V, I, and T, respectively.

In some humanized 16G7 antibodies, the H1, H7, H71, and H73 positions in the VH region are Q, P, V, and I, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: H1 occupies Q or E, H5 occupies V or Q, H7 occupies S or P, H9 occupies A or S, H10 occupies E or V , H11 occupies V or L, H12 occupies K or V, H13 occupies K or R, H20 occupies V or L, H31 occupies G or S, H37 occupies V or A, H 38 occupies R or K, H40 occupies A or R, H48 occupies M or I, H60 occupies A or N, H64 occupies Q or K, H66 occupies R or K, H67 occupies V or A, H69 occupies M or L, H71 occupies R or V, H73 occupies T or I, H75 occupies I, S, or A, H80 occupies M or V, H82a occupies S or T, H82b occupies R or S, H83 occupies R or T, and H85 occupies D or E.

In some humanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H37, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83 and H85 in the VH region occupy E, Q, P, S, V, L, V, R, L, A, K, R, I, K, A, L, V, I, S, V, T, S , T and E, respectively, as in hu16G7VHv1. In some humanized 16G7 antibodies, the positions H1, H5, H11, H12, H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 in the VH region are E, Q, L, V, L, I, L, V , A, T, S, T, and E, respectively, as in hu16G7VHv2. In some humanized 16G7 antibodies, the positions H1, H12, H40, H48, H66, H67, H69, H71, H73, and H83 in the VH region are E, V, R, I, K, A, L, V, I, and T, respectively, as in hu16G7VHv2a. In some humanized 16G7 antibodies, the positions H1, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73, and H83 in the VH region are E, V, G, R, I, A, K, A, L , V, I, and T, respectively, as in hu16G7VHv2aB7. In some humanized 16G7 antibodies, the positions H1, H12, H40, H48, H69, H73, and H83 in the VH region are E, V, R, I, L, I, and T, respectively, as in hu16G7VHv2b. In some humanized 16G7 antibodies, the positions H1, H12, H31, H40, H48, H60, H64, H69, H73, and H83 in the VH region are E, V, G, R, I, A, Q, L, I, and T, respectively, as in hu16G7VHv2bH7. In some humanized 16G7 antibodies, the H1, H5, H7, H9, H10, H11, H12, H13, H20, H48, H69, H71, H75, H82a, H82b, H83, and H85 positions in the VH region are occupied by E, Q, P, S , V, L, V, R, L, I, L, V, A, T, S, T, and E, respectively, as in hu16G7VHv3. In some humanized 16G7 antibodies, the positions H1, H5, H11, H12, H20, H48, H66, H67, H69, H71, H73, H75, H82a, H82b, H83, and H85 in the VH region are E, Q, L, V, L , I, K, A, L, V, I, S, T, S, T, and E, respectively, as in hu16G7VHv4. In some humanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H38, H40, H48, H66, H67, H69, H71, H73I, H75, H80, H82a, H82b, H83 and H85 in the VH region is occupied by E, Q, P, S, V, L, V, R, L, K, R, I, K, A, L, V, I, S, V, T, S, T and E respectively, as in hu16G7VHv5V. In some humanized 16G7 antibodies, positions H1, H5, H7, H9, H10, H11, H12, H13, H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82a, H82b, H83, and H85 c the VH regions occupy Q, P, S, V, L, V, R, L, R, I, K, A, L, V, I, S, V, T, S, T, and E, respectively, as in hu16G7VHv5VR. In some humanized 16G7 antibodies, the positions H7, H71, and H73 in the VH region are P, V, and I, respectively, as in hu16G7VHv6a. In some humanized 16G7 antibodies, the position of H71 in the VH region is V, as in hu16G7VHv6b. In some humanized 16G7 antibodies, the positions H1, H7, H71, and H73 in the VH region are E, P, V, and I, respectively, as in hu16G7VHv7.

In some humanized 16G7 antibodies, the heavy chain variable region has >85% human sequence identity. In some humanized 16G7 antibodies, the light chain variable region has >85% human sequence identity. In some humanized 16G7 antibodies, the heavy chain variable region and the light chain variable region each share >85% human germline sequence identity.

In some humanized 16G7 antibodies, three heavy chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOS: 8, 9 and 10) and three light chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOs: 12, 13 and 14); with the proviso that position H31 is S or G, position H60 is N or A, and position 64 is K or Q. In some humanized 16G7 antibodies, the CDR-H1 contains an amino acid sequence containing SEQ ID NO: 49. In some humanized antibodies, the 16G7 CDR -H2 contains an amino acid sequence containing SEQ ID NO: 50. In some humanized 16G7 antibodies, CDR-H2 contains an amino acid sequence containing

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SEQ ID NO: 51.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: L4 is occupied by L, L9 is occupied by S, L15 is occupied by V, L22 is occupied by S, and L43 is occupied by S. In some humanized 16G7 antibodies, positions L4, L9, L15,

L22 and L43 in the VL region occupy L, S, V, S and S, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VH region is occupied by an amino acid, as indicated: L4 occupies L, L9 occupies S, L15 occupies V, L18 occupies K, L19 occupies V, L21 occupies M, L22 occupies S, and L43 occupies S. In some humanized 16G7 antibodies, positions L4, L9, L15, L18, L19, L21, L22, and L43 in the VL region are occupied by L, S, V, K, V, M, S, and S, respectively.

In some humanized 16G7 antibodies, at least one of the following positions in the VL region is occupied by an amino acid, as indicated: L4 occupies M or L, L9 occupies D or S, L15 occupies L or V, L18 occupies R or K, L19 occupies A or V , L21 occupies I or M, L22 occupies N or S, L43 occupies P or S, and L48 occupies I or M.

In some humanized 16G7 antibodies, the L4, L9, L15, L18, L19, L21, L22, and L43 positions in the VL region are occupied by L, S, V, K, V, M, S, and S, respectively, as in hu16G7VLv1. In some humanized 16G7 antibodies, the L4, L9, L15, L22, and L43 positions in the VL region are occupied by L, S, V, S, and S, respectively, as in hu16G7VLv2. In some humanized 16G7 antibodies, the L4, L9, L15, L18, L19, L21, L22, and L43 positions in the VL region are occupied by L, S, V, K, V, M, S, and S, respectively, as in hu16G7VLv3.

In some humanized 16G7 antibodies, the heavy chain variable region has >85% human sequence identity. In some humanized 16G7 antibodies, the light chain variable region has >85% human sequence identity. In some humanized 16G7 antibodies, the heavy chain variable region and the light chain variable region each share >85% human germline sequence identity. In some humanized 16G7 antibodies, three heavy chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOS: 8, 9 and 10) and three light chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOs: 12, 13 and 14); with the proviso that position H31 is S or G, position H60 is N or A, and position 64 is K or Q. In some humanized 16G7 antibodies, CDR-H1 contains an amino acid sequence containing SEQ ID NO: 49. In some humanized 16G7 antibodies CDR-H2 contains an amino acid sequence containing SEQ ID NO: 50. In some humanized 16G7 antibodies, CDR-H2 contains an amino acid sequence containing SEQ ID NO: 51.

The CDR regions of such humanized antibodies may be identical or substantially identical to the CDR regions of 16G7. CDRs can be defined according to any conventional definition (eg, Chothia or the combined definition of Chothia and Cabot), but preferably they are defined according to the definition of Cabot.

The framework positions of the variable regions correspond to the Cabot numbering, unless otherwise indicated. Other such variants typically differ from the exemplary Hu16G7 heavy and light chain sequences by a small number (eg, typically no more than 1, 2, 3, 5, 10, or 15) substitutions, deletions, or insertions. Such differences are usually present in the framework region, but may also occur in the CDR.

The possibility of additional variation in humanized variants of 16G7 lies in additional backmutations in the framework regions of the variable region. Many framework residues that do not make contact with CDRs in a humanized MAT (monoclonal antibody) may contain amino acid substitutions from the corresponding positions of the donor mouse MAT or other mouse or human antibodies, and even many potential CDR-contacting residues are also amenable to replacement. Even amino acids within the CDR can be replaced, for example, by residues found at the appropriate position of the human acceptor sequence used to provide the variable region frameworks. In addition, alternative human acceptor sequences can be used, for example for heavy and/or light chains. If different acceptor sequences are used, one or more of the backmutations recommended above may be omitted because the corresponding donor and acceptor residues are already the same without backmutations.

Preferably, substitutions or backmutations in humanized 16G7 variants (whether conservative or not) have no significant effect on the binding affinity or activity of the humanized MAT, ie. regarding its ability to communicate with the tau.

Humanized 16G7 antibodies are further characterized by the ability to bind to both phosphorylated and non-phosphorylated tau and misfolded/aggregated forms of tau.

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D. Chimeric and venated antibodies.

The present invention also provides chimeric and veneered forms of antibodies other than human antibodies, in particular 16G7 antibodies according to the examples.

A chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human (eg, mouse) antibody are combined with the constant regions of the human light and heavy chains. Such antibodies essentially or completely retain the binding specificity of the mouse antibody and consist of about two-thirds of the human sequence. In an embodiment, the 16G7 chimeric antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 54 and the light chain amino acid sequence of SEQ ID NO: 55.

A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some of the non-human variable region framework residues of a non-human antibody, but in which other variable region framework residues that may contribute to B or T cell epitopes, such as exposed residues (Padlan, Mol. Immunol., 28:489, 1991), are replaced by residues from the corresponding positions in the human antibody sequence. The result is an antibody in which the CDR is derived wholly or essentially from a non-human antibody and the variable region framework regions of the non-human antibody are more human-like due to the substitutions. Veneered forms of the 16G7 antibody are included in the present invention.

E. Human antibodies.

Human antibodies against tau or a fragment thereof (for example, amino acid residues 55-78, 60-75, 61-70, 55-69 or 64-78 of SEQ ID NO: 3 corresponding to amino acid residues 113-136, 118-133, 119- 128, 113-127 or 122-136, respectively, SEQ ID NO: 1) proposed through a variety of techniques described below. Some human antibodies are selected by competitive binding assays, by the phage display method according to Winter, see above, or otherwise, so that these antibodies have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. . It is also possible to screen human antibodies for a particular epitope specificity by using only a tau fragment as the target antigen, such as a tau fragment containing only amino acid residues 55-78, 60-75, 61-70, 55-69, or 64-78 SEQ ID NO: 3 (corresponding to amino acid residues 113-136, 118-133, 119-128, 113-127 or 122-136, respectively, SEQ ID NO: 1), and/or by screening antibodies against a population of tau variants, such as tau variants containing various mutations within amino acid residues 55-78, 60-75, 61-70, 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to amino acid residues 113-136, 118-133, 119- 128, 113-127 or 122-136, respectively, SEQ ID NO: 1).

Methods for producing human antibodies include the trioma method according to Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, US patent No. 4634664; and Engleman et al., US Pat. No. 4,634,666, the use of transgenic mice containing human immunoglobulin genes (see, for example, Lonberg et al., WO 93/12227 (1993); US 5877397; US 5874299; US 5814318; US 5789650; US 5770429; US 5661016; US 5633425; US 5625126; US 5569825; US 5545806; Neuberger, Nat. Biotechnol., 14:826 (1996); and Kucherlapati, WO 91/10741 (1991)), phage display methods (see eg Dower et al., WO 91/17271; McCafferty et al., WO 92/01047; US 5877218; US 5871907; US 5858657; US 5837242; US 5733743; and US 5565332); and the methods described in WO 2008/081008 (e.g., immortalization of memory B cells isolated from a human, for example, with EBV (Epstein-Barr virus), screening for desired properties, and cloning and expression of recombinant forms).

F. Selection of the constant region.

The heavy and light chain variable regions of a chimeric, veneered, or humanized antibody can be fused to at least a portion of a human constant region. The choice of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, and/or complement-dependent cytotoxicity are desired. For example, human IgG1 and IgG3 isotypes are characterized by complement-dependent cytotoxicity, while human IgG2 and IgG4 isotypes are not. Human IgG1 and IgG3 also elicit more potent cell-mediated effector functions than human IgG2 and IgG4. The constant regions of the light chains can be lambda or kappa. Numbering systems for constant regions include EU numbering (Edelman, G.M. 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, ref. above).

One or more amino acids at the amino or carboxyl terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be absent or may be derivatized in some or all molecules. Constant regions can be replaced to reduce

- 27 041230 or increase in effector function, such as mediated by complement cytotoxicity or ADCC (see, for example, Winter et al., US patent No. 5624821; Tso et al., US patent No. 5834597; and Lazar et al., Proc. Natl. Acad. Sci., USA, 103:4005, 2006), or to increase the half-life in humans (see, for example, 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 the constant region) to increase the half-life of the antibody. A substitution at any or all positions 234, 235, 236 and/or 237 reduces affinity for Fcy receptors, in particular the FcyRI receptor (see, for example, US 6624821). Alanine substitution at positions 234, 235 and 237 of human IgG1 can be used to reduce effector functions. Some antibodies contain an alanine substitution at positions 234, 235 and 237 of human IgG1 to reduce effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, for example, US 5624821). Some antibodies use a mutation at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329 and 331 of human IgG1 according to EU numbering. Some antibodies use a mutation at one or more of positions 318, 320 and 322 of human IgG1 according to EU numbering. 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.

Antibodies can be expressed as tetramers containing two light and two heavy chains, as individual heavy chains, light chains, as Fab, Fab', F(ab') ₂ and Fv, or as single chain antibodies in which the mature variable domains the heavy and light chains are connected by a spacer.

Human constant regions show allotypic variation and isoallotypic variation between different individuals; this means that the constant regions in different individuals may differ in one or more polymorphic positions. Isoallotypes differ from allotypes in that the isoallotype-recognizing sera binds to a non-polymorphic region of one or more other isotypes. Thus, for example, another heavy chain constant region is derived from IgG1 G1m3 with or without a C-terminal lysine. Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying positions in natural allotypes.

G. Expression of recombinant antibodies.

There are many methods for producing chimeric and humanized antibodies using an antibody-expressing cell line (eg, hybridoma). For example, antibody immunoglobulin variable regions can be cloned and sequenced using well known techniques. In one method, the heavy chain VH variable region is cloned by RT-PCR (reverse transcription polymerase chain reaction) using mRNA obtained from hybridoma cells. The consensus primers are applied to the leader peptide of the VH region containing the translation initiation codon as the 5' primer and the g2b constant region specific 3' primer. Illustrative primers are described in US Patent Publication US 2005/0009150 by Schenk et al. (hereinafter Schenk). Sequences from multiple independently generated clones can be compared to ensure that no changes were introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing the VH fragment generated by the 5'-RACE RT-PCR methodology and the 3' g2b specific primer.

The light chain VL variable region can be cloned in a similar manner. In one approach, a consensus primer set is designed to amplify VL regions using a 5' primer designed to hybridize to the VL region containing a translation initiation codon and a 3' Ck region specific primer downstream of the V-J bridging region. According to the second approach, 5'-RACE RT-PCR methodology is used to clone the cDNA encoding VL. Illustrative primers are described in Schenk; link above. The cloned sequences are then combined with sequences encoding human (or other non-human) constant regions.

In one approach, the heavy and light chain variable regions are redesigned to encode splice donor sequences downstream of the respective VDJ or VJ junctions and cloned into a mammalian expression vector such as pCMV-hy1 for the heavy chain and pCMV-Mcl for the light chain. . These vectors encode human y1 and Ck constant regions as exon fragments downstream of the inserted variable region cassette. After sequence confirmation, CHO cells can be co-transfected with heavy chain and light chain expression vectors to generate chimeric antibodies. The conditioned medium is harvested 48 hours after transfection and analyzed by Western blot for antibody production or ELISA for antigen binding. Chimeric antibodies are humanized as described above.

