EA047155B1 - ANTIBODIES RECOGNIZING TAU - Google Patents

Description

Tau is a well-known human protein that can exist in phosphorylated forms (see, for example, Goedert, Proc. Natl. Acad. Sci. U.S.A. 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). , especially in the central nervous system. Total tau (t-tau, i.e. phosphorylated and non-phosphorylated forms) and phospho-tau (p-tau, i.e. phosphorylated tau) are released by the brain in response to neuronal damage and neurodegeneration, and their levels are reported to be elevated in the CSF of patients with Alzheimer's disease compared to the general population (Jack et al., Lancet Neurol 9: 119-28 (2010)).

Tau is a major component of neurofibrillary tangles, which, along with plaques, are a hallmark of Alzheimer's disease. Tangles are abnormal fibrils with a diameter of 10 nm, arranged in pairs in the form of a spiral with a regular periodicity of 80 nm. Tau in neurofibrillary tangles is abnormally phosphorylated (hyperphosphorylated) by phosphate groups attached to specific sites on the molecule. In Alzheimer's disease, severe damage to neurofibrillary tangles is observed in neurons of layer II of the entorhinal cortex, CA1 and subicular areas of the hippocampus, the amygdala, and deeper layers (layers III, V and superficial layer VI) of the neocortex. Hyperphosphorylated tau has also been reported to disrupt microtubule assembly, which may contribute to neural network disruption.

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

Summary of the invention

In one aspect, the invention provides an isolated monoclonal antibody that competes with the 9F5 antibody for binding to human tau. In some such antibodies, the heavy chain CDR-H3 has an amino acid sequence comprising SEQ ID NO: 10. In some such antibodies, the heavy chain CDR-H1 has an amino acid sequence comprising SEQ ID NO: 8. In some such antibodies, the light chain CDR-L1 , CDR-L2 and CDR-L3 have amino acid sequences comprising SEQ ID NO: 12, 13 and 14, respectively). In some such antibodies, the heavy chain CDR-H1 has the amino acid sequence comprising SEQ ID NO: 8. Some such antibodies bind to the same epitope on human tau as 9F5.

Some such antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody 9F5, where 9F5 is a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region having the amino acid sequence, comprising SEQ ID NO: 11.

In some such antibodies, the three heavy chain CDRs CDR-H1, CDR-H2 and CDR-H3 correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 8, 9 and 10, respectively), except that the H28 position may be occupied N or T, position H51 can be occupied by I or V, position H54 can be occupied by N or D, and position H56 can be occupied by D or E, and the three light chain CDRs CDR-L1, CDR-L2 and CDR-L3 correspond to the composite definition according to Kabat/Chothia (SEQ ID NO: 12, 13 and 14, respectively), except that position L27b is occupied by L, D, T or Q, position L27c is occupied by L, D, G, S, E, T, N, A, P or I, position L30 can be occupied by I, Y, E, K, G or Q, position L31 can be occupied by T, N or G, position L33 occupied by L N, T, S, R or G, position L51 can be occupied by M, G, E, D, K or I, position L54 can be occupied by L, R, G or T, position L89 occupied by A or G, position L92 occupied by L, D, E, G, Q, T or I, and position L93 is occupied by E or G.

In some antibodies, CDR-H1 has an amino acid sequence including SEQ ID NO: 50. In some antibodies, CDR-H2 has an amino acid sequence including SEQ ID NO: 51. In some antibodies, CDR-H2 has an amino acid sequence including SEQ ID NO: 52 In some antibodies, CDR-L1 has an amino acid sequence including any of SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 172-193. In some antibodies, CDR-L2 has an amino acid sequence including any of SEQ ID NO: 55 and SEQ ID NO: 194205. In some antibodies, CDR-L3 has an amino acid sequence including any of SEQ ID NO: 206-213. In some antibodies, CDR-H1 has an amino acid sequence

- 1 047155 comprising SEQ ID NO: 50, and CDR-H2 has the amino acid sequence comprising SEQ ID NO: 51. In some antibodies, CDR-L1 has the amino acid sequence comprising SEQ ID NO: 53, and CDR-L2 has the amino acid sequence, comprising SEQ ID NO: 55. In some antibodies, CDR-L1 has an amino acid sequence comprising SEQ ID NO: 54 and CDR-L2 has an amino acid sequence comprising SEQ ID NO: 55.

Some antibodies are 9F5 or a chimeric, venous, or humanized form of it. In some antibodies, the heavy chain variable region has >85% identity with the human sequence. In some antibodies, the light chain variable region has >85% identity with the human sequence. In some antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. Some antibodies are humanized antibodies.

Some antibodies are a humanized or chimeric antibody 9F5 that specifically binds to human tau, where 9F5 is a murine antibody characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11. Some antibodies contain a humanized mature heavy chain variable region containing three 9F5 heavy chain CDRs, and a humanized mature light chain variable region containing three 9F5 light chain CDRs. In some antibodies, the CDRs correspond to a definition selected from the group of Kabat, Chothia, the composite definition of Kabat/Chothia, AbM and Contact.

In some antibodies, the humanized mature heavy chain variable region contains three 9F5 heavy chain CDRs as defined by the Kabat/Chothia composite definition (SEQ ID NO: 8-10), and the humanized mature light chain variable region contains three 9F5 light chain CDRs as defined by the Kabat composite definition /Chothia (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Kabat heavy chain 9F5 CDRs (SEQ ID NO: 40, SEQ ID NO: 9 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 9F5 light chain CDRs by Kabat (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Chothia heavy chain 9F5 CDRs (SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 9F5 light chain CDRs by Chothia (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three AbM heavy chain 9F5 CDRs (SEQ ID NO: 8, SEQ ID NO: 43 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 9F5 light chain CDRs by AbM (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Contact 9F5 heavy chain CDRs (SEQ ID NOs: 44-46), and the humanized mature light chain variable region contains three Contact 9F5 light chain CDRs (SEQ ID NOs: 47-49 ).

For example, the antibody may be a humanized antibody, a venated antibody, or a chimeric antibody.

Some such antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to either of SEQ ID NOs: 15-22 and SEQ ID NOs: 109-129, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any of SEQ ID NO: 23-29, SEQ ID NO: 61-108 and SEQ ID NO: 130-171.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by E, H17 is occupied by T, H20 is occupied by I, H69 is occupied by M, H75 is occupied by T, H93 is occupied by T, H94 is occupied by T, and H109 is occupied by V. In some antibodies, positions H1, H17, H20, H69, H75, H94 and H109 are occupied by E, T, I, M, T, T, T and V, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H66 is occupied by R, and H81 is occupied by E. In some antibodies, positions H66 and H81 are occupied by R and E, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H23 is occupied by I, and H83 is occupied by R. In some antibodies, positions H23 and H83 are occupied by K and R, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H43 is occupied by K, H51 is occupied by V, H76 is occupied by D, M80 is occupied by M, and H108 is occupied by L. In some antibodies, positions H43, H51, H76, H80 and H108 is occupied by K, V, D, M and L, respectively.

In some antibodies, the H28 position in the VH region is occupied by T.

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

In some antibodies, the H40 position in the VH region is occupied by A.

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In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H5 is occupied by V, H11 is occupied by V, H12 is occupied by K, H38 is occupied by R, and H42 is occupied by G. In some antibodies, positions H5, H11, H12, H38, and H42 occupied by V, V, K, R and G respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H12 is occupied by V or K, H17 is occupied by S or T, H20 is occupied by L or I, H23 occupied by T or K, H28 occupied by N or T, H38 occupied by K, R or Q, H40 occupied by R or A, H42 occupied by E or G, H43 occupied by Q or K, H48 occupied by I or M, H51 occupied by I or V, H54 occupied by N or D, H56 occupied by D or E, H66 occupied by K or R, H69 occupied by I or M, H75 occupied by S or T, H76 occupied by N or D, H79 occupied by Y, Q, D, N or G , H80 occupied by L, M, P, D, G or E, H81 occupied by Q or E, H82 occupied by L, P, K, R, E or N, H82a occupied by S or G, H82c occupied by L, G, D or S , H83 occupied by T or R, H93 occupied by A or T, H94 occupied by S or T, H108 occupied by T or L, H109 occupied by L or V.

In some antibodies, positions H1, H17, H20, H69, H75, H93, H94 and H109 in the VH region are occupied by E, T, I, M, T, T, T and V, respectively. In some antibodies, positions H1, H17, H20, H66, H69, H75, H81, H93, H94 and H109 in the VH region are occupied by E, T, I, R, M, T, E, T, T and V, respectively. In some antibodies, positions H1, H17, H20, H23, H28, H66, H69, H75, H81, H83, H93, H94 and H109 in the VH region are occupied by E, T, I, K, T, R, M, T, E , R, T, T and V respectively. In some antibodies, positions H1, H17, H20, H23, H28, H43, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, T , I, K, T, K, V, D, E, R, M, T, D, M, E, R, T, T, L and V respectively. In some antibodies, positions H1, H17, H20, H23, H28, H40, H43, H48, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region occupied by E, T, I, K, T, A, K, M, V, D, E, R, M, T, D, M, E, R, T, T, L and V respectively. In some antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H40, H42, H43, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH area are occupied by E, V, V, K, T, I, K, R, A, G, K, V, D, E, R, M, T, D, M, E, R, T , T, L and V respectively. In some antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H40, H42, H43, H51, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in areas VH are occupied by E, V, V, K, T, I, K, R, A, G, K, V, R, M, T, D, M, E, R, T, T, L and V, respectively.

In some antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, V , V, K, T, I, K, Q, G, K, R, M, T, M, E, R, T, T, L and V respectively. In some antibodies, the heavy chain variable region contains the amino acid sequence SEQ ID NO: 127.