Chimeric, venated, humanized and human antibodies are typically prepared

- 28 041230 through recombinant expression. Recombinant polynucleotide constructs typically contain an expression control sequence operably linked to the coding sequences of the antibody chains, including naturally occurring or heterologous expression control elements such as a promoter. Expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been inserted into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and collection and purification of cross-reactive antibodies.

These expression vectors are typically replicable in host organisms as episomes or as part of the host's chromosomal DNA. Typically, expression vectors contain selectable markers, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells that have been transformed with the desired DNA sequences.

E. coli is one of the prokaryotic hosts suitable for the expression of antibodies, in particular antibody fragments. Microorganisms such as yeast are also suitable for expression. Saccharomyces is a yeast host with suitable vectors that optionally contain expression control sequences, an origin of replication, termination sequences, and the like. Exemplary promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and maltose and galactose utilization enzymes.

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 have been developed that are capable of secreting intact heterologous proteins, and such host cell lines 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 cells may be different from human cells. Expression vectors for these cells may contain expression control sequences such as an origin of replication, a promoter, an enhancer (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 contain promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol., 148:1149 (1992).

Alternatively, antibody-coding sequences can be inserted into transgenes for introduction into the genome of the transgenic animal and subsequent expression in the milk of the transgenic animal (see, for example, US Patent No. 5,741,957; US Patent No. 5,304,489; and US Patent No. 5,849,992). Suitable transgenes include light and/or heavy chain coding sequences operably linked to a promoter and enhancer from a breast-specific gene such as casein or beta-lactoglobulin.

Vectors containing the DNA segments of interest can be transferred into the host cell in ways that depend on the type of cellular host. For example, calcium chloride-based transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, ballistic transfection, or virus-based transfection can be used for other cell hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To obtain transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells and the nucleus of such cells can be transferred into enucleated oocytes.

After introducing the vector or vectors encoding the heavy and light chains of the antibody into the cell culture, cell pools can be screened for growth productivity and product quality in serum-free medium. The highly productive cell pools can then be subjected to single-cell FACS-based cloning to create monoclonal lines. You can use specific productivity more than 50 or 100 pg per cell per day, which corresponds to product titers of more than 7.5 g/l of culture. Antibodies produced by unicellular clones can also be examined for turbidity, filtration properties by PAAG (polyacrylamide gel electrophoresis), IEF (isoelectric focusing), UV scanning, high performance size exclusion chromatography (HEEC), carbohydrate-oligosaccharide mapping, mass spectrometry; and a binding assay such as ELISA or Biacore. The selected clone can then be used to create a cell bank in multiple flasks and stored frozen for later use.

Once expressed, antibodies can be purified according to standard methods known in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, and the like. (See Scopes, Protein Purification (SpringerVerlag, NY, 1982) for an overview).

- 29 041230

Methodologies for the commercial production of antibodies can be applied, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell bank generation, use of selectable markers for copy number amplification, CHO terminator, or improvement in protein titers (see, e.g., US 5786464; US 6114148; US 6063598; US 7569339; WO2004/050884; WO2008/012142; WO2008/012142; WO2005/019442; WO2008/107388; WO2009/020598; and US8).

IV. active immunogens.

The agent used for active immunization acts to induce in the patient the same types of antibodies as described above in relation to passive immunization. The agents used for active immunization may be the same types of immunogens used for the production of monoclonal antibodies in laboratory animals, for example a peptide of 3-15 or 3-12 or 5-12 or 5-8 contiguous amino acids from the region tau corresponding to residues 55-78, 60-75, 61-70, 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to amino acid residues 113-136, 118-133, 119-128, 113-127 or 122- 136, respectively, SEQ ID NO: 1), such as, for example, a peptide containing residues 55-78, 60-75, 61-70, 55-69 or 64-78 of SEQ ID NO: 3 (corresponding to amino acid residues 113- 136, 118-133, 119-128, 113-127 or 122-136, respectively, SEQ ID NO: 1). To induce antibodies that bind to the same or overlapping epitope as 16G7, epitope specificity for these antibodies can be mapped (eg, by assaying binding to a series of overlapping tau-spanning peptides). Then, a tau fragment consisting of an epitope, or containing an epitope, or overlapping with an epitope, can be used as an immunogen. Such fragments are generally used in non-phosphorylated form.

The heterologous carrier and adjuvant, if used, may be the same as those used to prepare the monoclonal antibody, but may also be selected for better pharmaceutical suitability for use in humans. Suitable carriers include serum albumins, fisurella hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid or other pathogenic bacterial toxoid such as diphtheria toxoid (e.g. CRM197), E. coli toxoid, cholera or H. pylori toxoid, or an attenuated toxin derivative. T cell epitopes are also suitable carrier molecules. Some conjugates can be formed by coupling agents of the present invention to an immunostimulatory polymer molecule (e.g. tripalmitoyl-S-glycerol with cysteine (Pam ₃ Cys), mannan (mannose polymer) or glucan (β 1 —>2 polymer)), cytokines (e.g. , IL-1, α- and β-peptides IL-1, IL-2, γ-IFN, IL-10, GM-CSF (granulocyte-macrophage colony stimulating factor)) and chemokines (for example, MIP1-a and β, and RANTES). Immunogens can be coupled to carriers with or without spacer amino acids (eg, gly-gly). Additional carriers include virus-like particles. Virus-Like Particles (VLPs), also referred to as pseudovirions or virus-derived particles, are subunit structures composed of multiple copies of the viral capsid and/or envelope protein capable of self-assembling in vivo into VLPs of defined spherical symmetry. (Powilleit et al. (2007), PLoS ONE, 2(5):e415.) Alternatively, peptidic immunogens can be attached to at least one artificial T-cell epitope capable of binding to a large proportion of MHC Class II molecules, such as a universal DR epitope (PADRE). PADRE is described in US 5736142, WO 95/07707; and Alexander J. et al., Immunity, 1:751-761 (1994). Active immunogens can be presented in multimeric form, in which multiple copies of the immunogen and/or its carrier are presented as a single covalent molecule.

The fragments are often administered with pharmaceutically acceptable adjuvants. The adjuvant increases the titer of the induced antibodies and/or the binding affinity of the induced antibodies compared to when the peptide is used alone. A variety of adjuvants can be used in combination with the tau immunogenic moiety to elicit an immune response. Preferred adjuvants increase the intrinsic 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, 3 De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa). Stimulon ™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree native to South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press) , NY, 1995); US 5057540), (Aquila BioPharmaceuticals, Framingen, MA; now Antigenics, Inc., New York, NY) Other adjuvants are oil-in-water emulsions (such as squalene or peanut butter), optionally in combination with immunostimulatory agents such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med., 336, 86-91 (1997)), pluronic polymers, and killed mycobacteria. Ribi adjuvants are an oil-in-water emulsion. contains a metabolizable oil (squalene) emulsified with saline containing Tween 80. Ribi also contains purified mycobacterial products that act as immunostimulants and bacterial monophosphoryl lipid A. Another adjuvant is CpG (WO

98/40100). Adjuvants may be administered as components of a therapeutic composition together with the active agent, or may be administered separately before, during or after administration of the therapeutic agent.

Analogues of naturally occurring tau fragments that induce anti-tau antibodies can also be used. For example, in such peptides, one or more or all of the L-amino acids can be replaced by D-amino acids. You can also change the order of amino acids to the opposite direction (retro-peptide). Optionally, the peptide contains all D-amino acids in reverse order (retro-inverted peptide). Peptides and other compounds that do not necessarily share significant amino acid sequence similarity with tau peptides, yet act as tau peptide mimetics and induce a similar immune response. You can also use anti-idiotypic antibodies against anti-tau monoclonal antibodies, as described above. Such anti-Id antibodies mimic an antigen and elicit an immune response against it (see Essential Immunology, Roit ed., Blackwell Scientific Publications, Palo Alto, CA ^6th ed., p. 181).

Peptides (and optionally a carrier fused to a peptide) can also be administered in the form of a nucleic acid encoding a peptide and expressed in situ in a patient. The nucleic acid segment encoding the immunogen is typically attached to regulatory elements, such as a promoter and enhancer, that allow expression of the DNA segment in the intended target cells of the patient. For expression in blood cells, which is desirable for inducing an immune response, promoter and enhancer elements from immunoglobulin light or heavy chain genes or CMV (cytomegalovirus) major immediate early promoter and enhancer are suitable for direct expression. Associated regulatory elements and coding sequences are often cloned into a vector. Antibodies can also be administered in the form of nucleic acids encoding the heavy and/or light chains of the antibody. If both heavy and light chains are present, the chains are preferably joined as a single chain antibody. Antibodies for passive administration can also be obtained, for example, by affinity chromatography from the sera of patients who have received peptide immunogens.

DNA can be delivered in naked form (ie without colloidal or encapsulating materials). Alternatively, several viral vector systems can be used, including retroviral systems (see, for example, Lawrie and Tumin, Cur. Opin. Genet. Develop., 3, 102-109 (1993)); adenovirus vectors (see, for example, Bett et al., J. Virol., 67, 591 1 (1993)); adeno-associated viral vectors (see, for example, Zhou et al., J. Exp. Med., 179, 1867 (1994)), viral vectors from the pox family, including vaccinia and avian poxviruses, viral vectors from the genus alpha viruses , such as those derived from Sindbis viruses and Semliki forest virus (see, for example, Dubensky et al., J. Virol., 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see US 5643576) 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)).

The DNA encoding the immunogen or the vector containing the DNA can be packaged into liposomes. Suitable lipids and related analogs are described in US Pat. , as well as poly(lactide-co-glycolides) (see, for example, McGee et al., J. Micro Encap., 1996).

H. Antibody screening assays.

Antibodies can be initially screened for intended binding specificity as described above. In a similar manner, active immunogens can be screened for their ability to induce antibodies with this binding specificity. In this case, the active immunogen is used to immunize a laboratory animal and the resulting serum is examined for the appropriate binding specificity.

Then, antibodies that have the desired binding specificity can be tested in cell and animal models. The cells that are used for such screening are preferably neuronal cells. A cellular model of tau pathology has been reported in which neuroblastoma cells are transfected with a four tau repeat domain, optionally with a mutation associated with tau pathology (eg, delta K280, see Khlistunova, Current Alzheimer Research, 4, 544-546 (2007)). In another model, tau is induced in the N2a neuroblastoma cell line by the addition of doxycycline. Cellular models allow us to study the toxicity of tau to cells in a soluble or aggregated state, the occurrence of tau aggregates after turning on tau gene expression, the dissolution of tau aggregates again after turning off gene expression, and the effectiveness of antibodies in inhibiting the formation of tau aggregates or their disaggregation.

It is also possible to screen for antibodies or active immunogens in disease models,

- 31 041230 associated with tau, in transgenic animals. Such transgenic animals may contain a tau transgene (eg, any of the human isoforms) and optionally a human APP transgene, among others, such as a kinase that phosphorylates tau, ApoE, presenilin, or alpha-synuclein. Such transgenic animals are predisposed to developing at least one sign or symptom of a disease associated with tau.

An exemplary transgenic animal is the K3 mouse strain (Itner et al., Proc. Natl. Acad. Sci., USA, 105(41):15997-6002 (2008)). These mice contain the human tau transgene with the K369 I mutation (the mutation is associated with Pick's disease) and the Thy 1.2 promoter. This model demonstrates the rapid course of neurodegeneration, a motor defect, and degeneration of afferent fibers and cerebellar granule cells. Another exemplary animal is the JNPL3 mouse strain. These mice contain the human tau transgene with the P301L mutation (the mutation is associated with frontotemporal dementia) and the Thy 1.2 promoter (Taconic, Germantown, NY, Lewis et al., Nat. Genet., 25:402-405 (2000)). These mice are characterized by a more gradual course of neurodegeneration. Mice develop neurofibrillary tangles in several regions of the brain and spinal cord, publication incorporated by reference in its entirety. This model is an excellent model for investigating the consequences of coil development and for screening therapy that can inhibit the formation of these aggregates. Another advantage of these animals is the relatively early manifestation of pathology. In the homozygous line, behavioral abnormalities associated with tau pathology can be observed as early as at least 3 months of age, but the animals remain relatively healthy until at least 8 months of age. In other words, at 8 months the animals are moving, eating and can perform behavioral tasks well enough to monitor the effects of treatment. Active immunization of these mice for 6-13 months with AI wI KLH-PHF-1 produced titers of approximately 1000 and demonstrated sparser neurofibrillary tangles, less pSer422, and reduced weight loss compared to untreated control mice.

Antibody or active agent activity can be assessed by various criteria including reduction in total tau or phosphorylated tau, reduction in other pathological characteristics such as Ae amyloid deposits, and inhibition or delay of behavioral deficits. Active immunogens can also be tested for serum antibody induction. Both passive and active immunogens can be tested for passage of antibodies across the blood-brain barrier into the brain of a transgenic animal. Antibodies or antibody-inducing fragments can also be tested in non-human primates that naturally or induce disease symptoms that are characterized by tau. Assays for an antibody or active agent are usually performed in conjunction with a control in which a parallel experiment is performed, except that the antibody or active agent is absent (eg, replaced by a vehicle). The reduction, delay, or inhibition of signs or symptoms of a disease induced by the antibody or active agent being tested can then be assessed in comparison to a control.

VI. Treatable patients.

The presence of neurofibrillary tangles has been found in several diseases, including Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia , frontotemporal lobar degeneration, argyrophilic granular disease, glial globular tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD), and progressive supranuclear palsy (PNS) . The regimens of the present invention may also be used in the treatment or prevention of any of these diseases. Because of the widespread association between neurological diseases and conditions, and tau, the regimens of the present invention can be used in the treatment or prophylaxis of any subject who has elevated levels of tau or phosphorylated tau (e.g., in CSF) compared to the mean in individuals without neurological diseases. The regimens of the present invention may also be used in the treatment or prevention of a neurological disease in individuals containing a tau mutation associated with a neurological disease. The methods of the present invention are particularly suitable for the treatment or prevention of Alzheimer's disease, especially in patients.

Treatable patients include individuals who are at risk of developing the disease but who do not have symptoms, as well as patients who currently have symptoms. Patients at risk of developing the disease include those who are at known genetic risk for developing the disease. Such individuals include those whose relatives have suffered from the disease and those whose risk has been determined by analysis of genetic or biochemical markers. Genetic risk markers

- 32 041230 include mutations in tau, such as those discussed above, as well as mutations in other genes associated with neurological disease. For example, the ApoE4 allele in the heterozygous, and even more in the homozygous form, is associated with the risk of developing Alzheimer's disease. Other risk markers for Alzheimer's disease include mutations in the APP gene, specifically mutations at position 717 and positions 670 and 671, which are referred to as Hardy and Swedish mutations, respectively, mutations in presenilin, PS1, and PS2 genes, a family history of AD (Alzheimer's disease), hypercholesterolemia, or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be recognized by PET imaging based on characteristic dementia as well as the presence of the risk factors described above. In addition, a variety of diagnostic tests are available to identify individuals who suffer from AD. These tests include the measurement of tau or phospho-tau and Ae42 levels in the CSF. Elevated levels of tau or phospho-tau and decreased levels of Ae42 are indicative of the presence of AD. Some mutations are associated with Parkinson's disease. Ala30Pro or Ala53 or mutations in other genes such as the leucine-rich repeat kinase, PARK8, are associated with Parkinson's disease. Any of the neurological diseases mentioned above can also be diagnosed in individuals based on DSM IV TR criteria (Diagnostic and Statistical Manual of Mental Disorders, fourth edition, text revision, Diagnostic and Statistical Manual of Mental Disorders, fourth edition, revised).

In asymptomatic patients, treatment can begin at any age (eg, 10, 20, 30). However, it is usually not necessary to start treatment until the patient is 40, 50, 60, or 70 years of age. Treatment usually involves multiple doses over a period of time. Treatment can be monitored by assessing antibody levels over time. If the response decreases, a booster dose is indicated. In the case of potential patients with Down's syndrome, treatment can be started before birth by administering a therapeutic agent to the mother or immediately after birth.

I. Nucleic acids.

The present invention also provides nucleic acids encoding any of the heavy and light chains described above (eg, SEQ ID NO: 7 and SEQ ID NO: 11). Optionally, such nucleic acids also encode a signal peptide and can be expressed with the signal peptide attached to a constant region. Nucleic acid coding sequences may be operably linked to regulatory sequences to allow expression of coding sequences such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. Nucleic acids encoding the heavy and light chains may exist in isolated form, or they may be cloned into one or more vectors. Nucleic acids can be synthesized by, for example, solid phase synthesis or NDP with overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be joined as a single contiguous nucleic acid, eg in an expression vector, or such nucleic acids may be separate, eg each cloned into its own expression vector.

J. Conjugated antibodies.

Conjugated antibodies that specifically bind to antigens such as tau are suitable for detecting the presence of tau; monitoring and evaluating the effectiveness of therapeutic agents used to treat patients diagnosed with Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granular disease, globular glial tauopathy, amyotrophic lateral sclerosis/Parkinsonism-Guam complex dementia, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD) ) or progressive supranuclear palsy (PNP); to inhibit or reduce tau aggregation; inhibiting or reducing the formation of tau fibers; reducing or eliminating tau deposits; stabilization of non-toxic tau conformations; or the treatment or prevention of Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, glial globular tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD), or progressive supranuclear palsy (PNP) in a patient.

For example, such antibodies can be conjugated to other therapeutic agents, other proteins, other antibodies, and/or detectable labels. See publications WO 03/057838; US 8455622. Such therapeutic agents can be any agent that can be used to treat, control, alleviate, prevent or improve an undesirable condition.

- 33 041230 or diseases in the patient such as Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia , frontotemporal lobar degeneration, argyrophilic granular disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD), or progressive supranuclear palsy ( PNP).

Conjugated therapeutic agents may include cytotoxic agents, cytostatic agents, neurotrophic agents, neuroprotective agents, radiotherapeutic agents, immunomodulatory agents, or any biologically active agent that facilitates or enhances the activity of an antibody.

A cytotoxic agent can be any agent that is toxic to a cell. The cytostatic agent can be any agent that inhibits cell proliferation. The neurotrophic agent can be any agent, including chemical or protein agents, that stimulates the maintenance, growth, or differentiation of neurons. A neuroprotective agent may be an agent, including chemical or protein agents, that protects neurons from acute stroke or degenerative processes. An immunomodulator may be any agent that stimulates or inhibits the development or maintenance of an immunological response. The radiotherapeutic agent can be any molecule or compound that emits radiation. When such therapeutic agents are coupled to a specific anti-tau antibody, such as the antibodies described herein, the coupled therapeutic agents will have specific affinity for cells affected by tau-induced disease compared to normal cells. As a consequence, administration of conjugated antibodies directly targets cancer cells with minimal damage to surrounding normal healthy tissue. This approach may be particularly suitable for therapeutic agents that are too toxic to be administered on their own. In addition, lower amounts of therapeutic agents can be used.

Some of these antibodies can be modified to act as immunotoxins. See, for example, US Pat. No. 5,194,594. For example, ricin, a cellular toxin derived from plants, can be combined with antibodies using the bifunctional reagents S-acetylmercaptosuccinic anhydride for the antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz et al., Cancer Res., 48(16):4469-4476 (1998). The association results in the loss of the binding activity of the ricin B chain, while not impairing either the toxic potential of the ricin A chain or the activity of the antibody. Similarly, saporin, an inhibitor of ribosome assembly, can be attached to antibodies via a disulfide bond between chemically introduced sulfhydryl groups. See Polito et al., Leukemia, 18:1215-1222 (2004).

Some of these antibodies can be attached to radioactive isotopes. Examples of radioactive isotopes include, for example, yttrium ⁹⁰ ( ⁹⁰ Y), indium ¹¹¹ ( ⁿ¹ In), ¹³¹ I, ⁹⁹ mTc, radioactive silver-111, radioactive silver-199 and bismuth ²¹³ .

Attachment of radioactive isotopes to antibodies can be accomplished by conventional bifunctional chelates. Sulfur-based linkers can be used to attach radioactive silver-111 and radioactive silver-199. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Attachment of radioactive isotopes of silver may involve the reduction of immunoglobulin with ascorbic acid. For radioactive isotopes such as ⁿ¹ In and ⁹⁰ Y, ibritutomab tiuxetan can be used, which will react with such isotopes to form ¹¹¹ In-ibritutomab tiuxetan and ⁹⁰ Y-ibritutomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48, Suppl., 1:S91-S95 (2001).

Some of these antibodies can be attached to other therapeutic agents. Such therapeutic agents may be, for example, cytotoxic, cytostatic, neurotrophic or neuroprotective. For example, antibodies can be conjugated to toxic chemotherapy drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (eg auristatins) or minor sulcus binding agents such as calicheamicin. Other exemplary therapeutic agents include those known to be useful in the treatment, management, or amelioration of Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic or boxer's dementia, Pick's disease, Niemann-Pick's disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Alzheimer's variant c Lewy bodies (LBVAD) or progressive supranuclear palsy (PNP).

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

The antibodies disclosed herein may also be combined or conjugated with one or more other antibodies (eg, to form antibody heteroconjugates). Such other antibodies may bind to different epitopes within the tau or may bind to a different target antigen.

Antibodies can also be combined with 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 parkinsonism, posttraumatic or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, fronto- temporal lobar degeneration, argyrophilic granular disease, glial globular tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD) or progressive supranuclear palsy (PNP), and/or to evaluate the effectiveness of treatment. Such antibodies are particularly suitable for making such a determination in subjects who suffer from or are predisposed to Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick type disease. C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism-dementia complex Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, variant Alzheimer's disease with bodies Levi disease (LBVAD) or progressive supranuclear palsy (PNP), or in appropriate biological samples obtained from such subjects. Typical detectable labels that can be combined with or attached to an antibody include various enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as streptavidin/biotin and avidin/biotin; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials such as luminol; bioluminescent materials such as luciferase, luciferin and aequorin; radioactive materials such as radioactive silver-111, radioactive silver-199, bismuth ²¹³ , iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ⁵ S), tritium ( ³ H) , indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In, ⁿ¹ In,), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh , ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn and ¹¹⁷ Tin; positron-active metals using various types of positron emission tomography; non-radioactive paramagnetic metal ions; and molecules that are radioactively labeled or conjugated to specific radioactive isotopes.

Attachment of radioactive isotopes to antibodies can be accomplished by conventional bifunctional chelates. Sulfur-based linkers can be used to attach radioactive silver-111 and radioactive silver-199. See Hazra et al., Cell Biophys., 24-25:1-7 (1994). Attachment of radioactive isotopes of silver may involve the reduction of immunoglobulin with ascorbic acid. For radioactive isotopes such as ⁿ¹ In and ⁹⁰ Y, ibritutomab tiuxetan can be used, which will react with such isotopes to form ¹¹¹ In-ibritutomab tiuxetan and ⁹⁰ Y-ibritutomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl., 1:S91-S95 (2001).

Therapeutic agents, other proteins, other antibodies, and/or detectable labels can be combined or conjugated directly or indirectly using an intermediary (eg, linker) with an antibody of the present invention. See, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), p. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., Antibodies For Drug Deliv

- 35 041230 ery, in Controlled Drug Delivery ( ^2nd Ed.), Robinson et al. (eds.), p. 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.), p. 475-506 (1985); Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), p. 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. You can use a variety of linkers that release attached therapeutic agents, proteins, antibodies and/or detectable labels under acidic or reducing conditions when exposed to specific proteases or under other specified conditions.

VI. Pharmaceutical compositions and methods of use.

In prophylactic uses, an antibody or antibody-inducing agent, or a pharmaceutical composition containing said antibody or agent, is administered to a patient predisposed or otherwise at risk for developing a disease (e.g., Alzheimer's disease) at a dosage, frequency, and route of administration that is effective to reduce the risk, reduce the severity or delay the manifestation of at least one sign or symptom of the disease. In particular, the regimen is preferably effective for inhibiting or delaying tau or phospho-tau and paired fibers formed therefrom in the brain and/or inhibiting or delaying their toxic effects and/or inhibiting/or delaying the development of a behavioral deficit. In therapeutic uses, the antibody or antibody-inducing agent is administered to a patient predisposed to or already suffering from a disease (e.g., Alzheimer's disease) at a regimen (dose, frequency, and route of administration) effective to alleviate or at least inhibit further deterioration in at least one sign or symptom of a disease. In particular, the regimen is preferably effective in reducing or at least inhibiting further increases in levels of tau, phospho-tau or paired fibers formed therefrom, associated toxicity and/or behavioral deficits.

A regimen is considered therapeutically or prophylactically effective if an individual patient receiving treatment achieves a better outcome than the mean outcome in a control population of comparable patients who have not been treated with the methods of the present invention, or if treated patients experience a better outcome than controls. patients in a controlled clinical trial (eg, a phase II, phase II/III, or phase III study) at a p<0.05, or 0.01, or even 0.001.

Effective doses will vary depending on many different factors such as routes of administration, target site, physiological condition of the patient, whether the patient is an ApoE carrier, whether the patient is a human or animal, other drugs administered, whether the treatment is prophylactic or therapeutic.

Exemplary dosage ranges for antibodies are from about 0.01 to 60 mg/kg, or from about 0.1 to 3 mg/kg, or 0.15-2 mg/kg, or 0.15-1.5 mg/kg body weight of the patient. The antibody can be administered at such doses daily, every other day, weekly, every two weeks, once a month, once every three months, or according to any other schedule determined through empirical analysis. An exemplary treatment involves the administration of multiple doses over an extended period of time, such as at least six months. Additional exemplary treatment regimens include administration once every two weeks, or once a month, or once every 3-6 months.

The amount of agent for active administration varies from 0.1-500 micrograms per patient, and more often from 1-100 or 1-10 micrograms per injection for administration to a human. The time between injections can vary greatly from once a day to once a year to once every ten years. A typical regimen consists of immunization followed by booster injections at intervals such as 6 weeks or two months. The other regimen consists of immunization followed by booster injections at 1, 2 and 12 months. Another regimen involves an injection once every two months for a lifetime. Alternatively, booster injections may be administered at irregular intervals as indicated by immune response monitoring.

Antibodies or antibody inducing agents are preferably administered peripherally (ie, the unit in which the antibody is administered or which induces crosses the blood-brain barrier to reach the intended site in the brain). Routes of administration include topical, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular or intramuscular. Preferred routes of administration of antibodies are intravenous and subcutaneous.

Preferred routes for active immunization are subcutaneous and intramuscular. This type of injection is most commonly given in the muscles of the shoulder or leg. In some methods, the agents are injected directly into the specific tissue in which the deposits have accumulated, such as by intracranial injection.

Pharmaceutical compositions for parenteral administration are preferably sterile

- 36 041230 nutritious and essentially isotonic, and they are produced under GMP (Good manufacturing practices, good manufacturing practice) conditions. Pharmaceutical compositions may be provided in unit dosage form (ie, single dose). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The composition depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain adjuvants such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies may be in lyophilized form for reconstitution prior to use in a suitable vehicle, such as sterile, pyrogen-free water.

The regimens of the present invention may be administered in combination with another agent effective in the treatment or prevention of the disease being treated. For example, in the case of Alzheimer's disease, the regimens of the present invention can be combined with anti-Άβ immunotherapy (WO 2000/072880), cholinesterase inhibitors or memantine, or in the case of Parkinson's disease with alpha-synuclein immunotherapy WO 2008/103472, levodopa, dopamine agonists, COMT inhibitors, MAO-B inhibitors, amantadine or anticholinergics.

Antibodies are administered in an effective manner, which means a dose, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and/or alleviates at least one sign or symptom of the disorder being treated. If the patient is already suffering from a disorder, the regimen may be referred to as a therapeutically effective regimen. If a patient is at an increased risk of developing a disorder compared to the general population but is not yet symptomatic, the regimen may be referred to as a prophylactically effective regimen. In some instances, therapeutic or prophylactic efficacy can be demonstrated in an individual patient compared to historical controls or past experience of the same patient. In other cases, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical study in a population of treated patients compared to a control population of untreated patients.

Exemplary antibody doses are 0.1-60 mg/kg (eg, 0.5, 3, 10, 30, or 60 mg/kg), or 0.5-5 mg/kg body weight (eg, 0.5, 1 , 2, 3, 4 or 5 mg/kg), or 10-4000 mg or 10-1500 mg as a fixed dose. The dose depends on the patient's condition and response to prior treatment, and if so, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other factors.

Administration may be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Some antibodies can be introduced into the systemic circulation via intravenous or subcutaneous administration. Intravenous administration can be carried out, for example, by infusion over a period of time, such as 30-90 minutes.

The frequency of administration depends on the half-life of the antibody in the vascular bed, the condition of the patient and the route of administration, among other factors. The frequency may be administration daily, weekly, once a month, once every three months, or administration at irregular intervals in response to changes in the patient's condition or progression of the disorder being treated. An exemplary frequency for intravenous administration is from weekly to once every three months for a continuous course of treatment, although more or less frequent dosing is also possible. For subcutaneous administration, exemplary dosing frequencies range from daily to once a month, although more or less frequent dosing is also possible.

The number of doses administered depends on whether the disorder is acute or chronic and on the response of the disorder to treatment. In the case of acute disorders or acute exacerbations of a chronic disorder, 1 to 10 doses are often sufficient. Sometimes a single bolus dose, optionally divided, is sufficient for an acute disorder or an acute exacerbation of a chronic disorder. Treatment can be repeated in case of recurrence of an acute disorder or acute exacerbation. For chronic disorders, the antibody can be administered at regular intervals, such as weekly, every two weeks, once a month, once every three months, once every six months for at least 1, 5 or 10 years, or for the patient's life.

Ά. Methods of diagnostics and monitoring.

Methods for visualization, in vivo diagnostics and optimization of immunotherapy.

The present invention provides methods for visualizing tau deposits (eg, neurofibrillary tangles and tau inclusions) in a patient in vivo. The methods are performed by administering a reagent, such as an antibody that binds to tau (e.g., mouse 16G7 antibody, gu

- 37 041230 manized, chimeric or veneered antibody 16G7), to the patient, and then by means of detection of the agent after its binding. In some methods, the antibody binds to the tau epitope within amino acid residues 55-78 of SEQ ID NO: 3 (corresponding to amino acid residues 113-136 of SEQ ID NO: 1). In some methods, the antibody binds to an epitope within amino acid residues 60-75 of SEQ ID NO: 3 (corresponding to amino acid residues 118-133 of SEQ ID NO: 1) or within amino acid residues 61-70 of SEQ ID NO: 3 (corresponding to amino acid residues 119 -128 SEQ ID NO: 1). Clearing response to injected antibodies can be avoided or reduced by using antibody fragments that lack the full length constant region, such as Fab. In some methods, the same antibody can act as a reagent for both treatment and diagnosis.