In some antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E, V , V, K, T, I, K, K, E, K, R, M, T, M, E, R, T, T, L and V, respectively. In some antibodies, the heavy chain variable region contains the amino acid sequence SEQ ID NO: 128.

In some antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H82c, H83, H93, H94, H108 and H109 in the VH region are occupied by E , V, V, K, T, I, K, K, E, K, R, M, T, M, E, G, R, T, T, L and V respectively.

In some antibodies, the H80 position in the VH region is occupied by P. In some antibodies, the H80 position in the VH region is occupied by D. In some antibodies, the H82c position in the VH region is occupied by G. In some antibodies, the H82c position in the VH region is occupied by D. In some antibodies, the H82 position in in the VH region is occupied by P. In some antibodies, the H80 position in the VH region is occupied by G. In some antibodies, the H82 position in the VH region is occupied by K. In some antibodies, the H82 position in the VH region is occupied by R. In some antibodies, the H82 position in the VH region is occupied by E. B In some antibodies, the position of H82 in the VH region is occupied by N.

In some antibodies, the H79 position in the VH region is occupied by D. In some antibodies, the H79 position in the VH region is occupied by N. In some antibodies, the H79 position in the VH region is occupied by G. In some antibodies, the H80 position in the VH region is occupied by E. In some antibodies, the H80 position in in the VH region is occupied by a G. In some antibodies, the H82c position in the VH region is occupied by an S. In some antibodies, the H79 position in the VH region is occupied by a Q. In some antibodies, the H82a position in the VH region is occupied by a G.

In some antibodies, at least one of the following positions in the VL region is occupied by the indicated amino acid: L7 is occupied by S, L8 is occupied by P, L15 is occupied by P, and L100 is occupied by Q. In some antibodies, positions L7, L8, L15 and L100 are occupied by S, P, P and Q respectively.

In some antibodies, the L66 position in the VL region is occupied by a G. In some antibodies, the L64 position in the VL region is occupied by an S.

In some antibodies, position L17 in the VL region is occupied by E. In some antibodies, at least one of the following positions in the VL region is occupied by the indicated amino acid: L11 is occupied by L, L51 is occupied by G, and L54 is occupied by R.

In some antibodies, positions L11, L51 and L54 are occupied by L, G and R, respectively. In some antibodies, the L30 position in the VL region is occupied by Y.

In some antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L3 is occupied by V or Q, L7 is occupied by A or S, L8 is occupied by A or P, L9 is occupied by F or L, L11 is occupied by N or L, L15 is occupied by L or P, L17 occupied by T or E, L18 occupied by S or P, L27b occupied by L, D, T or Q,

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L27c occupied by L, D, G, S, E, T, N, A, P or I, L30 occupied by I, Y, E, K, G, L31 occupied by T, N or G, L33 occupied by L, N, T, S, R or G, L37 occupied by L, Q, G or I, L39 occupied by R or K, L51 occupied by M, G, E, D, K or I, L54 occupied by R, G or T, L60 occupied by N or D, L64 occupied by G or S, L66 occupied by E or G, L73 occupied by L, P or G, L74 occupied by R or K, L75 occupied by I, D, P, Q or G, L76 occupied by S, P or G, L77 occupied by R or D, L78 occupied by V, R, D, E, P, K, G or Q, L85 occupied by V or G, L86 occupied by Y or T, L89 occupied by A or G, L92 occupied by L, D, E, G, Q, T or I, L93 occupied by E or G, L100 occupied by G or Q.

In some antibodies, positions L64 and L66 in the VL region are occupied by S and G, respectively. In some antibodies, positions L7, L8, L15, L64, L66 and L100 in the VL region are occupied by S, P, P, S, G and Q, respectively. In some antibodies, positions L7, L8, L15, L17, L66 and L100 in the VL region are occupied by S, P, P, E, G and Q, respectively. In some antibodies, positions L7, L8, L11, L15, L17, L51, L54, L66 and L100 in the VL region are occupied by S, P, L, P, E, G, R, G and Q, respectively.

In some antibodies, the light chain variable region contains any of the amino acid sequences of SEQ ID NO: 133, 135-137, 142-144, 149, 158, 159 and 168. In some antibodies, the light chain variable region contains the amino acid sequence of SEQ ID NO: 133. In some antibodies, the light chain variable region contains the amino acid sequence of SEQ ID NO: 137. In some antibodies, the light chain variable region contains the amino acid sequence of SEQ ID NO: 149. In some antibodies, the light chain variable region contains the amino acid sequence of SEQ ID NO: 159.

In some antibodies, positions L7, L8, L11, L15, L17, L30, L51, L54, L66 and L100 in the VL region are occupied by S, P, L, P, E, Y, G, R, G and Q, respectively. In some antibodies, positions L7, L8, L11, L15, L17, L30, L51, L54 and L100 in the VL region are occupied by S, P, L, P, E, Y, G, R and Q, respectively. In some antibodies, positions L7, L8, L9, L11, L15, L17, L18, L31, L39, L51, L54, L60, L66, L74 and L100 in the VL region are occupied by S, P, L, L, P, E, P , N, K, G, R, D, G, K and Q respectively.

In some antibodies, positions L7, L8, L11, L15, L17, L39, L60, L64, L66, L74 and L100 in the VL region are occupied by S, P, L, P, E, K, N, S, G, K and Q respectively. In some antibodies, position L3 in the VL region is occupied by Q. In some antibodies, position L27c in the VL region is occupied by D, G, I, L or S, position L37 in the VL region is occupied by G, I, L or Q, position L51 in the VL region is occupied E, G, I, K or M, position L54 in the VL region is occupied by G, L, R or T, and position L92 in the VL region is occupied by G, I or L. In some antibodies, position L27c in the VL region is occupied by D or S, VL region position L37 is occupied by G, L or Q, VL region position L51 is occupied by G or K, VL region position L54 is occupied by R, and VL region position L92 is occupied by I.

In some antibodies, position L27c in the VL region is occupied by D, position L37 in the VL region is occupied by G, and position L51 in the VL region is occupied by G. In some antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127, and the light chain variable region chain has an amino acid sequence comprising SEQ ID NO: 149.

In some antibodies, position L27c in the VL region is occupied by D, position L37 in the VL region is occupied by Q, and position L51 in the VL region is occupied by G. In some antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127, and the light chain variable region chain has an amino acid sequence comprising SEQ ID NO: 137.

In some antibodies, position L27c in the VL region is occupied by S, position L37 in the VL region is occupied by L, and position L51 in the VL region is occupied by G. In some antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127, and the light chain variable region chain has an amino acid sequence comprising SEQ ID NO: 159.

In some antibodies, position L27c in the VL region is occupied by D, position L37 in the VL region is occupied by Q, and position L51 in the VL region is occupied by K. In some antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127, and the light chain variable region chain has an amino acid sequence comprising SEQ ID NO: 138.

In some antibodies, position L27c in the VL region is occupied by S, position L37 in the VL region is occupied by Q, and position L51 in the VL region is occupied by G. In some antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127, and the light chain variable region chain has an amino acid sequence comprising SEQ ID NO: 133.

In some antibodies, positions L7, L8, L11, L15, L17, L39, L60, L64, L66, L74 and L100 in the VL region are occupied by S, P, L, P, E, K, D, S, G, K and Q respectively. In some antibodies, position L3 in the VL region is occupied by Q. In some antibodies, position L27c in the VL region is occupied by G or S, position L37 in the VL region is occupied by G, I, or Q, position L51 in the VL region is occupied by G, I, or K, position L54 in the VL region is occupied by G or R, and position L92 in the VL region is occupied by G, I or L.

In some antibodies, position L27c in the VL region is occupied by G, position L37 in the VL region is occupied by G, position L51 in the VL region is occupied by G, and position L54 in the VL region is occupied by R. In some antibodies, position L92 in the VL region is occupied by I. In some antibodies the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 129, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 168.

In some antibodies, the position L51 in the VL region is occupied by E. In some antibodies, the position

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L51 in the VL region is occupied by D. In some antibodies, the L27c position in the VL region is occupied by D. In some antibodies, the L27c position in the VL region is occupied by G. In some antibodies, the L27c position in the VL region is occupied by S. In some antibodies, the L27c position in the VL region is occupied by E In some antibodies, the L30 position in the VL region is occupied by E. In some antibodies, the L30 position in the VL region is occupied by K. In some antibodies, the L27c position in the VL region is occupied by T.

In some antibodies, position L27c in the VL region is occupied by N. In some antibodies, position L27b in the VL region is occupied by D. In some antibodies, position L30 in the VL region is occupied by G. In some antibodies, the proposed position L33 in the VL region is occupied by N. In some antibodies, position L27c in the VL region is occupied by A. In some antibodies, the L33 position in the VL region is occupied by T. In some antibodies, the L33 position in the VL region is occupied by S. In some antibodies, the L33 position in the VL region is occupied by R. In some antibodies, the L30 position in the VL region is occupied by Q. In some antibodies, the L27b position in the VL region is occupied by T.

In some antibodies, position L31 in the VL region is occupied by G. In some antibodies, position L27b in the VL region is occupied by Q. In some antibodies, position L33 in the VL region is occupied by G. In some antibodies, position L27c in the VL region is occupied by P. In some antibodies, position L78 in the VL region is occupied by VL is occupied by R. In some antibodies, position L75 in the VL region is occupied by D. In some antibodies, position L78 in the VL region is occupied by D. In some antibodies, position L78 in the VL region is occupied by E. In some antibodies, position L78 in the VL region is occupied by P. In some In antibodies, position L78 in the VL region is occupied by K.

In some antibodies, position L77 in the VL region is occupied by D. In some antibodies, position L78 in the VL region is occupied by G. In some antibodies, position L76 in the VL region is occupied by P. In some antibodies, position L75 in the VL region is occupied by P. In some antibodies, the proposed position is L75 in the VL region is occupied by Q. In some antibodies, position L75 in the VL region is occupied by G. In some antibodies, position L73 in the VL region is occupied by P. In some antibodies, position L73 in the VL region is occupied by G. In some antibodies, position L78 in the VL region is occupied by Q. In some antibodies, position L76 in the VL region is occupied by G.