Diagnostic reagents can be administered by intravenous injection into the patient's body or directly into the brain by intracranial injection or by drilling a hole in the skull. The dose of the reagent should be within the same ranges as for the treatments. Typically, the reagent is labeled, although in some methods the primary tau affinity reagent is unlabeled and a secondary labeling agent is used to bind to the primary reagent. The choice of label depends on the detection methods. For example, a fluorescent label is suitable for optical detection. The use of paramagnetic markers is suitable for non-surgical tomographic detection. Radioactive labels can also be detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

Methods for visualizing tau protein deposits in vivo are useful in diagnosing or confirming a diagnosis of tauopathy such as Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and Pick's disease, or a predisposition to such a disease. For example, the methods can be applied to a patient who has symptoms of dementia. If the patient has abnormal neurofibrillary tangles, then the patient is likely suffering from Alzheimer's disease. Alternatively, if the patient has abnormal tau inclusions, then, depending on the location of the inclusions, the patient may be suffering from frontotemporal lobar degeneration. The methods can also be applied to asymptomatic patients. The presence of abnormal tau deposits is indicative of a predisposition to subsequent symptomatic disease. The methods are also suitable for monitoring disease progression and/or response to treatment in patients previously diagnosed with tau-induced disease.

Diagnosis can be made by comparing the number, size and/or intensity of the labeled locus with the corresponding baseline values. Baseline values may represent mean levels in a population of non-diseased individuals. Baseline values may also represent prior levels determined in the same patient. For example, baseline values can be determined in a patient prior to initiation of tau immunotherapy treatment and then the measured values compared to baseline values. A decrease in values compared to baseline indicates a positive response to treatment.

In some patients, the diagnosis of tauopathy can be facilitated by performing a PET scan. The PET scan can be performed using, for example, a conventional PET imaging apparatus and ancillary equipment. The scan typically covers one or more areas of the brain known to be commonly associated with tau deposits and one or more areas that typically have few (if any) deposits and protrude as controls.

The signal detected by a PET scan can be represented as a multidimensional image. The multidimensional image can be represented in two dimensions representing a cross section of the brain, in three dimensions representing the three-dimensional brain, or in four dimensions representing changes in the three-dimensional brain over time. You can apply a color scale with different colors indicating different amounts of label, and inferred the detected deposition of tau protein. Scan results can also be presented numerically with numbers relating to the detected amount of label and, as a result, the amount of tau deposits. A label present in an area of the brain known to be associated with deposits in the case of a particular tauopathy (e.g., Alzheimer's disease) can be compared with a label present in an area known not to be associated with deposits to obtain a ratio, indicating the degree of deposits in the first area. For the same radiolabeled ligand, these ratios provide a comparable measure of tau deposits and changes between different patients.

In some methods, the PET scan is performed at the same time or during the same patient visit as the MPT (magnetic resonance imaging) or CT (computed tomography) scan. An MPT or CT scan provides more information about the anatomical details of the brain than a PET scan. However, the image from the PET scan can be superimposed on the image from the MPT or CT scan to more accurately indicate the location of the PET ligand and inferred tau deposits compared to anatomical structures in the brain. Some devices can perform both PET and MPT or CT scans without repositioning the patient between scans, making it easier to overlay images.

Suitable PET ligands include radiolabeled antibodies of the present invention (eg, mouse 16G7 antibody, humanized, chimeric, or veneered 16G7 antibody). The radioactive isotope used may be, for example, C ¹¹ , N ¹³ , O ¹⁵ , F ¹⁸ or I ¹²³ . The interval between the administration of the PET ligand and the performance of the scan may depend on the PET ligand and, in particular, its rate of uptake and clearance in the brain, as well as the half-life of its radioactive label.

PET scanning can also be performed as a prophylactic measure in asymptomatic patients or patients who have symptoms of mild cognitive impairment but have not yet been diagnosed with tauopathy, but who are at increased risk of developing tauopathy. In the case of asymptomatic patients, scanning is particularly appropriate for individuals who are considered to be at increased risk of developing tauopathy due to family history, genetic or biochemical risk factors, or advanced age. Prophylactic scanning can be started, for example, in patients aged 45 to 75 years. Some patients have their first scan at age 50.

Preventive scans can be performed at intervals of, for example, 6 months to 10 years, preferably 1-5 years. In some patients, preventive scans are performed annually. If a PET scan performed as a preventive measure indicates abnormally high levels of tau deposits, immunotherapy can be initiated and follow-up PET scans performed as for patients diagnosed with tauopathy. If PET scans performed as a preventive measure show normal levels of tau deposits, follow-up PET scans can be performed at intervals of 6 months to 10 years, preferably 1-5 years, as before, or in response to the appearance of signs and symptoms of tauopathy or mild cognitive impairment. By combining a prophylactic scan with the administration of tau-targeted immunotherapy, if and when tau deposits are found above normal levels, levels of tau deposits can be reduced to or close to normal levels, or at least further inhibited from increasing, and the patient may remain free of tauopathy for a longer period of time than if they do not receive prophylactic scanning and tau-targeted immunotherapy (eg, at least 5, 10, 15, or 20 years, or for the remainder of the patient's life).

Normal levels of tau deposits can be determined based on the number of neurofibrillary tangles or tau inclusions in the brain of a representative sample from individuals in the general population who have not been diagnosed with a particular tauopathy (eg, Alzheimer's disease) and who are not thought to be at increased risk the development of such a disease (eg, a representative sample of disease-free individuals under the age of 50 years). Alternatively, a normal level can be determined in an individual patient if the PET signal in accordance with the methods of the present invention in a region of the brain known to develop tau protein deposits does not differ (within the accuracy of the measurement) from the signal from the region brain, in which, as is known, such deposits normally do not develop. An elevated level in an individual can be determined by comparison with normal levels (e.g., outside the mean and standard deviation variation) or simply based on an increased signal outside experimental error in a region of the brain associated with tau deposits compared to an area which is known to be unrelated to deposits. For the purpose of comparing levels of tau deposits in an individual and a population, tau deposits should preferably be determined in the same region or regions of the brain, and these regions include at least one region known to form tau deposits. -protein associated with a particular taupopathy (eg, Alzheimer's disease). A patient who has an elevated level of tau deposits is a candidate for starting immunotherapy.

After the start of immunotherapy, a decrease in the level of tau deposits can first be observed as evidence that the treatment is producing the desired effect. The observed decrease may be, for example, in the range of 1-100, 1-50 or 1-25% of the original value. Such effects may be measured in one or more areas of the brain known to form deposits, or may be measured from the average of such areas. The net effect of treatment can be approximated by adding the percentage reduction from baseline to the increase in tau deposits that would otherwise occur in the average untreated patient.

Maintaining tau deposits at a roughly constant level or even negligible

- 39 041230 an increase in tau deposits may also indicate a response to treatment, even if a suboptimal response. Such responses can be compared to the time course of tau deposition levels in untreated patients with a particular tauopathy (eg, Alzheimer's disease) to determine if immunotherapy has an effect in inhibiting the subsequent increase in tau deposition.

Monitoring changes in tau deposits allows adjustment of immunotherapy or other treatment regimen in response to treatment. PET monitoring provides an indication of the nature and extent of response to treatment. It can then be determined if treatment needs to be adjusted and, if necessary, treatment can be adjusted in response to PET monitoring. Thus, PET monitoring allows adjustment of tau-targeted immunotherapy or other treatment regimen before other biomarkers, MPT, or cognitive measures have demonstrated detectable responses. A significant change means that a comparison of the parameter value after treatment compared to baseline provides some evidence that the treatment has or has not resulted in a beneficial effect. In some cases, changing the values of a parameter in a patient by itself provides evidence that the treatment has or has not resulted in a beneficial effect. In other cases, the change in values (if any) in a patient is compared with the change in values (if any) in a representative control population of patients who did not receive immunotherapy. The difference between a particular patient's response and a normal control patient's response (eg, the mean plus the standard deviation variance) can also provide evidence that the immunotherapy regimen achieved or failed to achieve a beneficial effect for the patient.

In some patients, monitoring indicates a detectable decrease in tau deposits, but this level of tau deposits remains above normal. For such patients, if there are no unacceptable side effects, the treatment regimen can be continued without changes or even with an increase in the frequency of administration and / or dose, if the treatment is no longer carried out at the maximum recommended dose.

If monitoring indicates levels of tau deposits in a patient that have already been reduced to normal levels or are near normal levels of tau deposits, the immunotherapy regimen can be adjusted from that for induction (i.e., which reduces tau deposits). ) to that for conservation (i.e., which maintains tau deposits at an approximately constant level). This regimen can be influenced by reducing the dose and/or frequency of administration of immunotherapy.

In other patients, monitoring may indicate that immunotherapy has some beneficial but suboptimal effect. Optimum effect can be defined as the percentage reduction in tau deposits within the top half or quartile of change in tau deposits (measured or calculated for the entire brain or a representative area or areas known to form tau deposits) observed in a representative sample from patients suffering from tauopathy who receive immunotherapy at a given time point after the start of therapy. A patient who experiences less of a decrease, or a patient whose tau deposits remain constant or even increase, but to a lesser extent than expected in the absence of immunotherapy (eg, as measured in a control group of patients who are not administered immunotherapy), can be classified as demonstrating a positive but suboptimal response. Such patients can optionally adjust the regimen, while increasing the dose and/or frequency of administration of the agent.

In some patients, tau deposits may increase to a similar or greater extent than tau deposits in patients who are not receiving immunotherapy. If such an increase continues for a period of time, such as 18 months or 2 years, even after any increase in the frequency or dose of the agent, immunotherapy can be discontinued, if necessary, in favor of other treatment options.

The above description of diagnosing, monitoring, and adjusting the treatment of tauopathy is primarily focused on the application of the PET scan. However, any other technique for visualizing and/or measuring tau deposits suitable for use with anti-tau antibodies of the present invention (e.g., mouse antibody, humanized, chimeric, or veneered 16G7 antibody) can be used to perform such methods, instead of PET scanning.

Also provided are methods for detecting an immune response against tau in a patient suffering from or predisposed to tau related diseases. The methods can be used to monitor the progress of therapeutic and prophylactic treatment with the agents provided herein. The antibody profile after passive immunization typically shows a rapid peak in antibody concentration followed by an exponential decline. In the absence of the next dose, the decline will reach pre-treatment levels over a period ranging from days to months, depending on the half-life of the antibody administered. For example, the half-life of some human antibodies is on the order of 20 days.

- 40 041230

In some methods, a measurement of anti-tau antibody at baseline in a subject is taken prior to administration, a second measurement is taken shortly thereafter to determine peak antibody levels, and one or more subsequent measurements are taken at time intervals to monitor decline in antibody levels. When the antibody level has dropped to baseline or a predetermined percentage of the peak below baseline (eg, 50%, 25%, or 10%), the next dose of antibody is administered. In some methods, peak or subsequent measured levels below background level are compared with reference levels previously determined to constitute a favorable prophylactic or therapeutic regimen for other subjects. If the measured antibody level is significantly less than the reference level (eg, less than the mean value minus one or preferably two standard deviations from the reference value in the population of subjects benefiting from treatment), an additional dose of antibody is indicated.

Also provided are methods for detecting tau in a subject, for example by measuring tau in a sample from the subject or by imaging tau in the subject in vivo. Such methods are suitable for diagnosing or confirming a diagnosis of tau-associated diseases or a predisposition to said diseases. The methods may also be applied to asymptomatic subjects. The presence of tau indicates a predisposition to future symptomatic disease. The methods are also suitable for monitoring disease progression and/or response to treatment in subjects previously diagnosed with Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic or boxer's dementia, Pick's disease, Niemann's disease. -Pica type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism-dementia complex Guam, corticobasal degeneration (CBD), dementia with Lewy bodies, disease variant Alzheimer's with Lewy bodies (LBVAD) or progressive supranuclear palsy (PNP).

Contact can be made with biological samples obtained from a subject suspected to be suffering from or at risk of developing Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick's disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, Alzheimer's variant c Lewy bodies (LBVAD) or progressive supranuclear palsy (PNP), with the antibodies disclosed herein, to assess the presence of tau. For example, tau levels in such subjects can be compared to those present in healthy subjects. Alternatively, tau levels in such subjects who are receiving treatment for the disease can be compared to those of subjects who have not received treatment for Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic or boxer's dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), Lewy body dementia, variant disease Alzheimer's with Lewy bodies (LBVAD) or progressive supranuclear palsy (PNP). Some such studies involve biopsy of tissue obtained from such subjects. ELISA assays may also be suitable methods, for example, for assessing tau in liquid samples.

IX. Sets.

The present invention also provides kits (eg, containers) containing the antibodies disclosed herein and related materials such as instructions for use (eg, leaflet). Instructions for use may include, for example, instructions for administering the antibody and optionally one or more additional agents. The antibody containers can be single doses, large volume packs (eg, multi-dose packs), or doses smaller than the single dose.

Package leaflet means instructions that are usually included in commercial packages of therapeutic products and which contain information regarding the indications, use, dose, administration, contraindications and / or warnings regarding the use of such therapeutic products.

The kits may also contain a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. Kits may also contain other materials commercially and consumerly desirable, including other

- 41 041230 buffers, diluents, filters, needles and syringes.

X. Other applications.

Antibodies can be used to detect tau or fragments thereof in the context of clinical diagnosis or treatment, or in research. For example, antibodies can be used to detect the presence of tau in a biological sample as evidence that the biological sample contains tau deposits. The binding of antibodies to a biological sample can be compared to the binding of antibodies to a control sample. The control sample and the biological sample may contain cells that originate from the same tissue. Control samples and biological samples can be obtained from the same individual or different individuals at the same time or at different time periods. If necessary, several biological samples and several control samples are evaluated at different time periods to protect against random variation, regardless of differences between samples. A direct comparison can then be made between the biological sample or samples and the control sample or samples to determine if the binding of the antibody (i.e. the presence of tau) to the biological sample or samples is increased, decreased or similar compared to the binding of the antibody to the control sample or samples. . Increased binding of the antibody to the biological sample or samples compared to the control sample or samples is indicative of the presence of tau in the biological sample or samples. In some cases, increased antibody binding is statistically significant. Optionally, the binding of the antibody to the biological sample is at least 1.5, 2, 3, 4, 5, 10, 20, or 100 times greater than the binding of the antibody to the control sample.

In addition, antibodies can be used to detect the presence of tau in a biological sample to monitor and evaluate the effectiveness of a therapeutic agent that is used to treat a patient who has been diagnosed with Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia. or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granular disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism complex, Guam dementia, corticobasal degeneration (CBD), dementia with Lewy bodies, a variant of Alzheimer's disease with Lewy bodies (LBVAD), or progressive supranuclear palsy (PNP). Biological sample from a patient diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic or boxer's dementia, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granular disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism-dementia complex Guam, corticobasal degeneration (CBD), Lewy body dementia, Lewy body variant of Alzheimer's disease (LBVAD), or progressive supranuclear palsy (PNP) ), assessed to establish the baseline level of binding of antibodies to the sample (ie, the baseline level of the presence of tau in the sample) prior to the start of therapy with a therapeutic agent. In some cases, several biological samples from a patient are evaluated at different time points to establish both a baseline and an indicator of random variation independent of treatment. Then the therapeutic agent is administered in the mode. The regimen may include multiple injections of the agent over a period of time. Optionally, antibody binding (ie, the presence of tau) is assessed at various times in several biological samples from the patient, both to establish a measure of random variation and to demonstrate a trend in response to immunotherapy. The various binding scores of the antibody to biological samples are then compared. If only two assessments are made, a direct comparison can be made between the two assessments to determine if antibody binding (ie, the presence of tau) is increased, decreased, or remains similar between the two assessments. If more than two measurements are taken, the measurements can be analyzed over time, starting from before treatment with a therapeutic agent and continuing throughout the course of therapy. In the case of patients who have decreased antibody binding to biological samples (i.e., the presence of tau), it will be concluded that the therapeutic agent was effective in the treatment of Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, posttraumatic dementia or boxer's disease, Pick's disease, Niemann-Pick type C disease, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granular disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism-dementia Guam complex, corticobasal degeneration ( CBD), Lewy body dementia, Lewy body variant Alzheimer's disease (LBVAD), or progressive supranuclear palsy (PNP) in a patient. The decrease in antibody binding may 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 performed in conjunction with assessment of other signs and symptoms of Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism complex, Guam dementia, corticobasal degeneration (CBD), Lewy body dementia, variant disease

Alzheimer's with Lewy bodies (LBVAD) or progressive supranuclear palsy (PNP).

Antibodies can also be used as research reagents for laboratory testing when detecting tau or its fragments. In such applications, antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioactive isotopes, and can be offered in kit form with all the necessary reagents to perform a detection assay. Antibodies can also be used to purify tau or tau binding partners, for example by affinity chromatography.

All patents, websites, other publications, account numbers, etc., referenced above or below are incorporated by reference in their entirety for all purposes to the same extent as if each of these documents were specifically and individually identified as incorporated by reference. If different versions of the sequence are associated with an account number at different times, then the version associated with the account number on the effective filing date of this application is assumed. Effective filing date means the earlier of the actual filing date or the filing date of the priority application referring to the accession number, if applicable. Likewise, if different versions of a publication, website, or the like are published at different times, the most recent version published on the actual date of filing is meant, unless otherwise indicated. Any feature, step, element, implementation variant or aspect of the present invention can be used in combination with any other, unless specifically indicated otherwise. While the present invention has been described in sufficient detail by way of illustration and example for the purposes of clarity and understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

Examples

Example 1 Identification of anti-tau monoclonal antibodies.

Monoclonal antibody 16G7 was obtained according to a modification of the method of Kohler and Milstein (G. Kohler, C. Milstein (1975), Nature, 256:495-497). For all injections and screening assays, human tau containing all 4 microtubule-binding repeats and lacking N-terminal splice variants (tau 4R0N, 383 amino acids) was used. Tau was purified from SF9 cells infected with a tau-containing baculovirus construct (J. Knops et al. (1991), J. Cell Biol., 115(5):725-33). Six week old A/J mice were injected with 100 μg of purified tau at two week intervals. Tau was emulsified in complete Freund's adjuvant for the first immunization and in incomplete Freund's adjuvant for all subsequent immunizations. Serum samples were taken three days after the third injection to assess the titer in these animals. The highest titer mice were intravenously injected with 100 µg tau in 500 µl PBS two weeks after they received their third injection. Myeloma fusion occurred three days later using SP2/0 as a fusion partner. Supernatants from wells containing hybridoma cells were screened for their ability to induce precipitation of labeled ¹²⁵ I tau. Tay was radioiodinated using immobilized glucose oxidase and lactoperoxidase according to the manufacturer's instructions (Bio-Rad). Briefly, 10 μg of purified recombinant tau was radiolabeled with 1 μCi Na I to a specific activity of 20 μCi/μg protein. 16G7 was identified as a high affinity tau-specific monoclonal antibody and cloned by limiting dilution. The 16G7 isotypes were determined to be gamma 1 kappa.

Example 2 Epitope mapping of the 16G7 antibody.

A range of overlapping biotinylated peptides spanning the entire 383 aa human 4R0N tau protein was used to map the mouse 16G7 antibody. (amino acids). Additional peptides were used to model potential post-translational modifications to the C- and N-terminal ends of the protein.

Biotinylated peptides were attached to individual wells of a streptavidin sensitized ELISA plate. The plate was blocked and treated with mouse 16G7 followed by incubation with horseradish peroxidase conjugated anti-mouse immunoglobulin antibody. After thorough washing, OPD was applied to the plate and the reaction was allowed to proceed. The plates were read at 450 nm absorbance. Background subtraction was performed using absorbance values from wells containing no primary antibody, and the threshold for positive binding was set at 0.2 absorbance units.

Positive binding was found for the peptide spanning amino acid residues 55-69 of SEQ ID NO: 3 and less binding was found for the peptide spanning amino acid residues 64-78 of SEQ ID NO: 3 (FIG. 1). Using the numbering of the full-length human tau protein 4R2N (441 amino acids) (SEQ ID NO: 1), these peptides correspond to amino acid residues 113-127 and 122-136, respectively, of SEQ ID NO: 1.

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Example 3 Design of Humanized 16G7 Antibodies.

The starting point or donor antibody for humanization was the mouse 16G7 antibody. The amino acid sequence of the mature m16G7 heavy chain variable region is shown as SEQ ID NO:7. The amino acid sequence of the mature m16G7 light chain variable region is shown as SEQ ID NO:11. as SEQ ID NO: 8-10, respectively. The amino acid sequences of the light chain CDR1, CDR2 and CDR3 according to Cabot are shown as SEQ ID NOs: 12-14, respectively. Cabot numbering is used throughout.

The kappa variable region (Vk) of 16G7 belongs to mouse subgroup 1 according to Cabot, which corresponds to human subgroup 4 according to Cabot, and the heavy chain variable region (Vh) belongs to mouse subgroup 2b according to Cabot, which corresponds to human subgroup 1 according to Cabot [Kabat E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. CDR-L1 according to Chothia of 17 residues belongs to canonical class 3, CDR-L2 according to Chothia of 7 residues - class 1, CDR-L3 according to Chothia of 9 residues - class 1 in Vk [Martin A.C., Thornton J.M. (1996), J. Mol. Biol., 263:800-15. CDR-H1 according to Chothia of 10 residues belongs to class 1, CDR-H2 according to Chothia of 17 residues belongs to class 2 [Martin & Thornton, 1996]. CDR-H3 contains no canonical classes, but the 9-residue loop likely contains a bent base according to the rules of Shirai et al. [Shirai H. et al. (1999), FEBS Lett., 455:188-97]. The protein sequences were searched in the PDB database [Deshpande N. et al. (2005), Nucleic Acids Res., 33:D233-7] to find structures that would provide an exemplary structural model of 16G7. To build the Fv model 16G7, the FAB structure was used against the O-antigen of Francisella tularenis (code pdb 3UJT) [Rynkiewicz, M.J. et al. (2012), Biochemistry, 51:5684-5694] with a resolution of 2.1 A. This model retains the same canonical loop structure as 16G7. For VLv1, VLv2, and VLv3, the human VL acceptors AAB24404.1 and AEK69364.1 from the NCBI database non-redundant protein sequence search (according to the previous INN humanization rule) were also used. For VHv1, VHv2, VHv3, VHv4, VHv5V and VHv5VR, acceptors AAA18265.1 and ADW08092.1 were also used from the NCBI non-redundant protein sequence search (according to the previous INN humanization rule).

According to the 2015 INN antibody humanization rule [Jones, T.D. et al. (2016), The INNs and outs of antibody nonproprietary names. mAbs (http://dx.doi.org/10.1080/19420862.2015.1114320)], a search in the IMGT database allowed selection of suitable human germline framework regions into which mouse CDRs could be transferred. For Vk, a human kappa light chain with IMGT#IGKV4-1*01 was selected. It is characterized by the same canonical classes for CDR-L1, CDR-L2 and L3 and belongs to subgroup 1 of human kappa. For Vh, a human heavy chain with IMGT#IGHV1-2*02, which belongs to subgroup 1 of the human heavy chain, was selected. It is characterized by the canonical form CDR-H1 and H2 16G7. VHv2a, VHv2b, VHv2aB7, VHv2bH7, VHv6a, VHv6b and VHv7 were designed using IMGT# IGHV1-2*02 as an acceptor.

Heavy and light chain sequence variants resulting from the antibody humanization process were then aligned with human germline sequences using the IMGT Domain GapAlign tool to assess the degree of heavy and light chain humanization as outlined in the WHO INN committee guidelines (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet), 2014. Available at: http://www.who.int/medicines/services/inn/BioRev2014.pdf). Residues were changed to align with the corresponding human germline sequence whenever possible to enhance the degree of humanization.

Конструировали 13 вариантов гуманизированной вариабельной области тяжелой цепи и 3 варианта гуманизированной вариабельной области легкой цепи, содержащие различные пер мутации замен (hu16G7VHv1, hu16G7VHv2, hu16G7VHv2a, 16G7VHv2aB7, hu16G7VHv2b, hu16G7VHv2bH7, hu16G7VHv3, hu16G7VHv4, hu16G7VHv5V, hu16G7VHv5VR, hu16G7VHv6a, hu16G7VHv6b и hu16G7VHv7 (SEQ ID NOs: 15-27, respectively) and hu16G7VLv1, hu16G7VLv2, and hu16G7VLv3 (SEQ ID NOs: 28-30, respectively) (Tables 4 and 3).Illustrative designs of humanized Vk and Vh with backmutations and other mutations based on selected human framework regions are shown in Tables 3 and 4, respectively.The gray shaded areas in Tables 3 and 4 mark CDRs according to the Cabot/Chothia pooled definition.SEQ ID NOs: 15-27 and SEQ ID NOs: 28-30 contain backmutations and other mutations as shown in Table 5. Amino acids at positions

H1, H5, H7, H9, NU, NI, H12, H13,

H20, H31, H37, H38, H40, H48, H60, H64, H66, H67, H69, H71, H73, H75, H80, H82s, H82b,

Н83 и Н85 в hul6G7VHvl, hul6G7VHv2), hul6G7VHv2a, 16G7VHv2aB7, hul6G7VHv2b, hul6G7VHv2bH7, hul6G7VHv3, hul6G7VHv4, hul6G7VHv5V, hul6G7VHv5VR, hul6G7VHv6a, hul6G7VHv6b и hul6G7VHv7 перечислены в табл. 6. Amino acids at positions at L4, L9, L15, L18, L19, L21, L22, L43, and L48 of hu16G7VLv1, hu16G7VLv2, and hu16G7VLv3 are listed in Table 6. 7. Percentage degree of humanization for gu

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VH hul6G7VHvl, hul6G7VHv2, hul6G7VHv2a, 16G7VHv2aB7, hul6G7VHv2b, hul6G7VHv2bH7, hul6G7VHv3, hul6G7VHv4, hul6G7VHv5V, hul6G7VHv5VR, hul6G7VHv6a, hul6G7VHv6b и hu!6G7VHv7 (SEQ ID NO: 15-27 соответственно) и гуманизированных цепей VL hu16G7VLv1, hu16G7VLv2 и hu16G7VLv3 (SEQ ID NO: 28-30, respectively) is presented in table. 8.

Table 3

Residue No. according to Cabot Linear Residue No. FR or CDR VL 16G7 mice (SEQ ID NO: 11) Accounts. Human Fr VL acceptor IMGT #IGKV4-l*01 (SEQ ID NO:36) Hul6G7VLvl (SEQ ID NO:28) Hul6G7VLv2 (SEQ ID NO:29) Hul6G7VLv3 (SEQ ID NO:30) 1 1 frl D D D D D 2 2 frl I I I I I 3 3 frl V V V V V 4 4 frl L M L L L 5 5 frl S T T T T 6 6 frl Q Q Q Q Q 7 7 frl s s s s s 8 8 frl P P P P P 9 9 frl s D s s s 10 10 frl s s s s s eleven eleven frl L L L L L 12 12 frl A A A A A 13 13 frl V V V V V 14 14 frl S S s s s 15 15 frl V L V V V 16 16 frl G G G G G 17 17 frl E E E E E 18 18 frl TO R TO R TO 19 19 frl V A V A V 20 20 frl T T T T T 21 21 frl M I M I M 22 22 frl s N s s s 23 23 frl c C c c c 24 24 CDR-L1 To TO To To To 25 25 CDR-L1 s s s s s 26 26 CDR-L1 s s s s s 27 27 CDR-L1 Q Q Q Q Q 27A 28 CDR-L1 s s s s s 27B 29 CDR-L1 L V L L L 27С thirty CDR-L1 L L L L L

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27D 31 CDR-L1 D Y D D D 27E 32 CDR-L1 G s G G G 27F 33 CDR-L1 N s N N N 28 34 CDR-L1 D N D D D 29 35 CDR-L1 Q N Q Q Q thirty 36 CDR-L1 TO TO TO TO TO 31 37 CDR-L1 N N N N N 32 38 CDR-L1 Y Y Y Y Y 33 39 CDR-L1 L L L L L 34 40 CDR-L1 A A A A A 35 41 Fr2 W W W W W 36 42 Fr2 Y Y Y Y Y 37 43 Fr2 Q Q Q Q Q 38 44 Fr2 Q Q Q Q Q 39 45 Fr2 To To To To To 40 46 Fr2 P P P P P 41 47 Fr2 G G G G G 42 48 Fr2 Q Q Q Q Q 43 49 Fr2 s P s s s 44 50 Fr2 P P P P P 45 51 Fr2 To To To To To 46 52 Fr2 L L L L L 47 53 Fr2 L L L L L 48 54 Fr2 M I M I I 49 55 Fr2 Y Y Y Y Y 50 56 CDR-L2 w w w w w 51 57 CDR-L2 A A A A A 52 58 CDR-L2 S S S S S 53 59 CDR-L2 T T T T T 54 60 CDR-L2 R R R R R

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55 61 CDR-L2 E E E E E 56 62 CDR-L2 S S S S S 57 63 Fr3 G G G G G 58 64 Fr3 V V V V V 59 65 Fr3 P P P P P 60 66 Fr3 D D D D D 61 67 Fr3 R R R R R 62 68 Fr3 F F F F F 63 69 Fr3 T S S S S 64 70 Fr3 G G G G G 65 71 Fr3 S S S S S 66 72 Fr3 G G G G G 67 73 Fr3 S S S S S 68 74 Fr3 G G G G G 69 75 Fr3 T T T T T 70 76 Fr3 D D D D D 71 77 Fr3 F F F F F 72 78 Fr3 T T T T T 73 79 Fr3 L L L L L 74 80 Fr3 T T T T T 75 81 Fr3 I I I I I 76 82 Fr3 S S S S S 77 83 Fr3 s s s s s 78 84 Fr3 L L L L L 79 85 Fr3 TO Q Q Q Q 80 86 Fr3 A A A A A 81 87 Fr3 E E E E E 82 88 Fr3 D D D D D 83 89 Fr3 L V V V V 84 90 Fr3 A A A A A

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85 91 Fr3 V V V V V 86 92 Fr3 Y Y Y Y Y 87 93 Fr3 Y Y Y Y Y 88 94 Fr3 c c c c c 89 95 CDR-L3 Q Q Q Q Q 90 96 CDR-L3 Q Q Q Q Q 91 97 CDR-L3 F Y F F F 92 98 CDR-L3 Y Y Y Y Y 93 99 CDR-L3 D s D D D 94 100 CDR-L3 Y T Y Y Y 95 101 CDR-L3 P P P P P 95A CDR-L3 95V CDR-L3 95C CDR-L3 95D CDR-L3 95E CDR-L3 95F CDR-L3 96 102 CDR-L3 w w w w 97 103 CDR-L3 T T T T 98 104 Fr4 F F F F 99 105 Fr4 G G G G 100 106 Fr4 G G G G 101 107 Fr4 G G G G 102 108 Fr4 T T T T 103 109 Fr4 TO TO TO TO 104 BY Fr4 L L L L 105 111 Fr4 E E E E 106 112 Fr4 I I I I 106A 113 Fr4 TO TO TO TO 107 114 Fr4 R R R R