In some antibodies, position L92 in the VL region is occupied by D. In some antibodies, position L86 in the VL region is occupied by T. In some antibodies, position L92 in the VL region is occupied by E. In some antibodies, position L92 in the VL region is occupied by G. In some antibodies, position L92 in in the VL region is occupied by a Q. In some antibodies, the position L93 in the VL region is occupied by a G. In some antibodies, the proposed position L85 in the VL region is occupied by a G. In some antibodies, the position L92 in the VL region is occupied by a T. In some antibodies, the position L89 in the VL region is occupied by a G.

In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, G and I, respectively. In some antibodies, positions L3Q, L27c, L37, L51 L54 and L92 in the VL region are occupied by Q, S, Q, G, R and I. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q , S, Q, G, T and I respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, R and G, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, G, R and I, respectively.

In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, Q, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, Q, K, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, K, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q. G, Q, K, G and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, K, G and I, respectively.

In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, R and G, respectively. In some antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, G, G, G and R, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, T and I. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied Q, G, G, G, T and G respectively.

In some antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, G, G, G and T, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, G, G, T and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, G, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, I, I, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, I, G and I, respectively.

In some antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, S, Q, I and G, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, E, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, E, G and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92L in the VL region are occupied by Q, G, I, E, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, I, E, R and G, respectively.

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In some antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, I, I, E and R, respectively. In some antibodies, positions L3, L37, L51, L54 and L92 in the VL region are occupied by Q, Q, G, R and I, respectively. In some antibodies, positions L3, L27c, L51, L54 and L92 in the VL region are occupied by Q, S, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L54 and L92 in the VL region are occupied by Q, S, Q, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51 and L92 in the VL region are occupied by Q, S, Q, G and I, respectively.

In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, G and I. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied Q, S, Q, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, R and G, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, K, R and I, respectively.

In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, K, G and I, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, R and I, respectively. In some antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, G, G, G and R, respectively. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, I, I, R and I. In some antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied Q, S, Q, I, G and I respectively.

Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 95% identical to either of SEQ ID NOs: 15-22 and SEQ ID NOs: 109-129, and a mature light chain variable region having an amino acid sequence of at least 95% identical to any of SEQ ID NO: 23-29, SEQ ID NO: 61-108 and SEQ ID NO: 130-171. Some antibodies contain a mature heavy chain variable region having an amino acid sequence that is at least 98% identical to either of SEQ ID NOs: 15-22 and SEQ ID NOs: 109-129, and a mature light chain variable region having an amino acid sequence of at least at least 98% identical to any of SEQ ID NO: 23-29, SEQ ID NO: 61-108 and SEQ ID NO: 130-171. In some antibodies, the mature heavy chain variable region has any amino acid sequence of SEQ ID NO: 15-22 and SEQ ID NO: 109-129, and the mature light chain variable region has any amino acid sequence of SEQ ID NO: 23-29, SEQ ID NO: 61-108 and SEQ ID NO: 130-171.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 15, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 15, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 15, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 15 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 15 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 15. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 15, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 28. has the amino acid sequence of SEQ ID NO: 15, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 16, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 16, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 16, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 16 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 16 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 16 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 16. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 16, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 16, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

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In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 17, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 17, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 17, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 17 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 17 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 17 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 17, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 28. has the amino acid sequence of SEQ ID NO: 17, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 18 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 18 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 18. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 18. has the amino acid sequence of SEQ ID NO: 18, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 19, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 19, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 19, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 19 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 19 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 19 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 19, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 19, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 20, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 20, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 20, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 20 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 20 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 20. chain has the amino acid sequence of SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 20, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 28. In some antibodies, the mature variable region

- 7 047155 the yellow chain has the amino acid sequence of SEQ ID NO: 20, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 21, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 21, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 21, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence SEQ ID NO: 21 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 21 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 21. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 21, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 21. has the amino acid sequence of SEQ ID NO: 21, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 22, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 23. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 22, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 24. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 22, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. In some antibodies, the mature light chain variable region has the amino acid sequence SEQ ID NO: 25. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 22 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 26. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 22 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 22. chain has the amino acid sequence SEQ ID NO: 27. In some antibodies, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 22, and the mature light chain variable region has the amino acid sequence SEQ ID NO: 28. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 22, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 29.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 149. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 142. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 159. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 159. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 148.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 137. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 145. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 136. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 136. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 138.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 158. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 143. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 144. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 144. the heavy chain variable region has the amino acid sequence SEQ

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ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 133.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 160. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 161. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 139. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 139. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 128, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 168.

Some such antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody YC 12, where YC 12 is a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 11.

In some such antibodies, three heavy chain CDRs CDR-H1, CDR-H2 and CDR-H3 correspond to the Kabat/Chothia composite definition (SEQ ID NO: 8, 9 and 10, respectively), and three light chain CDRs CDR-L1, CDR -L2 and CDR-L3 correspond to the Kabat/Chothia composite definition (SEQ ID NO: 12, 13 and 14, respectively).

Some antibodies are 10C12 or a chimeric, venous, or humanized form of it. In some antibodies, the heavy chain variable region has >85% identity with the human sequence. In some antibodies, the light chain variable region has >85% identity with the human sequence. In some antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. Some antibodies are humanized antibodies.

Some antibodies are a humanized or chimeric antibody 10C12 that specifically binds to human tau, where 10C12 is a murine antibody characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11. Some antibodies contain a humanized mature heavy chain variable region comprising three 10C12 heavy chain CDRs, and a humanized mature light chain variable region comprising three 10C12 light chain CDRs. In some antibodies, the CDRs correspond to a definition selected from the group of Kabat, Chothia, the composite definition of Kabat/Chothia, AbM and Contact.

In some antibodies, the humanized mature heavy chain variable region contains three 10C12 heavy chain CDRs as defined by the Kabat/Chothia composite definition (SEQ ID NO: 8-10), and the humanized mature light chain variable region contains three 10C12 light chain CDRs as defined by the Kabat composite definition /Chothia (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Kabat heavy chain 10C12 CDRs (SEQ ID NO: 40, SEQ ID NO: 9 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 10C12 light chain CDRs by Kabat (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Chothia heavy chain 10C12 CDRs (SeQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 10C12 light chain CDRs by Chothia (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three AbM heavy chain 10C12 CDRs (SEQ ID NO: 8, SEQ ID NO: 43 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 10C12 light chain CDRs by AbM (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Contact heavy chain 10C12 CDRs (SEQ ID NOs: 44-46), and the humanized mature light chain variable region contains three Contact 10C12 light chain CDRs (SEQ ID NOs: 47-49 ).

For example, the antibody may be a humanized antibody, a venated antibody, or a chimeric antibody.

Some such antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to any of SEQ ID NOs: 214-215, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any from SEQ ID NO: 216-217.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, and H94 is occupied by T. In some antibodies, positions H24, H48, H67, H69, H93 and H94 are occupied by A, I, A, M, T and T,

- 9 047155 respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, H94 is occupied by T.

In some antibodies, positions H24, H48, H67, H69, H93 and H94 are occupied by A, I, A, M, T and T, respectively. In some antibodies, positions H1, H24, H48, H67, H69, H93 and H94 are occupied by E, A, I, A, M, T and T, respectively.

In some antibodies, the L64 position in the VL region is occupied by S.

In some antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L64 is occupied by S, L104 is occupied by V or L. In some antibodies, position L64 is occupied by S. In some antibodies, positions L64 and L104 in the VL region are occupied by S and L, respectively .

Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 214-215, and a mature light chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 216-217. Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 214-215, and a mature light chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 216217. In some antibodies, the mature heavy chain variable region has any of the amino acid sequences of SEQ ID NOs: 214-215 and the mature light chain variable region has any of the amino acid sequences of SEQ ID NOs: 216-217.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 214, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 216. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 214, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 217. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 215, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 216. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 216. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 215, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 217.

Some such antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody 12C4, where 12C4 is a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 219, and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 11.

In some of these antibodies, three heavy chain CDRs CDR-H1, CDR-H2 and CDR-H3 correspond to the Kabat/Chothia composite definition (SEQ ID NO: 8, 220 and 10, respectively), and three light chain CDRs CDR-L1, CDR- L2 and CDR-L3 correspond to the Kabat/Chothia composite definition (SEQ ID NO: 12, 13 and 14, respectively).

Some antibodies are 12C4 or its chimeric, venous or humanized form. In some antibodies, the heavy chain variable region has >85% identity with the human sequence. In some antibodies, the light chain variable region has >85% identity with the human sequence. In some antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. Some antibodies are humanized antibodies.

Some antibodies are a humanized or chimeric antibody 12C4 that specifically binds to human tau, where 12C4 is a murine antibody characterized by a mature heavy chain variable region of SEQ ID NO: 219 and a mature light chain variable region of SEQ ID NO: 11. Some antibodies contain a humanized mature heavy chain variable region containing three 12C4 heavy chain CDRs, and a humanized mature light chain variable region containing three 12C4 light chain CDRs. In some antibodies, the CDRs correspond to a definition selected from the group of Kabat, Chothia, the composite definition of Kabat/Chothia, AbM and Contact.

In some antibodies, the humanized mature heavy chain variable region contains three 12C4 heavy chain CDRs according to the Kabat/Chothia composite definition (SEQ ID NOs: 8, 220 and 10), and the humanized mature light chain variable region contains three 12C4 light chain CDRs according to the composite definition by Kabat/Chothia (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Kabat heavy chain 12C4 CDRs (SEQ ID NO: 40, SEQ ID NO: 220 and SEQ ID NO: 10), and the humanized mature heavy chain variable region

- 10 047155 light chain layer contains three Kabat light chain 12C4 CDRs (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Chothia heavy chain 12C4 CDRs (SEQ ID No: 41, SeQ ID NO: 42 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 12C4 light chain CDRs by Chothia (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three AbM heavy chain 12C4 CDRs (SEQ ID NO: 8, SEQ ID NO: 257 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 12C4 light chain CDRs by AbM (SEQ ID NO: 12-14). In some antibodies, the humanized mature heavy chain variable region contains three Contact heavy chain 12C4 CDRs (SEQ ID NOs: 44, 258 and 46), and the humanized mature light chain variable region contains three Contact 12C4 light chain CDRs (SEQ ID NO: 47 -49).

For example, the antibody may be a humanized antibody, a venated antibody, or a chimeric antibody.

Some such antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 221-222, and a humanized mature light chain variable region having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 221-222. identical to any of SEQ ID NO: 223-224.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H48 is occupied by M or I, H93 is occupied by A or T, H94 is occupied by R or T. In some antibodies, positions H1, H48, H93 and H94 in the VH region are occupied by E, I, T and T, respectively.

In some antibodies, positions in the VL region are occupied by the indicated amino acid: L64 is occupied by G or S, L104 is occupied by V or L. In some antibodies, positions L64 and L104 in the VL region are occupied by S and L, respectively.

Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 221-222, and a mature light chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 223-224. Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 221-222, and a mature light chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 223224. In some antibodies, the mature heavy chain variable region has any of the amino acid sequences of SEQ ID NOs: 221-222 and the mature light chain variable region has any of the amino acid sequences of SEQ ID NOs: 223-224.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 221, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 223. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 221, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 224. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 222, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 223. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 223. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 222, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 224.

Some such antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody 17C12, where 17C12 is a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 225, and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 228.

In some of these antibodies, three heavy chain CDRs CDR-H1, CDR-H2 and CDR-H3 correspond to the Kabat/Chothia composite definition (SEQ ID NO: 226, 227 and 10, respectively), and three light chain CDRs CDR-L1, CDR- L2 and CDR-L3 correspond to the Kabat/Chothia composite definition (SEQ ID NO: 229, 230 and 231, respectively).

Some antibodies are 17C12 or its chimeric, venous or humanized form. In some antibodies, the heavy chain variable region has >85% identity with the human sequence. In some antibodies, the light chain variable region has >85% identity with the human sequence. In some antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. Some antibodies are humanized antibodies.

Some antibodies are a humanized or chimeric antibody 17C12 that specifically binds to human tau, where 17C12 is a murine antibody characterized by the mature heavy chain variable region of SEQ ID NO: 225 and the mature heavy chain variable region

- 11 047155 light chain region SEQ ID NO: 228. Some antibodies contain a humanized mature heavy chain variable region containing three 17C12 heavy chain CDRs and a humanized mature light chain variable region containing three 17C12 light chain CDRs. In some antibodies, the CDRs correspond to a definition selected from the group of Kabat, Chothia, the composite definition of Kabat/Chothia, AbM and Contact.

In some antibodies, the humanized mature heavy chain variable region contains three 17C12 heavy chain CDRs according to the Kabat/Chothia composite definition (SEQ ID NOs: 226, 227 and 10), and the humanized mature light chain variable region contains three 17C12 light chain CDRs according to the composite definition by Kabat/Chothia (SEQ ID NO: 229-231). In some antibodies, the humanized mature heavy chain variable region contains three Kabat heavy chain 17C12 CDRs (SEQ ID NO: 40, SEQ ID NO: 227 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 17C12 light chain CDRs by Kabat (SEQ ID NO: 229-231). In some antibodies, the humanized mature heavy chain variable region contains three Chothia 17C12 heavy chain CDRs (SEQ ID NO: 259, SEQ ID NO: 42 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 17C12 light chain CDRs by Chothia (SEQ ID NO: 229-231). In some antibodies, the humanized mature heavy chain variable region contains three 17C12 heavy chain CDRs of AbM (SEQ ID NO: 226, SEQ ID NO: 260 and SEQ ID NO: 10), and the humanized mature light chain variable region contains three 17C12 light chain CDRs by AbM (SEQ ID NO: 229-231). In some antibodies, the humanized mature heavy chain variable region contains three Contact heavy chain 17C12 CDRs (SEQ ID NO: 44, SEQ ID NO: 261 and SEQ ID NO: 46), and the humanized mature light chain variable region contains three 17C12 light chain CDRs by Contact (SEQ ID NO: 262-264).

For example, the antibody may be a humanized antibody, a venated antibody, or a chimeric antibody.

Some such antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 232-233, and a humanized mature light chain variable region having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 232-233. identical to any of SEQ ID NO: 234-235.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H2 is occupied by I, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, and H94 is occupied by T. In some antibodies, the positions H2, H24, H48, H67, H69, H93 and H94 are occupied by E, A, I, A, M, T and T, respectively.

In some antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H1 is occupied by Q or E, H2 is occupied by I, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, H94 is occupied by T , H108 is occupied by T or L, H113 is occupied by R or S. In some antibodies, positions H2, H24, H48, H67, H69, H93 and H94 in the VH region are occupied by E, A, I, A, M, T and T, respectively. In some antibodies, positions H1, H2, H24, H48, H67, H69, H93, H94, H108 and H113 in the VH region are occupied by E, I, A, I, A, M, T, T, L and S, respectively.

In some antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is occupied by V, and L36 is occupied by L. In some antibodies, positions L2 and L36 are occupied by V and L, respectively.

In some antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is occupied by V, L36 is occupied by L, L43 is occupied by P or S. In some antibodies, positions L2 and L36 in the VL region are occupied by V and L, respectively. In some antibodies, positions L2, L36 and L43 in the VL region are occupied by V, L and S, respectively.

Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 232-233, and a mature light chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 234-235. Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 232-233, and a mature light chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 234235. In some antibodies, the mature heavy chain variable region has any of the amino acid sequences of SEQ ID NOs: 232-233 and the mature light chain variable region has any of the amino acid sequences of SEQ ID NOs: 234-235.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 232, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 234. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 232, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 235. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 233, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 234. In some

- 12 047155 antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 233, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 235.

Some such antibodies contain three light chain CDRs and three heavy chain CDRs of the monoclonal antibody 14H3, where 14H3 is a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 240, and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 244.

In some of these antibodies, the three heavy chain CDRs CDR-H1, CDR-H2 and CDR-H3 correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 241, 242 and 243, respectively), except that the H35B position may be occupied by G or S, and the three light chain CDRs CDR-L1, CDR-L2 and CDR-L3 correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 245, 246 and 247, respectively). In some antibodies, CDR-H1 has the amino acid sequence comprising SEQ ID NO: 277.

Some antibodies are 14H3 or its chimeric, venous or humanized form. In some antibodies, the heavy chain variable region has >85% identity with the human sequence. In some antibodies, the light chain variable region has >85% identity with the human sequence. In some antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. Some antibodies are humanized antibodies.

Some antibodies are a humanized or chimeric 14H3 antibody that specifically binds to human tau, where 14H3 is a murine antibody characterized by a mature heavy chain variable region of SEQ ID NO: 240 and a mature light chain variable region of SEQ ID NO: 244. Some antibodies contain a humanized mature heavy chain variable region containing three 14H3 heavy chain CDRs, and a humanized mature light chain variable region containing three 14H3 light chain CDRs. In some antibodies, the CDRs correspond to a definition selected from the group Kabat, Chothia, the composite definition of Kabat/Chothia, AbM and Contact.

In certain antibodies, wherein the humanized mature heavy chain variable region contains three 14H3 heavy chain CDRs as defined by the Kabat/Chothia composite definition (SEQ ID NO: 241243) and the humanized mature light chain variable region contains three 14H3 light chain CDRs as defined by the Kabat composite definition /Chothia (SEQ ID NO: 245-247). In some antibodies, the humanized mature heavy chain variable region contains three Kabat heavy chain 14H3 CDRs (SEQ ID NO: 265, SEQ ID NO: 242 and SEQ ID NO: 243), and the humanized mature light chain variable region contains three 14H3 light chain CDRs by Kabat (SEQ ID NO: 245-247). In some antibodies, the humanized mature heavy chain variable region contains three Chothia 14H3 heavy chain CDRs (SEQ ID NO: 266, SEQ ID NO: 267 and SEQ ID NO: 243), and the humanized mature light chain variable region contains three 14H3 light chain CDRs by Chothia (SEQ ID NO: 245247). In some antibodies, the humanized mature heavy chain variable region contains three AbM heavy chain 14H3 CDRs (SEQ ID NO: 241, SEQ ID NO: 268 and SEQ ID NO: 243), and the humanized mature light chain variable region contains three 14H3 light chain CDRs by AbM (SEQ ID NO: 245-247). In some antibodies, the humanized mature heavy chain variable region contains three Contact 14H3 heavy chain CDRs (SEQ ID NOs: 269-271), and the humanized mature light chain variable region contains three Contact 14H3 light chain CDRs (SEQ ID NOs: 272-274 ).

For example, the antibody may be a humanized antibody, a venated antibody, or a chimeric antibody.

Some such antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 248-249 and a humanized mature light chain variable region having an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 248-249. identical to any of SEQ ID NO: 250-251.

In some antibodies, the position of H35B in the VH region is occupied by S.

In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H35B is occupied by S, H108 is occupied by M or L, H113 is occupied by L or S. In some antibodies, the position H35B in the VH region is occupied by S. In some antibodies, the positions H35B, H108 and H113 in the VH region are occupied by S, L and S, respectively.

In some antibodies, at least one of the following positions in the VL region is occupied by the indicated amino acid: L2 is occupied by V, and L87 is occupied by F. In some antibodies, positions L2 and L87 are occupied by V and F, respectively.