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Table 4

Residue No. according to Cabot Linear Residue No. FR or CDR VH 16G7 mouse (SEQ ID NO: 7) Human FR VH Acceptor IMGT Accession #IGHV1-2*O2 (SEQ (ID NO: 33) hu!6G7VHvl (SEQ ID NO: 15) hu!6G7VHv2 (SEQ ID NO: 16) hu!6G7VHv2a (SEQ ID NO: 17) 16G7VHv2aB7 (SEQ ID NO: 18) hu!6G7VHv2b (SEQ ID NO: 19) hu!6G7VHv2bH7 (SEQ ID NO: 20) hu!6G7VHv3 (SEQ ID NO:21) hu!6G7VHv4 (SEQ ID NO:22) hu!6G7VHv5V (SEQ ID NO:23) hu!6G7VHv5VR (SEQ ID NO:24) hu!6G7VHv6a (SEQ ID NO:25) hu!6G7VHv6b (SEQ ID NO:26) hu!6G7VHv7 (SEQ ID NO:27) 1 1 frl Q Q E E E E E E E E E E Q Q E 2 2 frl V V V V V V V V V V V V V V V 3 3 frl Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q 4 4 frl L L L L L L L L L L L L L L L 5 5 frl Q V Q Q V V V V Q Q Q Q V V V 6 6 frl Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q 7 7 frl P s P s s s s s P s P P P s P 8 8 frl G G G G G G G G G G G G G G G 9 9 frl S A S A A A A A S A S S A A A 10 10 frl V E V E E E E E V E V V E E E eleven eleven frl L V L L V V V V L L L L V V V 12 12 frl V TO V V V V V V V V V V TO TO TO 13 13 frl R To R TO TO TO TO TO R TO R R To To To 14 14 frl P P P P P P P P P P P P P P P 15 15 frl G G G G G G G G G G G G G G G 16 16 frl A A A A A A A A A A A A A A A 17 17 frl S S S S S S S S S S S S S S S 18 18 frl V V V V V V V V V V V V V V V 19 19 frl To To To To To To To To To To To To To To To 20 20 frl L V L L V V V V L L L L V V V 21 21 frl s s s s s s s s s s s s s s s 22 22 frl c c c c c c c c c c c c c c c 23 23 frl To To To To To To To To To To To To To To To 24 24 frl A A A A A A A A A A A A A A A 25 25 frl s s s s s s s s s s s s s s s 26 26 frl G G G G G G G G G G G G G G G 27 27 frl Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

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28 28 frl T T T T T T T T T T T T T T T 29 29 frl F F F F F F F F F F F F F F F thirty thirty frl T T T T T T T T T T T T T T T 31 31 CDR- H1 S G S S S G S G S S S s S S S 32 32 CDR- H1 S Y s s s S s S s s s s s s s 33 33 CDR- H1 w Y w w w w w w w w w w w w w 34 34 CDR- H1 I M I I I I I I I I I I I I I 35 35 CDR- H1 H H H H H H H H H H H H H H H 35A CDR- H1 35V CDR- H1 36 36 Fr2 w w w w w w w w w w w w w w w 37 37 Fr2 A V A V V V V V V V V V V V V 38 38 Fr2 TO R TO R R R R R R R To R R R R 39 39 Fr2 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q 40 40 Fr2 R A R A R R R R A A R R A A A 41 41 Fr2 P P P P P P P P P P P P P P P 42 42 Fr2 G G G G G G G G G G G G G G G 43 43 Fr2 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q 44 44 Fr2 G G G G G G G G G G G G G G G 45 45 Fr2 L L L L L L L L L L L L L L L 46 46 Fr2 E E E E E E E E E E E E E E E 47 47 Fr2 W W W W W W W W W W W W W W W

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48 48 Fr2 I M I I I I I I I I I I M M M 49 49 Fr2 G G G G G G G G G G G G G G G 50 50 CDR- H2 E W E E E E E E E E E E E E E 51 51 CDR- H2 I I I I I I I I I I I I I I I 52 52 CDR- H2 Y N Y Y Y Y Y Y Y Y Y Y Y Y Y 52A 53 CDR- H2 P P P P P P P P P P P P P P P 52V 52C 53 54 CDR- H2 N N N N N N N N N N N N N N N 54 55 CDR- H2 S S S S S S S S S S S S S S S 55 56 CDR- H2 G G G G G G G G G G G G G G G 56 57 CDR- H2 N G N N N N N N N N N N N N N 57 58 CDR- H2 T T T T T T T T T T T T T T T 58 59 CDR- H2 N N N N N N N N N N N N N N N 59 60 CDR- H2 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 60 61 CDR- H2 N A N N N A N A N N N N N N N 61 62 CDR- E Q E E E E E E E E E E E E E

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H2 62 63 Fr3 TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO 63 64 Fr3 F F F F F F F F F F F F F F F 64 65 Fr3 TO Q TO TO TO TO TO Q TO TO TO TO TO TO TO 65 66 Fr3 G G G G G G G G G G G G G G G 66 67 Fr3 TO R TO R TO TO R R R TO TO TO R R R 67 68 Fr3 A V A V A A V V V A A A V V V 68 69 Fr3 T T T T T T T T T T T T T T T 69 70 Fr3 L M L L L L L L L L L L M M M 70 71 Fr3 T T T T T T T T T T T T T T T 71 72 Fr3 V R V V V V R R V V V V V V V 72 73 Fr3 D D D D D D D D D D D D D D D 73 74 Fr3 I T I T I I I I T I I I I T I 74 75 Fr3 S s S s S S S S s S S S s s s 75 76 Fr3 s I s A I I I I A s s s I I I 76 77 Fr3 s s s S s s s s S s s s s s s 77 78 Fr3 T T T T T T T T T T T T T T T 78 79 Fr3 A A A A A A A A A A A A A A A 79 80 Fr3 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 80 81 Fr3 V M V M M M M M M M V V M M M 81 82 Fr3 D E E E E E E E E E E E E E E 82 83 Fr3 L L L L L L L L L L L L L L L 82A 84 Fr3 T S T T S S S S T T T T S S S 82V 85 Fr3 S R S S R R R R S S S S R R R 82С 86 Fr3 L L L L L L L L L L L L L L L 83 87 Fr3 T R T T T T T T T T T T R R R 84 88 Fr3 S S S S S S S S S S S S S S S 85 89 Fr3 E D E E D D D D E E E E D D D 86 90 Fr3 D D D D D D D D D D D D D D D

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87 91 Fr3 S T s T T T T T T T T T T T T 88 92 Fr3 A A A A A A A A A A A A A A A 89 93 Fr3 V V V V V V V V V V V V V V V 90 94 Fr3 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 91 95 Fr3 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 92 96 Fr3 c c c c c c c c c c c c c c c 93 97 Fr3 A A A A A A A A A A A A A A 94 98 CDR- NZ R R R R R R R R R R R R R R 95 99 CDR- H3 L L L L L L L L L L L L L L 96 100 CDR- H3 G G G G G G G G G G G G G G 97 101 CDR- H3 W W W W W W W W W W W W W W 98 102 CDR- H3 L L L L L L L L L L L L L L 99 103 CDR- H3 I I I I I I I I I I I I I I 100 104 CDR- NZ P P P P P P P P P P P P P P 100A 105 L L L L L L L L L L L L L L 100V 100C 100D 100E 100F 100G 100H 1001 100J 100K 101 106 CDR- H3 D D D D D D D D D D D D D D 102 107 CDR- H3 Y Y Y Y Y Y Y Y Y Y Y Y Y Y 103 108 Fr4 W W W W W W W W W W W W W W 104 109 Fr4 G G G G G G G G G G G G G G 105 110 Fr4 Q Q Q Q Q Q Q Q Q Q Q Q Q Q 106 111 Fr4 G G G G G G G G G G G G G G 107 112 Fr4 T T T T T T T T T T T T T T 108 113 Fr4 T T T T T T T T T T T T T T 109 114 Fr4 L V V V V V V V V V V V V V 110 115 Fr4 I T T T T T T T T T T T T T 111 116 Fr4 V V V V V V V V V V V V V V 112 117 Fr4 s s s s s s s s s s s s s s 113 118 Fr4 s s s s s s s s s s s s s s

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Table 5

Backmutations and other VH, VL mutations for humanized 16G7

VH or VL variant VH acceptor sequence exon or VL Changes from acceptor framework residues and CDRs (based on CDRs according to the Cabot/Chothia pooled definition) hul6G7VHvl (SEQ ID NO: 15) Account Code NCBIIMGT# IGHV1-2*O2 (SEQ (ID NO: 33) H1, H5, H7, H9, NU, NI, H12, H13, H20, H37, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82A, H82B, H83, H85 hul6G7VHv2 (SEQ ID NO: 16) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H5, NI, H12, H20, H48, H69, H71, H75, H82A, H82B, H83, H85 hul6G7VHv2a (SEQ ID NO: 17) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H12, H40, H48, H66, H67, H69, H71, H73, H83 16G7VHv2aB7 (SEQ ID NO: 18) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H12, H31, H40, H48, H60, H66, H67, H69, H71, H73, H83 hul6G7VHv2b (SEQ ID NO: 19) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H12, H40, H48, H69, H73, H83 hul6G7VHv2bH7 (SEQ ID NO: 20) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H12, H31, H40, H48, H60, H64, H69, H73, H83 hul6G7VHv3 (SEQ ID NO:21) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) H1, H5, H7, H9, NU, NI, H12, H13, H20, H48, H69, H71, H75, H82A, H82B, H83, H85 hul6G7VHv4 (SEQ ID NO:22) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H5, NI, H12, H20, H48, H66, H67, H69, H71, H73, H75, H82A, H82B, H83, H85 hul6G7VHv5V (SEQ ID NO:23) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) H1, H5, H7, H9, NYU, NI, H12, H13, H20, H38, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82A, H82B, H83, H85

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hu!6G7VHv5VR (SEQ ID NO:24) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) Hl, H5, H7, H9,H1O,H11, H12, H13, H20, H40, H48, H66, H67, H69, H71, H73, H75, H80, H82A, H82B, H83, H85 hul6G7VHv6a (SEQ ID NO:25) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) H7, H71, H73 hul6G7VHv6b (SEQ ID NO:26) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) H71 hu!6G7VHv7 (SEQ ID NO:27) NCBI account code IMGT# IGHV1-2*O2 (SEQ (ID NO: 33) H1,H7,H71,H73 Hul6G7VLvl (SEQ ID NO:28) NCBI account code IMGT#IGKV4-l*01 (SEQ ID NO:36) L4,L9,L15,L18,L19, L21, L22, L43, L48 Hul6G7VLv2 (SEQ ID NO:29) NCBI account code IMGT#IGKV4-l*01 (SEQ ID NO:36)) L4,L9,L15, L22, L43 Hul6G7VLv3 (SEQ ID NO:30) NCBI account code IMGT#IGKV4-l*01 (SEQ ID NO:36) L4,L9,L15, L18, L19, L21, L22, L43

Table 6

Cabot numbering of framework region residues (or CDRs) (based on CDRs according to the Cabot/Chothia summary definition) for backmutations and other mutations in heavy chains of humanized 16G7 antibodies

Remainder (heavy chain) 16G7 mice hu!6G7VHvl hu!6G7VHv2 hu!6G7VHv2a 16G7VHv2aB7 hu!6G7VHv2b hu!6G7VHv2bH7 hu!6G7VHv3 hu!6G7VHv4 hu!6G7VHv5V hu!6G7VHv5VR hu!6G7VHv6a I hu!6G7VHv7 Hl Q Q E E E E E E E E E E Q Q E H5 V Q Q Q V V V V Q Q Q Q V V V H7 s P P s s s s s P s P P P s P H9 A s s A A A A A s A s s A A A H10 E V V E E E E E V E V V E E E hll V L L L V V V V L L L L V V V H12 TO V V V V V V V V V V V TO TO TO H13 To R R TO To To To To R TO R R TO To To H20 V L L L V V V V L L L L V V V H31 G S S S s G s G S S S S S s s H37 V A A V V V V V V V V V V V V H38 R TO TO R R R R R R R To R R R R H40 A R R A R R R R A A R R A A A H48 M I I I I I I I I I I I M M M

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H60 A N N N N A N A N N N N N N N H64 Q TO TO TO TO TO TO Q TO TO TO TO TO TO TO H66 R TO TO R TO TO R R R TO TO TO R R R H67 V A A V A A V V V A A A V V V H69 m L L L L L L L L L L L m m m H71 R V V V V V R R V V V V V V V H73 T I I T I I I I T I I I I T I H75 I S S A I I I I A S S S I I I H80 m V V M m m M M M m V V m m m H82A S T T T S S S S T T T T S S S H82V R S S S R R R R S S S S R R R H83 R T T T T T T T T T T T R R R H85 D E E E D D D D E E E E D D D

Table 7

Cabot numbering of framework residues (based on the CDR according to the Cabot/Chothia summary definition) for backmutations and other mutations in light chains of humanized 16G7 antibodies

Remainder IMGT#IGKV41*01(light chain) 16G7 mice Hul6G7VLvl Hul6G7VLv2 Hul6G7VLv3 L4 M L L L L L9 D S S S S L15 L V V V V L18 R To To R To L19 A V V A V L21 I m m I m L22 N S S S S L43 R S S S S L48 I m m I I

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Table 8

Percentage degree of humanization of the heavy and light chains of humanized 16G7 antibodies

VH or VL variant % degree humanization hul6G7VHvl (SEQ GO NO: 15) 64.30% hul6G7VHv2 (SEQ ID NO: 16) 76.50% hul6G7VHv2a (SEQ ID NO: 17) 79.60% 16G7VHv2aB7 (SEQ ID NO: 18) 81.60% hul6G7VHv2b (SEQ ID NO: 19) 82.70% hul6G7VHv2bH7 (SEQ ID NO: 20) 85.70% hu!6G7VHv3 (SEQ ID NO:21) 72.40% hu!6G7VHv4 (SEQ ID NO:22) 73.50% hu!6G7VHv5V (SEQ ID NO:23) 66.30% hu!6G7VHv5VR (SEQ ID NO:24) 67.30% hu!6G7VHv6a (SEQ ID NO:25) 86.70% hu!6G7VHv6b (SEQ ID NO:26) 88.80% hu!6G7VHv7 (SEQ ID NO:27) 85.70% hu!6G7VLvl (SEQ ID NO:28) 82.20% hu!6G7VLv2 (SEQ ID NO:29) 86.10% hu!6G7VLv3 (SEQ ID NO:30) 83.20%

Positions at which canonical, vernier or interaction surface residues differ between mouse and human acceptor sequences are candidates for substitution. Examples of canonical/CDR-interacting residues include Cabot residues H24, H26, H27, H29, H34, H52a, H55, H70, H71, H74, H94, L2, L4, L25, L27b, L33, L48, L64, L71, L90 and L95 in the table. 4. Examples of interaction surface/packaging residues (VH+VL) include Cabot residues H35, H37, H39, H45, H47, H91, H93, H95, H100a, H103, L34, L36, L38, L44, L46, L87, L89, L91, L96, L98 in the table. 3.

Reasons for choosing the provisions indicated in Table. 3 in the light chain variable region as replacement candidates are as follows.

M4L is a mutation of a residue that makes contact with LCDR1 and LCDR3. P43S is a mutation of a residue that makes contact with two interaction surface residues (Y91 and W103) in VH. I48M is a mutation of the remainder of the interaction surface. D9S, L15V, R18K, A19V, 121M and N22S are frequency based backmutations or frequency based/germline alignment mutations.

Reasons for choosing the provisions indicated in Table. 4 in the heavy chain variable region as replacement candidates are as follows.

Q1E is a stability enhancing mutation to reduce the potential for pyroglutamate formation (Liu, 2011, ref. above).

S7P is a proline mutation for improved folding. R71V is a mutation of a residue that can make contact with HCDR2 via hydrogen bonding. T73I is a residue mutation that can make contact with HCDR1 and HCDR2.

V5Q, E10V, V11L, K12V, K13R, V20L, S31G, V37A, R38K, A40R, M48I, N60A, K64Q, R66K, V67A. M69L, I75S, I75A, M80V, S82a-T, R83b-S, R83T and D85E are backmutations based on frequency or germline alignment mutations.