In some antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L2 is occupied by V, L7 is occupied by T or S, L37 is occupied by L or Q, L87 is occupied by F, L100 is occupied by G

- 13 047155 or Q, L104 is occupied by V or L. In some antibodies, positions L2 and L87 in the VL region are occupied by V and F, respectively. In some antibodies, positions L2, L7, L37, L87, L100 and L104 in the VL region are occupied by V, S, Q, F, Q and L, respectively.

Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 248-249, and a mature light chain variable region having an amino acid sequence at least 95% identical to any of SEQ ID NO: 250-251. Some antibodies contain a mature heavy chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 248-249, and a mature light chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NO: 250251. In some antibodies, the mature heavy chain variable region has any of the amino acid sequences of SEQ ID NOs: 248-249 and the mature light chain variable region has any of the amino acid sequences of SEQ ID NOs: 250-251.

In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 248, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 250. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 248, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 251. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 249, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 250. In some antibodies, the mature light chain variable region has the amino acid sequence of SEQ ID NO: 250. the heavy chain variable region has the amino acid sequence of SEQ ID NO: 249, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 251.

For example, the antibody may be a chimeric antibody. For example, the antibody may be a venated antibody.

The antibody may be an intact antibody. The antibody may be a binding moiety. In one embodiment, the binding fragment is a single chain antibody, Fab fragment, or Fab'2. The antibody may be a Fab fragment or a single chain Fv. Some antibodies are of the human IgG1 isotype, while others may be of the human IgG2 or IgG4 isotype. Some antibodies have a mature light chain variable region fused to a light chain constant region and a mature heavy chain variable region fused to a heavy chain constant region. The heavy chain constant region of some antibodies is a mutant form of the natural human antibody heavy chain constant region that has reduced binding to the Fcy receptor compared to the natural human antibody heavy chain constant region. In some antibodies, the heavy chain constant region is of the IgG1 isotype.

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

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

In another aspect, the 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. Some nucleic acids have a sequence comprising any of SEQ ID NOs: 38-39.

In another aspect, the invention provides a vector comprising a nucleic acid encoding a mature heavy chain variable region and a mature light chain variable region operably linked to one or more regulatory sequences for causing expression in a mammalian cell of any of the antibodies disclosed herein, a recombinant expression vector containing a nucleic acid, a host cell transformed with a recombinant expression vector, and a host cell transformed with the nucleic acid. Some nucleic acids further encode a heavy chain constant region fused to a mature heavy chain variable region and a light chain constant region fused to a mature light chain variable region. In some vectors, the antibody is a scFv. In some vectors, the antibody is a Fab fragment. In some vectors, one or more regulatory sequences include one or more of a promoter, an enhancer, a ribosome binding site, and a transcription termination signal. In some vectors, the nucleic acid further encodes signal peptides fused to the mature heavy and light chain variable regions. IN

- 14 047155 In some vectors, the nucleic acid is codon optimized for expression in the host cell. In some vectors, one or more regulatory sequences include a eukaryotic promoter. In some vectors, the nucleic acid additionally encodes the gene to be selected.

In yet another aspect, the invention provides methods for expressing an antibody in a mammalian cell, comprising incorporating nucleic acids disclosed herein into the genome of a transgenic animal, thereby resulting in expression of the antibody.

In yet another aspect, the invention provides first and second vectors, respectively comprising nucleic acids encoding a mature heavy chain variable region and a mature light chain variable region, each of which is operably linked to one or more regulatory sequences for effecting expression in a mammalian cell of any of the disclosed herein, antibodies, and a host cell containing nucleic acids. In some first and second vectors, the nucleic acids, respectively, further encode a heavy chain constant region fused to a mature heavy chain variable region and a light chain constant region fused to a mature light chain variable region.

In yet another aspect, the invention provides methods for expressing an antibody in a mammalian cell, comprising incorporating any of the nucleic acids disclosed herein into the genome of a transgenic animal, thereby resulting in expression of the antibody.

In yet another aspect, the invention provides methods for humanizing any non-human antibody described herein, for example, the murine antibody 9F5, wherein 9F5 is characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11, for example , mouse antibody 10C12, wherein 10C12 is characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11; for example, murine antibody 2D11, wherein 2D11 is characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11; for example, mouse antibody 12C4, wherein 12C4 is characterized by a mature heavy chain variable region of SEQ ID NO: 219 and a mature light chain variable region of SEQ ID NO: 11; for example, murine antibody 17C12, wherein 17C12 is characterized by a mature heavy chain variable region of SEQ ID NO: 225 and a mature light chain variable region of SEQ ID NO: 228; for example, the mouse antibody 14H3, wherein 14H3 is characterized by a mature heavy chain variable region of SEQ ID NO: 240 and a mature light chain variable region of SEQ ID NO: 244. Such methods may include selecting one or more acceptor antibodies, determining the amino acid residues of the mouse antibody that are desired save; 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 expressing the nucleic acids in a host cell to produce a humanized antibody.

Also provided are methods for producing antibodies, such as humanized, chimeric or venated antibodies, for example, humanized, chimeric or venated forms of 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. In such methods, cells transformed with nucleic acids encoding the heavy and light chains of an antibody are cultured such 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 the cells a vector encoding the heavy and light chains of the antibody and a selectable marker, expanding the cells under conditions of selecting cells with increased copy number of the vector, isolating single cells from the selected cells, and creating banks of cells cloned from a single cell selected on the basis of antibody yield.

Some cells can be propagated under selective conditions and screened for cell lines that naturally express and secrete at least 100 mg/l/10 ⁶ cells/24 h. Single cells can be isolated from the selected cells. Cells cloned from a single cell can then be placed in a bank. Individual cells can be selected based on desired properties, such as antibody yield. Examples of cell lines are cell lines expressing 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3.

The invention also provides methods for inhibiting or reducing tau aggregation in a subject having or at risk of developing tau-mediated amyloidosis, comprising administering to the subject an antibody disclosed herein in an effective regimen, thereby inhibiting or reducing tau aggregation in the subject. Examples of antibodies include humanized versions of 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3.

Also provided are methods of treating or preventing tau-associated disease in a subject, comprising administering an antibody disclosed herein in an effective regimen, thereby treating or preventing the disease. Examples of such disease are 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 disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar

- 15 047155 ny degeneration, disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP). In some methods, the tau-related disease is Alzheimer's disease. In some methods, the patient is a carrier of ApoE4.

Methods of reducing aberrant tau transmission are also provided, including administering an antibody disclosed herein in an effective regimen, thereby reducing tau transmission.

Methods of inducing tau phagocytosis are also provided, comprising administering an antibody disclosed herein in an effective regimen, thereby inducing tau phagocytosis.

Methods of inhibiting tau aggregation or precipitation are also provided, comprising administering an effective antibody disclosed herein in an effective regimen and thereby inhibiting tau aggregation or precipitation.

Methods of inhibiting the formation of tau tangles are also provided, including administering an antibody disclosed herein in an effective regimen.

The invention also provides a method for detecting tau protein deposits in a subject having or at risk of a disease associated with tau aggregation or deposition, comprising administering to the subject an antibody disclosed herein and detecting the tau associated antibody in the subject. Examples of such disease are 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 disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration , disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP). In some embodiments, the antibody is administered by intravenous injection into a subject. In some embodiments, the antibody is administered directly to the subject's brain by intracranial injection or by drilling a hole into the subject's skull. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, paramagnetic label, or radioactive label. In some embodiments, the radioactive tracer is detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

The invention also provides a method of measuring the effectiveness of a treatment of a subject being treated for a disease associated with tau aggregation or deposition, 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 antibody bound with tau in the subject, administering treatment to the subject, measuring a second level of tau protein deposits in the subject after treatment by administering the antibody to the subject and detecting an antibody associated with tau in the subject, wherein a decrease in the level of tau protein deposits indicates a positive response to treatment.

The invention also provides a method of measuring the effectiveness of a treatment of a subject being treated for a disease associated with tau aggregation or deposition, 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 antibody bound with tau in the subject, administering treatment to the subject, measuring a second level of tau protein deposits in the subject after treatment by administering the antibody to the subject and detecting a second amount of antibody associated with tau in the subject, wherein no change in the level of tau protein deposits or a slight increase in tau protein deposits indicates for a positive response to treatment.

The invention also provides an isolated monoclonal antibody that specifically binds to a peptide consisting of (Q/E)IVYK(S/P) residues (SEQ ID NO: 56). The invention also provides an isolated monoclonal antibody that specifically binds to a peptide consisting of QIVYKP residues (SEQ ID NO: 57). The invention also provides an isolated monoclonal antibody that specifically binds to a peptide consisting of IVYKSP residues (SEQ ID NO: 58). The invention also provides an isolated monoclonal antibody that specifically binds to a peptide consisting of EIVYKS residues (SEQ ID NO: 277).

The invention also provides an isolated monoclonal antibody that specifically binds to a polypeptide of SEQ ID NO: 1 on an epitope comprising at least one residue within residues 307-312 of SEQ ID NO: 1. Some such antibodies bind to an epitope within residues 307-312 312 SEQ ID NO: 1. The invention also provides an isolated monoclonal antibody that specifically binds to the polypeptide SEQ ID NO: 1 at an epitope comprising at least

- 16 047155 at least one residue within residues 391-397 of SEQ ID NO: 1. Some such antibodies bind to an epitope within residues 391-397 of SEQ ID NO: 1. The invention also provides an isolated monoclonal antibody that specifically binds to the polypeptide of SEQ ID NO: 1 on an epitope comprising at least one residue within residues 391-396 of SEQ ID NO: 1. Some such antibodies bind to the epitope within residues 391-396 of SEQ ID NO: 1. The invention also provides an isolated monoclonal antibody that specifically binds to a polypeptide of SEQ ID NO: 1 at an epitope comprising at least one residue within residues 307-312 of SEQ ID NO: 1 and at least one residue within residues 391-397 of SEQ ID NO: 1. B The invention also provides an isolated monoclonal antibody that specifically binds to a polypeptide of SEQ ID NO: 1 on an epitope comprising at least one residue within residues 307312 of SEQ ID NO: 1 and at least one residue within residues 391-396 of SEQ ID NO: : 1.

The invention also provides a method of treating or preventing tau-associated disease in a subject, comprising administering an immunogen comprising a tau peptide containing up to 20 contiguous amino acids of SEQ ID NO: 1, to which antibody 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3, where the peptide causes the formation of antibodies that specifically bind to tau in the subject. In some such methods, the tau peptide consists of 4-7 contiguous amino acids from residues 307-312 of SEQ ID NO: 1 or from residues 391-397 of SEQ ID NO: 1 or from residues 391-396 of SEQ ID NO: 1. In some such methods tau peptide consists of (Q/E)IVYK(S/P) residues (SEQ ID NO: 56). In some such methods, the tau peptide consists of QIVYKP residues (SEQ ID NO: 57). In some such methods, the tau peptide consists of EIVYKSP residues (SEQ ID NO: 58). In some such methods, the tau peptide consists of EIVYKS residues (SEQ ID NO: 277). In some such methods, the tau peptide is attached to a heterologous conjugate molecule.

The invention also provides a method for producing an antibody that specifically binds to an epitope containing (Q/E)IVYK(S/P) (SEQ ID NO: 56), comprising immunizing an animal with tau or a fragment thereof, and screening for antibodies that specifically bind to the epitope. In some such methods, the animal is immunized with human tau, consisting of 383 amino acids (4R0N). In some such methods, human tau contains the P301S mutation. In some such methods, human tau is recombinantly tagged with a His tag at the N-terminus.

In some such methods, screening is performed against 15 amino acid peptides containing QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or any other consensus motif provided by (Q/E )IVYK(S/P) (SEQ ID NO: 56). In some such methods, the peptides contain QIVYKP (SEQ ID NO: 57) or EIVYKSP (SEQ ID NO: 58), or EIVYKS (SEQ ID NO: 277).

In some such methods, an animal is immunized with a tau fragment containing a peptide represented by (Q/E)IVYK(S/P) (SEQ ID NO: 56) linked to a carrier. In some such methods, the peptide is QIVYKP (SEQ ID NO: 57) or EIVYKSP (SEQ ID NO: 58), or EIVYKS (SEQ ID NO: 277).

Brief description of graphic materials

In fig. 1A and 1B depict an alignment of the heavy chain variable regions of the murine antibody 9F5 (SEQ ID NO: 7) and the humanized versions of the 9F5 antibody (hu9F5VHvl, hu9F5VHv2, hu9F5VHv3, hu9F5VHv4, hu9F5VHv5, hu9F5VHv6, hu9F5VHv7 and hu9 F5VHv8) with the human germline heavy chain variable region sequence IGHV1-69-2*01 (SEQ ID NO: 33) with the human acceptor heavy chain variable region sequence AAN16432-VH_huFrwk (AAN16432_H; SEQ ID NO: 31) and with the human acceptor heavy chain variable region sequence 2RCS-VH_huFrwk (2RCSH; SEQ ID NO: 32). hu9F5VHv1 is SEQ ID NO: 15, hu9F5VHv2 is SEQ ID NO: 16, hu9F5VHv3 is SEQ ID NO: 17, hu9F5VHv4 is SEQ ID NO: 18, hu9F5VHv5 is SEQ ID NO: 19, hu9F5VHv6 is SEQ ID NO: 20, hu9F5VHv7 is SEQ ID NO: 21, and hu9F5VHv8 is SEQ ID NO: 22. The mouse 9F5 VH CDRs according to the Kabat/Chothia composite definition are shown in bold.

In fig. 2A and 2B depict an alignment of the heavy chain variable regions of the mouse antibody 9F5 (SEQ ID NO: 11) and humanized versions of the 9F5 antibody (hu9F5VLv1, hu9F5VLv2, hu9F5VLv3, hu9F5VLv4, hu9F5VLv5, hu9F5VLv6 and hu9F5VLv7) with sequential ity of the human germline light chain variable region IGKV2- 28*01 u_IGKJ2*01 (IGKV2-28*01_IGKJ2*01; SEQ ID NO: 37) with human acceptor CAB51297-VL_huFrwk (CAB51297L; SEQ ID NO: 35) and with human acceptor 1911357B-VL_huFRwk (1911357B_L; SEQ ID NO: 36). hu9F5VLv1 is SEQ ID NO: 23, hu9F5VLv2 is SEQ ID NO: 24, hu9F5VLv3 is SEQ ID NO: 25, hu9F5VLv4 is SEQ ID NO: 26, hu9F5VLv5 is SEQ ID NO: 27, hu9F5VLv6 is SEQ ID NO: 28, and hu9F5VLv7 is SEQ ID NO: 29. The mouse 9F5 VL CDRs, as defined by Kabat, are in bold.

In fig. 3 depicts the results of an assay showing that the mouse antibody 9F5 blocks the internalization of tau into neurons.

- 17 047155

In fig. 4A and 4B depict an alignment of the heavy chain variable regions of the murine antibody 9F5 (SEQ ID NO: 7) and humanized versions of the 9F5 antibody (hu9F5VHv1, hu9F5VHv2, hu9F5VHv3, hu9F5VHv4, hu9F5VHv5, hu9F5VHv6, hu9F5VHv7, F5VHv8, hu9F5VHv9 and hu9F5VHv10) with the severe variable region sequence human germline chain IGHV1-692*01 (SEQ ID NO: 33) with the human acceptor heavy chain variable region sequence AAN16432-VH_huFrwk (AAN16432_H; SEQ ID NO: 31) and with the human acceptor heavy chain variable region sequence 2RCS-VH_huFrwk (2RCS_H ; SEQ ID NO: 32). hu9F5VHv1 is SEQ ID NO: 15, hu9F5VHv2 is SEQ ID NO: 16, hu9F5VHv3 is SEQ ID NO: 17, hu9F5VHv4 is SEQ ID NO: 18, hu9F5VHv5 is SEQ ID NO: 19, hu9F5VHv6 is SEQ ID NO: 20, and hu9F5VHv7 is SEQ ID NO: 21, hu9F5VHv8 is SEQ ID NO: 22, hu9F5VHv9 is SEQ ID NO: 127, and hu9F5VHv10 is SEQ ID NO: 128. CDR VH of mouse 9F5 by composite definition by Kabat/Chothia, in bold. Residues identical to the VH residues of mouse 9F5 are indicated by “.”

In fig. 5A and 5B depict an alignment of the heavy chain variable regions of the murine antibody 9F5 (SEQ ID NO: 11) and humanized versions of the 9F5 antibody (hu9F5VLv1, hu9F5VLv2, hu9F5VLv3, hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, 9F5VLv8 and hu9F5VLv9) with the germline light chain variable region sequence human line IGKV2-28*01 u_IGKJ2*01 (IGKV2-28*01_IGKJ2*01; SEQ ID NO: 37) with human acceptor CAB51297-VL_huFrwk (CAB51297_L; SEQ ID NO: 35) and with human acceptor 1911357В-VL_huFRwk (191 1357B_L; SEQ ID NO: 36). hu9F5VLv1 is SEQ ID NO: 23, hu9F5VLv2 is SEQ ID NO: 24, hu9F5VLv3 is SEQ ID NO: 25, hu9F5VLv4 is SEQ ID NO: 26, hu9F5VLv5 is SEQ ID NO: 27, hu9F5VLv6 is SEQ ID NO: 28, hu9F5VLv7 is SEQ ID NO: 2, hu9F5VLv8 is SEQ ID NO: 130, and hu9F5VLv9 is SEQ ID NO: 131. The mouse 9F5 VL CDRs, as defined by Kabat, are in bold. Residues identical to those of mouse 9F5 VL are indicated by “.”

In fig. 6A, 6B and 6C depict an alignment of the hu9F5VLv8 light chain variable region with the light chain variable regions of the humanized variants of the 9F5 antibody: hu9F5VLv8_DIM1 (SEQ ID NO: 132), hu9F5VLv8_DIM2 (SEQ ID NO: 133), hu9F5VLv8_DIM3 (SEQ ID NO: 134), hu9F5VLv8_DIM4 (SEQ ID NO: 135), hu9F5VLv8_DIM5 (SEQ ID NO: 136), hu9F5VLv8_DIM6 (SEQ ID NO: 137), hu9F5VLv8_DIM7 (SEQ ID NO: 138), hu9F5VLv8_DIM8 (SEQ ID NO: 139), hu9F5VLv8_DIM9 ID NO: 140), hu9F5VLv8_DIM10 (SEQ ID NO: 141), hu9F5VLv8_DIM11 (SEQ ID NO: 142), hu9F5VLv8_DIM12 (SEQ ID NO: 143), hu9F5VLv8_DIM13 (SEQ ID NO: 144), hu9F5VLv8_DIM14 (SEQ ID NO: 145), hu9F5VLv8_DIM15 ( SEQ ID NO: 146), hu9F5VLv8_DIM16 (SEQ ID NO: 147), hu9F5VLv8_DIM17 (SEQ ID NO: 148), hu9F5VLv8_DIM18 (SEQ ID NO: 149), hu9F5VLv8_DIM19 (SEQ ID NO: 150), hu9F5VLv8_DIM2 0 (SEQ ID NO: 151 ), hu9F5VLv8_DIM21 (SEQ ID NO: 152), hu9F5VLv8_DIM22 (SEQ ID NO: 153), hu9F5VLv8_DIM23 (SEQ ID NO: 154), hu9F5VLv8_DIM24 (SEQ ID NO: 155), hu9F5VLv8_DIM25 (SEQ ID NO: 1 56), hu9F5VLv8_DIM26 (SEQ ID NO: 157), hu9F5VLv9_DIM1 (SEQ ID NO: 162), hu9F5VLv9_DIM2 (SEQ ID NO: 163), hu9F5VLv9_DIM4 (SEQ ID NO: 164), hu9F5VLv9_DIM5 (SEQ ID NO: 165), hu9F5VLv9_DIM8 (SEQ ID NO: 166) , hu9F5VLv9_DIM 10 (SEQ ID NO: 167), hu9F5VLv9_DIM11 (SEQ ID NO: 168), hu9F5VLv9_DIM13 (SEQ ID NO: 169), hu9F5VLv9_DIM19 (SEQ ID NO: 170), hu9F5VLv9_DIM20 (SEQ ID NO: 171), hu9F5VLv8_DIM27 (SEQ ID NO: 158), hu9F5VLv8_DIM28 (SEQ ID NO: 159), hu9F5VLv8_DIM29 (SEQ ID NO: 160), hu9F5VLv8_DIM30 (SEQ ID NO: 161). CDRs hu9F5VLv8, as defined by Kabat, are in bold.