Humanized sequences were generated using a two-step PCR protocol that allows multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis [Wang, W., Malcolm, B.A. (1999), BioTechniques, 26:680-682).

The designs based on human frame data were as follows.

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Variable region kappa.

hul6G7VLvl (SEQ ID NO:28):

DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWAS TRESGVPDRF SGSGSGTDFTLTIS SLQ AED VAVYYCQQF YDYPWTFGGGTKLEIKR hul6G7VLv2 (SEQ ID NO: 29):

DIVLTQSPSSLAVSVGERATISCKSSQSLLDGNDQKNYLAWYQQ

KPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTF GGGTKLEIKR hul6G7VLv3 (SEQ ID NO: 30):

DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRF SGSGSGTDFTLTIS SLQ AED VAVYYCQQF YDYPWTFGGGTKLEIKR VARIABLE HEAVY hul6G7VHvl (SEQ ID NO: 15):

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSS hul6G7VHv2 (SEQ ID NO: 16):

EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTN YNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYCARLGWLIPLDYWGQGTTVTVS S hul6G7VHv2a (SEQ ID NO: 17):

EVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNT NYNEKFKGKATLTVDISISTAYMELSRLTSDDTAVYYCARLGWLIPLDYWGQGTTVTVS S hul6G7VH2aB7 (SEQ ID NO: 18)

(SEQ ID NO: 19)

EVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNT NYNEKFKGRVTLTRDISISTAYMELSRLTSDDTAVYYCARLGWLIPLDYWGQGTTVTVS S hul6G7VH2bH7 (SEQ ID NO: 20)

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EVQLVQSGAEVVKPGASVKVSCKASGYTFTGSWIHWVRQRPGQGLEWIGEIYPNSGNT NYAEKFQGRVTLTRDISISTAYMELSRLTSDTAVYYCARLGWLIPLDYWGQGTTVTVS S hul6G7VHv3(SEQ ID NO: 21):

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTN YNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVS S hul6G7VHv4 (SEQ ID NO: 22):

EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDIS ST AYMELTSLTSEDT AVYYC ARLGWLIPLDYWGQGTTVT VS hul6G7VHv5V (SEQ ID NO: 23):

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVKQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSS hul6G7VHv5VR (SEQ ID NO: 24):

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSS hul6G7VHv6a (SEQ ID NO: 25):

QVQLVQPGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGN TNYNEKFKGRVTMTVDISISTAYMELSRLRSDDTAVYYCARLGWLIPLDYWGQGTTVT vss hul6G7VHv6b (SEQ ID NO: 26):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGN TNYNEKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYCARLGWLIPLDYWGQGTTVT VSS hul6G7VHv7 (SEQ ID NO: 27):

VSS

Example 4 16G7 immune-capture tau from Alzheimer's diseased human tissue.

Ways.

In FIG. 4A shows temporal cortex tissue from an Alzheimer's brain that was used to extract protein fractions with different solubility. Frozen tissue was homogenized in 5 volumes of reassembly buffer (RAB) (100 mM MES, 1 mM EGTA, 0.5 mM MgSO ₄ , 750 mM NaCl, pH 6.8) and centrifuged to obtain a RAB soluble fraction. The pellet was then homogenized in 5 volumes of radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.5) and centrifuged to obtain a fraction soluble in RIPA. This pellet was homogenized in 5 volumes of sarcosyl buffer (25 mM Tris, 1 mM EGTA, 1% sarcosyl, 10% sucrose, 1 mM DTT, 500 mM NaCl, pH 7.5) and centrifuged to obtain a sarcosyl-soluble fraction. This final sarcosyl-insoluble precipitate was homogenized in 2% SDS. Samples were prepared in reducing/denaturing sample buffer and separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Western blotting was performed using mouse 16G7 antibody.

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In FIG. 4B shows RAB soluble protein fractions prepared at 1 mg/mL. For each immunoprecipitation, 300 μg of sample was used. 10 μg of the indicated antibody (isotype control, 16G7 or anti-tau antibody 16B5) was added to RAB-soluble sample preparations and incubated for 2 h. Protein G magnetic beads were then added to the mixtures and incubated for an additional 1 h to capture the complexes antibody/antigen. The samples were thoroughly washed with 1x PBS and the beads were boiled in reducing/denaturing sample buffer to release trapped proteins. The resulting samples were separated by SDS-PAGE and Western blotted using a polyclonal anti-tau antibody (Dako, No. A0024).

Results.

In FIG. 4A 16G7 recognizes sarcosyl-soluble and sarcosyl-insoluble tau from human Alzheimer's tissue. Protein fractions with different solubility were separated by SDS-PAGE and blotted with 16G7. A variety of tau isoforms are observed in soluble and insoluble fractions, and a shift in electrophoretic mobility was found in the insoluble fractions, indicating a pathological form of tau. In the fraction insoluble in sarcosyl, a noticeable accumulation of tau is observed. Left: molecular weight markers. Lane designations: 1 - soluble in RAB, 2 - soluble in RIPA, 3 - soluble in sarcosyl, 4 - insoluble in sarcosyl.

In FIG. 4B 16G7 immunoprecipitated tau from tissue affected by Alzheimer's disease. RAB-soluble fractions immunoprecipitated with the indicated antibody and were detected with a polyclonal anti-tau antibody directed against a specific region of the tau molecule from the binding sites for 16G7 and anti-tau antibody 16B5. 16G7 reliably captures tau from this fraction. Inlet samples (RAB soluble sample) are shown on the right.

Example 5 Reactivity with human tissue affected by Alzheimer's disease (immunohistochemical analysis).

The frontotemporal cortex was obtained from patients not suffering from a neurodegenerative disease or suffering from Alzheimer's disease, which was confirmed during post-mortem evaluation. Immunohistochemical study was carried out on 10 μm thick cryosections lightly fixed with acetone, mounted on glass. All staining steps were performed using a Leica BOND Rx automatic stainer using Leica consumables. Mouse or human 16G7 forms were incubated with tissue sections followed by the addition of species-specific secondary antibodies conjugated to the HRP polymer. To prevent non-specific binding of endogenous immunoglobulins when humanized antibodies are applied to human tissue, the antibodies were non-covalently labeled with a biotin-conjugated monovalent Fab fragment against human immunoglobulins in vitro prior to tissue incubation. Tissue labeled with primary antibody-Fab-biotin complex was then amplified using an avidin-biotin amplification system (Vector Laboratories, Burlingame, CA). Staining was visualized with the DAB chromogen, which caused a brown precipitate to form. The negative control consisted of performing the entire immunohistochemical procedure on adjacent sections with an isotype control IgG antibody.

The antibodies tested were mouse 16G7, chimeric 16G7 (which contained the VH and VL from mouse antibody with human constant regions, heavy chain of SEQ ID NO: 54 and light chain of SEQ ID NO: 55), and humanized variants of 16G7 hu16G7VHv7/hu16G7VLv2 and hu16G7VHv2bH7/hu16G7VLv2 .

Staining performed with mouse 16G7 antibody, chimeric 16G7 and humanized forms of 16G7 (hu16G7VHv7/hu16G7VLv2 and hu16G7VHv2bH7/hu16G7VLv2) was qualitatively compared and evaluated for stain stability and intensity, as well as localization of immunoreactivity. Staining intensity was similar for the chimeric and humanized forms of 16G7 and showed a similar localization pattern compared to the mouse antibody form. Tau has been found in neurofibrillary tangles, fibers, filaments of neuropils, and in degenerating axons. Noticeable somatic staining was also observed.

Example 6 Disaggregation activity.

Ways.

Aggregation of recombinant tau. Purified recombinant tau with a 6xHis N-terminal tag was combined with equimolar amounts of low molecular weight heparin in 1x PBS (pH 7.4) and incubated at 37°C for 96 h on a rocking shaker. Sample aggregation was confirmed by binding to thioflavin T.

Incubation with antibodies. Antibodies were incubated with aggregated recombinant tau at the indicated molar ratios, incubated at 37° C. for 96 hours without rotation or shaking. At the end of the experiment, aggregation was measured by incubating the samples with 25 mM thioflavin T and measuring the emitted fluorescence (excitation/emission 450/482). The background of the buffer samples was subtracted from the signals.

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Results.

As shown in FIG. 5, 16G7 preferentially disassembles intact tau fibers. Varying molar ratios of 16G7 (triangles), isotype control (circles), and anti-tau antibody 16B5 (squares) were incubated with amyloid-containing tau fibers for 96 h. the thioflavin T signal present in the sample compared to both the isotype control antibody and anti-tau antibody 16B5, which binds to a different tau region.

Example 7 Affinity of mouse 16G7 antibody for tau.

SNR (surface plasmon resonance) analysis was performed using a Biacore T200 instrument to determine the kinetics of 16G7 binding to recombinant human tau. To prepare the sensor surface, an anti-mouse immunoglobulin antibody (GE Life Sciences) was immobilized onto the CM5 sensor probe via an amine coupling and 16G7 was captured at a level that allowed for maximum binding of 50 RU. (units of response). Various concentrations of recombinant tau, ranging from 10-0.14 nM, were passed over the captured ligand at a flow rate of 50 µl/min in assay buffer (HBS+0.05% P-20, 1 mg/ml BSA) for 180 s association and 900 with dissociation. The data were correlated twice to both an irrelevant sensor containing no antibody ligand and an analyte concentration of 0 nM to account for dissociation of the ligand from the capture molecule. The data were then analyzed using a 1:1 global fit. The binding kinetics is shown in Fig. 6.

Example 8 Tau Affinity of Chimeric 16G7 Antibody and Humanized Variants.

SPR analysis was performed using a Biacore T200 instrument to determine the kinetics of 16G7 binding to recombinant human tau. To prepare the sensor surface, an anti-human immunoglobulin antibody (GE Life Sciences) was immobilized on the CM5 sensor via amine coupling and the chimeric or humanized antibodies were captured at a level that provides maximum binding of 50 RU. Various concentrations of recombinant tau ranging from 10-0.14 nM were passed over the captured ligands in parallel at a flow rate of 50 µl/min in assay buffer (HBS+0.05% P-20, 1 mg/ml BSA) for 180 s for association and 900 s for dissociation. The data were correlated twice to both an irrelevant sensor containing no antibody ligand and an analyte concentration of 0 nM to account for dissociation of the ligand from the capture molecule. The data were then analyzed using a 1:1 global fit.

The antibodies tested were chimeric 16G7 (containing the VH and VL regions from a mouse antibody with human constant regions, heavy chain SEQ ID NO: 54, light chain SEQ ID NO: 55) and humanized variants (hu16G7VHv2a/hu16G7VLv2, hu16G7VHv2b/hu16G7VLv2 and hu16G7VHv7/ hu16G7VLv2).

In FIG. 7 the affinity of the studied antibodies was determined by analysis by the SPR method. Varying concentrations of recombinant tau were passed over the immobilized antibody and the resulting sensograms were analyzed using a 1:1 global fit. The affinity kinetics, association and dissociation rates are presented in Fig. 7.

Example 9 Avidity for aggregated tau.

Ways.

Getting Fab fragments. 16G7 Fab fragments were prepared using the Fab Micro Preparation kit according to the manufacturer's instructions (Pierce). Removal of released Fc and confirmation of intact end product was monitored by SDS-PAGE and concentration was determined using the bicinchoninic acid method (Pierce).

Aggregation of recombinant tau. Purified recombinant tau with a 6xHis N-terminal tag was combined with equimolar amounts of low molecular weight heparin in 1x PBS (pH 7.4) and incubated at 37°C for 96 h on a rocking shaker. Sample aggregation was confirmed by binding to thioflavin T.

Analysis of Fab fragments and intact antibody by SPR. Comparative studies of the binding of Fab fragments and intact antibodies were performed on a Biacore T200 instrument. For sensor surface preparation, anti-His antibodies (GE Life Sciences) were immobilized onto a CM3 sensor via amine coupling and aggregated recombinant tau (prepared as described above) was passed over the channel of interest, which acted as the assay ligand. 16G7 (intact antibody or Fab fragments) was passed over captured aggregated tau at a flow rate of 50 μl/min in assay buffer (FIBS+0.05% P-20, 1 mg/ml BSA) in single cycle mode, at concentrations varying from 10-0.016 nM, during the association phases lasting 180 s and the final dissociation phase lasting 900 s. The association/dissociation curves were correlated twice with the subtraction of both the irrelevant sensor data containing no antibody ligand and the 0 nM analyte concentration to account for the dissociation of the ligand from the capture molecule.

Results.

Avidity is indicative of a variety of antigen-antibody interaction affinities and more accurately represents the functional affinity of an antibody for an immobilized antigen such as tau aggregates or neurofibrillary tangles. In measurements by the method

The SPR difference between affinity (single interaction) and avidity (multiple interactions) is obtained by comparing the binding kinetics of a Fab fragment containing one binding domain and an intact antibody with two binding domains.

In FIG. 8 16G7 Fab binding measurements were compared to intact antibody. The phases of association (solid horizontal lines) and dissociation (dashed horizontal lines) are presented as diagrams. The difference between the affinity of a single binding site and the avidity of two binding sites is most evident in the final phase of dissociation, starting from 1300 s. There is no apparent dissociation of the antigen/antibody complex for the intact antibody, while the Fab fragments definitely dissociate from the aggregated tau.

Sequence listing

P10636-8 (SEQIDNO:1)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDE AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAK TPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKD NIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSST GSIDMVDSPQLATLADEVSASLAKQGL

P10636-7 (SEQIDNO:2)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKG ADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPG TPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTEN LKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGS LGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAK AKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

P10636-6 (human tau 4RON) (SEQ ID NO:3)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLE DEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIP AKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGS KDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQ SKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPWSGDTSPRHLSNV SSTGSIDMVDSPQLATLADEVSASLAKQGL

P10636-5 (SEQIDNO 4)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDE AAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAK TPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGSLG

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NIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAK TDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

P10636-4 (SEQIDNO:5)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSE EPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKG ADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPG TPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTEN LKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSST GSIDMVDSPQLATLADEVSASLAKQGL

Pl0636-2 (SEQ ID NO:6)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDTPSLE DEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIP AKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPK SPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTSKCGS LGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAK AKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL SEQ ID NO: 7; VH protein sequence:

QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVDLTSLTSEDSAVYYCARLGWLIPLDYWGQGTTLIVSS

SEQ ID NO: 8; HCDR1 mice: GYTFTSSWIH

SEQ ID NO: 9; HCDR2 mice: EIYPNSGNTNYNEKFKG

SEQ ID NO: 10; HDDR3 mice:

LGWLIPLDY

SEQ ID NO: 11; mouse VL protein sequence:

DIVLSQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWAS TRESGVPDRFTGSGSGTDFTLTISSLKAEDLAVYCQQFYDYPWTFGGGTKLEIKR

SEQ ID NO: 12; Mouse LCDR1: KSSQSLLDGNDQKNYLA

SEQ ID NO: 13; LCDR2 mice:

WASTRES

SEQ ID NO: 14; LCDR3 mice:

QQFYDYPWT

- 63 041230

SEQ ID NO: 15; >hul6G7VHvl

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSS

SEQ ID NO: 16; > hul6G7VHv2

EVQLQQSGAELVKPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTN YNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVS S

SEQ ID NO: 17; > hul6G7VHv2a

EVQLVQSGAEVVKPGASVKVSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNT NYNEKFKGKATLTVDISISTAYMELSRLTSDTAVYYCARLGWLIPLDYWGQGTTVTVS

SEQ ID NO: 18; > hul6G7VHv2aB7

S

SEQ ID NO: 19; > hul6G7VHv2b

S

SEQ ID NO:20; > hul6G7VHv2bH7

S

SEQ ID NO:21; > hul6G7VHv3

- 64 041230

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQAPGQGLEWIGEIYPNSGNTN YNEKFKGRVTLTVDTSASTAYMELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVS S