In fig. 7 depicts an alignment of the heavy chain variable regions of the mouse antibody 10C12 (SEQ ID NO: 7, designated H10C12 VH in FIG. 7) and the humanized variants of the 10C12 antibody (hu10C12VHv1 and hu10C12VHv2) with the human germline heavy chain variable region sequence IGHVl-69-2* 01 (SEQ ID NO: 33) and with the human acceptor heavy chain variable region sequence CAC20421 VH (SEQ ID NO: 218). hu10C12VHv1 is SEQ ID NO: 214 and hu10C12VHv2 is SEQ ID NO: 215. The mouse 10C12 VH CDRs according to the Kabat/Chothia composite definition are shown in bold.

In fig. 8 depicts an alignment of the murine 10C12 light chain variable regions (SEQ ID NO: 11) and humanized variants of the 10C12 antibody (hu10C12VLv1 and hu10C12VLv2) with the human germline light chain variable region sequence IGKV2-28*01 u_IGKJ2*01 (SEQ ID NO: 37) and with the human acceptor CAB51297-VL_huFrwk (SEQ ID NO: 35). hu10C12VLv1 is SEQ ID NO: 216 and hu10C12VLv2 is SEQ ID NO: 217. The mouse 10C12 VL CDRs, as defined by Kabat, are in bold.

In fig. 9 depicts an alignment of the mouse heavy chain variable regions of 12C4 (SEQ ID NO: 219) and humanized variants of the 12C4 antibody (hu12C4VHv1 and hu12C4VHv2) with the human germline heavy chain variable region sequence IGHV1-69-2*01 (SEQ ID NO: 33) and with the human acceptor heavy chain variable region sequence

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CAC20421 VH (SEQ ID NO: 218). hu12C4VHv1 is SEQ ID NO: 221 and hu12C4VHv2 is SEQ ID NO: 222. The mouse 12C4 VH CDRs according to the Kabat/Chothia composite definition are shown in bold.

In fig. 10 depicts an alignment of the murine 12C4 antibody light chain variable regions (SEQ ID NO: 11) and humanized variants of the 12C4 antibody (hu12C4VLv1 and huvVLv2) with the human germline light chain variable region sequence IGKV2-28*01 u_IGKJ2*01 (SEQ ID NO: 37 ) and with human acceptor CAB51297 (SEQ ID NO: 35). hu12C4VLv1 is SEQ ID NO: 223 and hu12C4VLv2 is SEQ ID NO: 224. Murine 12C4 VL CDRs as defined by Kabat are shown in bold.

In fig. 11 depicts an alignment of the murine heavy chain variable regions of the 17C12 antibody (SEQ ID NO: 225) and the humanized variants of the 17C12 antibody (hu17C12VHv1 and hu17C12VHv2) with the human germline heavy chain variable region sequence IGHV1-69-2*01 (SEQ ID NO: 33) and with the human acceptor heavy chain variable region sequence CAC20421 VH (SEQ ID NO: 218). hu17C12VHv1 is SEQ ID NO: 232 and hu17C12VHv2 is SEQ ID NO: 233. The mouse 17C12 VH CDRs according to the Kabat/Chothia composite definition are shown in bold.

In fig. 12 depicts an alignment of the murine 17C12 antibody light chain variable regions (SEQ ID NO: 228) and the humanized variants of the 17C12 antibody (hu17C12VLv1 and hu17C12VLv2) with the human germline light chain variable region sequence IGKV2-29*02 and IGKJ4*01 (SEQ ID NO: 239) and with human acceptor QDO16713 VL (SEQ ID NO: 238). hu17C12VLv1 is SEQ ID NO: 234 and hu17C12VLv2 is SEQ ID NO: 235. The mouse 17C12 VL CDRs as defined by Kabat are shown in bold.

In fig. 13 depicts an alignment of the murine heavy chain variable regions of the mouse 14H3 antibody (SEQ ID NO: 240) and the humanized variants of the 14H3 antibody (hu14H3VHv1 and hu14H3VHv2) with the human germline heavy chain variable region sequence of IGHV2-70*04 and IGHJ4*01 (SEQ ID NO: 254) and with the human acceptor heavy chain variable region sequence QDJ57937VH hFrwk (SEQ ID NO: 253). hu14H3VHv1 is SEQ ID NO: 248 and hu14H3VHv2 is SEQ ID NO: 249. The mouse 14H3 VH CDRs according to the Kabat/Chothia composite definition are shown in bold.

In fig. 14 shows an alignment of the murine 14H3 antibody light chain variable regions (SEQ ID NO: 244) and the humanized 14H3 antibody variants (hu14H3VLv1 and hu14H3VLv2) with the human germline light chain variable region sequence IGKV2-28*01 u_IGKJ2*01 (IGKV2-28*01_IGK J2 *01; SEQ ID NO: 37) and with human acceptor ABC66914VL_hFwrk (SEQ ID NO: 256). hu14H3VLv1 is SEQ ID NO: 250 and hu14H3VLv2 is SEQ ID NO: 251. The mouse 14H3 VL CDRs as defined by Kabat are shown in bold.

In fig. 15 depicts the results of an assay showing that mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11, mouse antibody 17C12, mouse antibody 14H3 and mouse antibody 9F5 block the internalization of tau by neurons.

In fig. 16 depicts the results of an assay showing that mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11 and mouse antibody 9F5 prevent tau toxicity in primary neurons (neuronal viability).

In fig. 17 depicts the results of an assay showing that mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11 and mouse antibody 9F5 prevent tau toxicity in primary neurons (LDH release).

In fig. 18 depicts the results of a Western blot analysis showing that mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11, mouse antibody 17C12, mouse antibody 14H3 and mouse antibody 9F5 detect tau in brain samples from patients with Alzheimer's disease.

In fig. 19 shows the results of an immunoprecipitation assay using mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11, mouse antibody 17C12, mouse antibody 14H3 and mouse antibody 9F5, and a sample from the brain of a patient with Alzheimer's disease.

In fig. 20 depicts the results of an assay to measure the ability of humanized 9F5 variants to resist aggregation caused by agitation stress.

In fig. 21 depicts the results of an assay to measure the ability of humanized 9F5 variants to withstand low pH exposure.

In fig. 22 depicts the results of an assay to measure the ability of humanized 9F5 variants to aggregate under simulated high concentration conditions.

In fig. 23A-F depict the results of immunohistochemical analyzes using control, mouse 2D11, mouse 9F5, mouse 12C4, mouse 14H3 and mouse 17C12.

In fig. 24 shows an alignment of the heavy chain variable regions of mouse antibody 9F5 (SEQ ID NO: 7), mouse antibody 10C12 (SEQ ID NO: 7), mouse antibody 2D11 (SEQ ID NO: 7),

- 19 047155 mouse antibody 12C4 (SEQ ID NO: 219), mouse antibody 14H3 (SEQ ID NO: 240) and mouse antibody 17C12 (SEQ ID NO: 225). The mouse 9F5 VH CDRs according to the Kabat/Chothia composite definition are shown in bold.

In fig. 25 shows an alignment of the light chain variable regions of mouse antibody 9F5 (SEQ ID NO: 11), mouse antibody 10C12 (SEQ ID NO: 11), mouse antibody 2D11 (SEQ ID NO: 11), mouse antibody 12C4 (SEQ ID NO: 11) , mouse antibody 14H3 (SEQ ID NO: 244) and mouse antibody 17C12 (SEQ ID NO: 228). Murine 9F5 VL CDRs as defined by Kabat are shown in bold.

Brief description of sequences

B SEQ ID NO: 1 provides the amino acid sequence of the human tau isoform (Swiss-Prot P10636-8).

B SEQ ID NO: 2 provides the amino acid sequence of the human tau isoform (Swiss-Prot P10636-7).

B SEQ ID NO: 3 provides the amino acid sequence of the human tau isoform (Swiss-Prot P10636-6), (human tau 4R0N).

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

B SEQ ID NO: 5 provides the amino acid sequence of the human tau isoform (Swiss-Prot P10636-4).

B SEQ ID NO: 6 provides the amino acid sequence of the human tau isoform (Swiss-Prot P10636-2).

B SEQ ID NO: 7 provides the amino acid sequence of the heavy chain variable region of the murine antibody 9F5.

B SEQ ID NO: 8 shows the amino acid sequence of CDR-H1 as determined by the Kabat/Chothia composite determination of mouse antibody 9F5.

B SEQ ID NO: 9 provides the Kabat amino acid sequence CDR-H2 of mouse antibody 9F5.

B SEQ ID NO: 10 shows the amino acid sequence of Kabat CDR-H3 of mouse antibody 9F5.

B SEQ ID NO: 11 sets forth the amino acid sequence of the light chain variable region of the murine antibody 9F5.