SEQ ID NO: 22; > hul6G7VHv4

YNEKFKGKATLTVDIS ST

SEQ ID NO:23; > hul6G7VHv5V

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVKQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSS

SEQ ID NO:24; > hul6G7VHv5VR

EVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWVRQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVELTSLTSEDTAVYYCARLGWLIPLDYWGQGTTVTVSS

SEQ ID NO: 25; > hul6G7VHv6a

QVQLVQPGAEVKKPGASVKVSCKASGYTFTSSWIHWVRQAPGQGLEWMGEIYPNSGN TNYNEKFKGRVTMTVDISISTAYMELSRLRSDDTAVYYCARLGWLIPLDYWGQGTTVT vss

SEQ ID NO:26; > hul6G7VHv6b

VSS

SEQ ID NO:27; > hul6G7VHv7

VSS

SEQ ID NO: 28; > hul6G7VLvl

DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWAS TRESGVPDRF SGSGSGTDFTLTIS SLQAEDVAVYYCQQF YDYPWTFGGGTKLEIKR

SEQ ID NO: 29; >hul6G7VLv2

DIVLTQSPSSLAVSVGERATISCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWAST RESGVPDRF SGSGSGTDFTLTIS SLQAEDVAVYYCQQF YDYPWTFGGGTKLEIKR

SEQ ID NO:30; >hul6G7VLv3

DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRF SGSGSGTDFTLTIS SLQAEDVAVYYCQQF YDYPWTFGGGTKLEIKR

SEQ ID NO: 31; human VH acceptor AAA18265.1

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGRGLEWMGRINPNSG GTNYAQKFQGRVTMTRDTSIRTAYVELSRLTSDDTAVYYCASLADDDPEDFWGQGTLV TVSS

SEQ ID NO: 32; human VH acceptor ADW08092.1

QVQLVESGAEVKKPGASVKLSCKASGYTFSSYWMHWVRQAPGQRLEWMGEINPDNG HTNYNEKFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCAREADYSYGAFDIWGPGTT VTVSS

SEQ ID NO: 33; human VH acceptor IMGT#IGHV1-2*02

- 65 041230

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSG GTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC

SEQ ID NO: 34; human VL acceptor AAB24404.1

DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNNKNYLAWYQQKPGQPPKLLIYWAST

RESGVPDRF SGSGSGTDFTLTIS SLQAED VAVYYCQQYYSTPPMFGQGTKVEIKR

SEQ ID NO: 35; human VL acceptor AEK69364.1

DIVLTQSPDSLAVSLGERATIKCKSSQSVLYGSDSKNYLAWYQQKPGQPPKLLIYWAST

RESGVPDRFSGSGSGTEFTLTISSLQAADVAVYYCQEYYSSLCTFGQGTKLEIKR

SEQ ID NO: 36; human VL acceptor IMGT#IGKV4-1*01

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWAST

RESGVPDRF SGSGSGTDFTLTIS SLQAED VAVYYCQQYYSTP

SEQ ID NO: 37; mouse VH nucleotide sequence:

CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAA

GCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTCCTGGATACACTGGGCGAA

GCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTTATCCTAATAGTGGTA

ATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACATATCC

TCCAGCACAGCCTACGTGGATCTCACCAGCCTGACATCTGAGGACTCTGCGGTCTAT TATTGTGCAAGATTGGGATGGTTAATACCCCTTGACTACTGGGCCAAGGCACCACT CTCATAGTCTCCTCA

SEQ ID NO: 38; mouse VL nucleotide sequence:

GACATTGTGCTGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTT ACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTAGATGGTAATGATCAAAAGAACTA CTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATGTACTGGGC ATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAG ATTTCACTCTCACCATCAGCAGTTTGAAGGCTGAAGACCTGGCAGTTTATTACTGTC AGCAGTTTTATGACTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA CGT

SEQ ID NO: 39; CDR-H1 according to Cabot

SSWIH

SEQ ID NO: 40; CDR-H1 according to Chothia GYTFTSS

SEQ ID NO: 41; CDR-H2 according to Chothia

YPNSGN

SEQ ID NO: 42; CDR-H2 according to AbM

EIYPNSGTN

SEQ ID NO: 43; CDR-L1 according to KNYLAWY pin definition

SEQ ID NO: 44; CDR-L2 according to LLMYWASTRE contact definition

SEQ ID NO: 45; CDR-L3 according to pin definition QQFYDYPW

SEQ ID NO: 46; CDR-H1 according to TSSWIH pin definition

SEQ ID NO: 47; CDR-H2 according to WIGEIYPNSGNTN pin definition

SEQ ID NO: 48; CDR-H3 according to ARRGWLIPLD pin definition

SEQ ID NO: 49; Alternative CDR-H1

GYTFGSWIH

SEQ ID NO: 50; Alternative CDR-H2

EIYPNSGNTNYAEKFKG

SEQ ID NO: 51; Alternative CDR-H2

EIYPNSGNTNYAEKFQG

SEQ ID NO: 52; consensus amino acid sequence of variable ob-

Claims (39)

heavy chain regions of mouse 16G7 antibody and humanized 16G7 antibodies (designated Most in Fig. 2) EVQLXQXGAEXVKPGASVKXSCKASGYTFTSSWfflWVRQRPGQGLEWIGEIYPNSGNT NYNEKFKGXXTLTVDISISTAYMELXXLTSXDTAVYYCARLGWLIPLDYWGQGTTVTV SS SEQ ID NO: 53; consensus amino acid sequence among light chain variable regions of mouse 16G7 antibody and humanized 16G7 antibodies (labeled Most in Figure 3) DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLXYWAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYDYPWTFGGGTKLEIKR SEQ ID NO: 54; 16G7 chimeric antibody heavy chain QVQLQQPGSVLVRPGASVKLSCKASGYTFTSSWIHWAKQRPGQGLEWIGEIYPNSGNTN YNEKFKGKATLTVDISSSTAYVDLTSLTSEDSAVYYCARLGWLIPLDYWGQGTTLIVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 55; 16G7 chimeric antibody light chain DIVLSQSPSSLAVSVGEKVTMSCKSSQSLLDGNDQKNYLAWYQQKPGQSPKLLMYWAS TRESGVPDRFTGSGSGTDFTLTISSLKAEDLAVYYCQQFYDYPWTFGGGTKLEIKRKTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSNTTLSKTHQPVEKVACE CLAIM 1. An isolated monoclonal antibody that binds to human tau protein, containing three heavy chain CDRs as set forth in SEQ ID NO: 7 and three light chain CDRs as set forth in SEQ ID NO: 11, provided that position 31 the heavy chain occupies S or G, position 60 of the heavy chain occupies N or A, and position 64 of the heavy chain occupies K or Q, where the positions are numbered according to Cabot's definition. 2. An antibody according to claim 1, wherein said three heavy chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOS: 8, 9 and 10) and said three light chain CDRs are as defined according to the Cabot/Chothia summary definition (SEQ ID NOS: 12, 13 and 14). 3. An antibody according to claim 1, which is a murine, chimeric, veneered, or humanized antibody. 4. An antibody according to claim 3, wherein said antibody is humanized and contains a humanized heavy chain mature variable region and a humanized light chain mature variable region. 5. An antibody according to claim 4, wherein said three heavy chain CDRs are as defined according to the Cabot/Chothia pooled definition (SEQ ID NOs: 8, 9 and 10) and said three light chain CDRs are as defined according to the Cabot/Chothia pooled definition (SEQ ID NOS: 12, 13 and 14), provided that heavy chain position 31 is S or G, heavy chain position 60 is N or A, and heavy chain position 64 is K or Q, where positions are numbered according to Cabot's definition. 6. An antibody according to claim 5, characterized in that CDR-H1 contains an amino acid sequence containing SEQ ID NO: 49, CDR-H2 contains an amino acid sequence containing SEQ ID NO: 50 or SEQ ID NO: 51. 7. An antibody according to claim 4, which is humanized, wherein said CDRs correspond to a definition selected from the group consisting of definitions according to the definition of Cabot, Chothia, Cabot/Chothia summary definition, AbM, and contact definition. 8. An antibody according to claim 7 which is humanized, wherein said humanized mature heavy chain variable region comprises three heavy chain CDRs according to the Cabot/Chothia summary definition as set forth in SEQ ID NOs: 8-10 and said humanized mature variable region light chain contains three CDRs of the light chain according to the summary definition of Cabot/Chothia, as indicated in SEQ ID NO: 12-14. - 67 041230 9. The antibody of claim 7 that is humanized, wherein said humanized mature heavy chain variable region contains three heavy chain CDRs as defined by Cabot as set forth in SEQ ID NO: 39, SEQ ID NO: 9, and SEQ ID NO: 10 , and said humanized mature light chain variable region contains three light chain CDRs as defined by Cabot as set forth in SEQ ID NOs: 12-14. 10. An antibody according to claim 7 that is humanized, wherein said humanized mature heavy chain variable region contains three heavy chain CDRs as defined by Chothia as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 10 , and said humanized mature light chain variable region contains three light chain CDRs as defined by Chothia as set forth in SEQ ID NOs: 12-14. 11. An antibody according to claim 7 which is humanized, wherein said humanized mature heavy chain variable region contains three heavy chain CDRs as defined by AbM as indicated in SEQ ID NO: 8, SEQ ID NO: 42, and SEQ ID NO: 10 , and said humanized mature light chain variable region contains three light chain CDRs as defined by AbM as set forth in SEQ ID NOs: 12-14. 12. The antibody of claim 7, which is humanized, wherein said humanized mature heavy chain variable region comprises three heavy chain CDRs according to the contact definition as set forth in SEQ ID NOs: 46-48, and said humanized mature light chain variable region comprises three light chain CDRs according to the pin definition as set forth in SEQ ID NOs: 43-45. 13. An antibody according to any one of claims 7-12, which is humanized, containing a humanized mature heavy chain variable region that contains an amino acid sequence at least 90% identical to any of SEQ ID NOs: 15-27, and a humanized mature variable region a light chain region that contains an amino acid sequence at least 90% identical to any of SEQ ID NOs: 28-30. 14. An antibody according to claim 13, which is humanized, containing a mature heavy chain variable region containing an amino acid sequence at least 95% identical to any of SEQ ID NOs: 15-27, and a mature light chain variable region containing an amino acid sequence , at least 95% identical to any of SEQ ID NO: 28-30. 15. An antibody according to claim 14, which is humanized, containing a mature heavy chain variable region containing an amino acid sequence at least 98% identical to any of SEQ ID NOs: 15-27, and a mature light chain variable region containing an amino acid sequence , at least 98% identical to any of SEQ ID NO: 28-30. 16. The antibody of claim 15, which is humanized, wherein said mature heavy chain variable region comprises the amino acid sequence of any of SEQ ID NOs: 15-27 and said mature light chain variable region comprises the amino acid sequence of any of SEQ ID NOs: 28- thirty. 17. An antibody according to claim 16, which is humanized, wherein said mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 17 and said mature light chain variable region contains the amino acid sequence as indicated in SEQ ID NO: 29. 18. An antibody according to claim 16, which is humanized, wherein said mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 19 and said mature light chain variable region contains the amino acid sequence as indicated in SEQ ID NO: 29. 19. An antibody according to claim 16, which is humanized, wherein said mature heavy chain variable region contains the amino acid sequence of SEQ ID NO: 27 and said mature light chain variable region contains the amino acid sequence as indicated in SEQ ID NO: 29. 20. An antigen-binding fragment of an antibody according to any one of claims 1 to 19 that binds to a human tau protein. 21. An antibody according to any one of claims 1 to 19, characterized in that said antibody has the human IgG1 isotype. 22. An antibody according to any one of claims 4-19 and 21, which is humanized, wherein said mature light chain variable region is fused to a light chain constant region and said heavy chain mature variable region is fused to a heavy chain constant region. 23. The antibody of claim 22, which is humanized, wherein said heavy chain constant region is a mutant form of a natural human heavy chain constant region that has reduced binding to the Fcy receptor compared to the natural human heavy chain constant region. 24. An antibody according to claim 22 or 23 that is humanized, wherein said heavy chain constant region is of the IgG1 isotype. - 68 041230 25. An antibody according to claim 22, which contains at least one mutation in the constant region, in which the specified mutation reduces the fixation or activation of complement by the constant region, where the mutation is in one or more of positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331 according to EU numbering. 26. An antibody according to any one of claims 1 to 19, characterized in that said antibody belongs to the human IgG2 or IgG4 isotypes. 27. A pharmaceutical composition comprising an antibody as defined in any one of claims 1-19, 21-26, or an antigen-binding fragment as defined in claim 20, and a pharmaceutically acceptable carrier. 28. A nucleic acid encoding an antibody heavy chain as defined in any one of claims 1-19, 21-26, or an antigen-binding fragment as defined in claim 20. 29. A nucleic acid encoding an antibody light chain as defined in any one of claims 1-19, 21-26, or an antigen-binding fragment as defined in claim 20. 30. A nucleic acid encoding a heavy chain and a light chain of an antibody as defined in any one of claims 1-19, 21-26, or an antigen-binding fragment as defined in claim 20. 31. A recombinant expression vector containing a nucleic acid according to any one of claims 28-30. 32. Host cell transformed with the recombinant expression vector of claim 31. 33. A method for humanizing a mouse antibody according to claim 3, said method comprising (a) selecting one or more acceptor antibody sequences; (b) identifying amino acid residues of the mouse antibody to be retained; (c) synthesizing a nucleic acid encoding a humanized heavy chain containing a mouse antibody heavy chain CDR and a nucleic acid encoding a humanized light chain containing a mouse antibody light chain CDR; and (d) expressing said nucleic acids in a host cell to produce a humanized antibody, said mouse antibody comprising a mature heavy chain variable region as set forth in SEQ ID NO: 7 and a mature light chain variable region as set forth in SEQ ID NO. : eleven. 34. A method for producing a humanized, chimeric, or veneered antibody according to claim 3, said method comprising (a) culturing cells transformed with nucleic acids encoding heavy and light chains of an antibody such that said cells secrete an antibody; and (b) purifying said antibody from the cell culture medium. 35. A method for producing a cell line producing a humanized, chimeric or veneered antibody according to claim 3, the method comprising (a) introducing a vector encoding the heavy and light chains of said antibody and a selectable marker into cells; (b) propagating said cells under conditions to select cells that contain an increased number of copies of the vector; (c) isolating individual cells from selected cells; and (d) creating a cell line cloned from a single cell selected based on antibody yield. 36. A method of inhibiting or reducing tau protein aggregation in a subject who suffers from tau-mediated amyloidosis or is at risk of developing tau-mediated amyloidosis, said method comprising administering to said subject an antibody as defined in any one of claims 1-19 , 21-26, or an antigen-binding fragment according to claim 20, and thereby inhibiting or reducing tau protein aggregation in said subject. 37. A method of treating or preventing a tau protein-induced disease in a subject, said method comprising administering an antibody as defined in any one of claims 1-19, 21-26, or an antigen-binding fragment of claim 20, and thereby treating or performing prevention of this disease. 38. The method of claim 37, wherein said tau protein-induced disease is Alzheimer's disease, Down's syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia or boxer's dementia, Pick's disease, Niemann-Pick's disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophilic granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/parkinsonism-guam dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, Alzheimer's disease variant with Lewy bodies (LBVAD) or progressive supranuclear palsy (PNP). 39. A method for detecting tau protein deposits in a subject suffering from or at risk of developing a disease associated with tau protein aggregation or accumulation, said method comprising administering to said subject an antibody as defined in any of claims 1-19, 21- 26, or an antigen-binding fragment according to claim 20 and detecting an antibody bound to the tau protein in said subject. -