B SEQ ID NO: 12 shows the amino acid sequence of Kabat CDR-L1 of mouse antibody 9F5.

B SEQ ID NO: 13 sets forth the amino acid sequence of Kabat CDR-L2 of mouse antibody 9F5.

B SEQ ID NO: 14 sets forth the amino acid sequence of Kabat CDR-L3 of mouse antibody 9F5.

B SEQ ID NO: 15 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv1.

B SEQ ID NO: 16 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv2.

B SEQ ID NO: 17 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv3.

B SEQ ID NO: 18 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv4.

B SEQ ID NO: 19 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VHv5.

B SEQ ID NO: 20 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv6.

B SEQ ID NO: 21 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv7.

B SEQ ID NO: 22 sets forth the amino acid sequence of the humanized heavy chain variable region of hu9F5VHv8.

B SEQ ID NO: 23 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VLv1.

B SEQ ID NO: 24 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VLv2.

B SEQ ID NO: 25 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VLv3.

B SEQ ID NO: 26 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VLv4.

B SEQ ID NO: 27 shows the amino acid sequence of the lung variable region

- 20 047155 chains of the humanized antibody hu9F5VLv5 9F5.

B SEQ ID NO: 28 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VLv6.

B SEQ ID NO: 29 sets forth the amino acid sequence of the humanized light chain variable region of hu9F5VLv7.

B SEQ ID NO: 30 provides the amino acid sequence of the PDB heavy chain variable region structural model. No. 50BF-VH_mSt.

B SEQ ID NO: 31 provides the amino acid sequence of the heavy chain variable region acceptor, GenBank accession number AAN16432-VH_huFrwk.

B SEQ ID NO: 32 sets forth the amino acid sequence of the heavy chain variable region acceptor PDB No. 2RCS-VH_huFrwk.

B SEQ ID NO: 33 sets forth the amino acid sequence of the germline heavy chain variable region of IMGT No. IGHV1-69-2*01.

B SEQ ID NO: 34 presents the amino acid sequence of the structural model of the light chain variable region PDB No. 50BF-VL_mSt.

B SEQ ID NO: 35 provides the amino acid sequence of the light chain variable region acceptor, GenBank accession number CAB51297-VL_huFrwk.

B SEQ ID NO: 36 provides the amino acid sequence of the light chain variable region acceptor, GenBank accession number 1911357B-VL_huFrwk.

B SEQ ID NO: 37 provides the amino acid sequence of the germline light chain variable region of IMGT No. IGKV2-28*01 and IGKJ2*01.

B SEQ ID NO: 38 provides the nucleic acid sequence encoding the heavy chain variable region of the murine antibody 9F5.

B SEQ ID NO: 39 provides the nucleic acid sequence encoding the light chain variable region of the murine antibody 9F5.

B SEQ ID NO: 40 sets forth the amino acid sequence of Kabat CDR-H1 of mouse antibody 9F5.

B SEQ ID NO: 41 sets forth the amino acid sequence of Chothia CDR-H1 of mouse antibody 9F5.

B SEQ ID NO: 42 sets forth the amino acid sequence of Chothia CDR-H2 of mouse antibody 9F5.

B SEQ ID NO: 43 provides the amino acid sequence of CDR-H2 from AbM of mouse antibody 9F5.

B SEQ ID NO: 44 provides the amino acid sequence of Contact CDR-H1 of mouse antibody 9F5.

B SEQ ID NO: 45 provides the amino acid sequence of Contact CDR-H2 of mouse antibody 9F5.

B SEQ ID NO: 46 provides the amino acid sequence of Contact CDR-H3 of mouse antibody 9F5.

B SEQ ID NO: 47 provides the amino acid sequence of Contact CDR-L1 of mouse antibody 9F5.

B SEQ ID NO: 48 provides the amino acid sequence of Contact CDR-L2 of mouse antibody 9F5.

B SEQ ID NO: 49 provides the amino acid sequence of Contact CDR-L3 of mouse antibody 9F5.

B SEQ ID NO: 50 provides the amino acid sequence of the alternative CDR-H1 as determined by the Kabat-Chothia composite definition of the humanized antibody 9F5 (present in hu9F5VHv4, hu9F5VHv5 and hu9F5VHv6).

SEQ ID NO: 51 provides the amino acid sequence of the alternative Kabat CDR-H2 of the humanized antibody 9F5 (present in hu9F5VHv5, hu9F5VHv6 and hu9F5VHv7).

SEQ ID NO: 52 provides the amino acid sequence of the alternative Kabat CDR-H2 of the humanized antibody 9F5 (present in hu9F5VHv8).

B SEQ ID NO: 53 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv5 and hu9F5VLv6).

B SEQ ID NO: 54 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv7).

SEQ ID NO: 55 represents the amino acid sequence of the Kabat alternative CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, hu9F5VLv8_DIM2, hu9F5VLv8_DIM4, hu9F5VLv8_DIM5, 9F5VLv8_DIM6, hu9F5VLv8_DIM11, hu9F5VLv8_DIM12, hu9F5VLv8_DIM13, hu9F5VLv8_DIM18, hu9F5VLv8_DIM27, hu9F5VLv8_DIM28, hu9F5VLv9_DIM2, hu9F5VLv9_DIM4, hu9F5VLv9_DIM5, hu9F5VLv9_DIM11 and hu9F5VLv9_DIM13).

B SEQ ID NO: 56 provides the amino acid sequence of the 9F5 antibody epitope.

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B SEQ ID NO: 57 provides the amino acid sequence of the 9F5 antibody-binding peptide consensus motif.

B SEQ ID NO: 58 provides the amino acid sequence of the 9F5 antibody-binding peptide consensus motif.

B SEQ ID NO: 59 shows the amino acid sequence of the linker.

B SEQ ID NO: 60 shows the amino acid sequence of the control peptide HA.

B SEQ ID NO: 61 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_M51E).

B SEQ ID NO: 62 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_M51D).

B SEQ ID NO: 63 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cD).

B SEQ ID NO: 64 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cG).

B SEQ ID NO: 65 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cS).

B SEQ ID NO: 66 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cE).

B SEQ ID NO: 67 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I30E).

B SEQ ID NO: 68 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I30K).

B SEQ ID NO: 69 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cT).

B SEQ ID NO: 70 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cN).

B SEQ ID NO: 71 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27bD).

B SEQ ID NO: 72 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I30G).

B SEQ ID NO: 73 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L33N).

B SEQ ID NO: 74 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cA).

B SEQ ID NO: 75 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L33T).

B SEQ ID NO: 76 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L33S).

B SEQ ID NO: 77 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L33R).

B SEQ ID NO: 78 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I30Q).

B SEQ ID NO: 79 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27bT).

B SEQ ID NO: 80 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_T31G).

B SEQ ID NO: 81 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27bQ).

B SEQ ID NO: 82 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L33G).

B SEQ ID NO: 83 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L27cP).

B SEQ ID NO: 84 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78R).

B SEQ ID NO: 85 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I75D).

B SEQ ID NO: 86 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78D).

B SEQ ID NO: 87 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78E).

B SEQ ID NO: 88 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78P).

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B SEQ ID NO: 89 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78K).

B SEQ ID NO: 90 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_R77D).

B SEQ ID NO: 91 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78G).

B SEQ ID NO: 92 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_S76P).

B SEQ ID NO: 93 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I75P).

B SEQ ID NO: 94 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I75Q).

B SEQ ID NO: 95 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_I75G).

B SEQ ID NO: 96 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L73P).

B SEQ ID NO: 97 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L73G).

B SEQ ID NO: 98 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V78Q).

B SEQ ID NO: 99 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_S76G).

B SEQ ID NO: 100 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L92D).

B SEQ ID NO: 101 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_Y86T).

B SEQ ID NO: 102 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L92E).

B SEQ ID NO: 103 provides the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L92G).

B SEQ ID NO: 104 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L92Q).

B SEQ ID NO: 105 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L93G).

B SEQ ID NO: 106 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_V85G).

B SEQ ID NO: 107 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_L92T).

B SEQ ID NO: 108 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv2 (also known as hu9F5VLv2_A89G).

B SEQ ID NO: 109 provides the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L80P).

B SEQ ID NO: 110 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L80D).

B SEQ ID NO: 111 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82cG).

B SEQ ID NO: 112 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82cD).

B SEQ ID NO: 113 provides the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82P).

B SEQ ID NO: 114 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L80G).

B SEQ ID NO: 115 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82K).

B SEQ ID NO: 116 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82R).

[B SEQ ID NO: 117 provides the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82E).

B SEQ ID NO: 118 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82N).

B SEQ ID NO: 119 provides the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_Y79D).

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B SEQ ID NO: 120 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_Y79N).

B SEQ ID NO: 121 provides the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_Y79G).

B SEQ ID NO: 122 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv5 (also known as hu9F5VHv5_M80E).

B SEQ ID NO: 123 provides the amino acid sequence of the heavy chain variable region variant hu9F5VHv5 (also known as hu9F5VHv5_M80G).

B SEQ ID NO: 124 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_L82cS).

B SEQ ID NO: 125 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_Y79Q).

B SEQ ID NO: 126 sets forth the amino acid sequence of the heavy chain variable region variant hu9F5VHv4 (also known as hu9F5VHv4_S82aG).

B SEQ ID NO: 127 sets forth the amino acid sequence of the heavy chain variable region of hu9F5VHv9.

B SEQ ID NO: 128 sets forth the amino acid sequence of the heavy chain variable region of hu9F5VHv10 (also known as hu9F5VHv9_Q38K_G42E).

SEQ ID NO: 129 provides the amino acid sequence of the heavy chain variable region hu9F5VHv10_L82cG.

B SEQ ID NO: 130 sets forth the amino acid sequence of the light chain variable region of hu9F5VLv8.

B SEQ ID NO: 131 sets forth the amino acid sequence of the heavy chain variable region of hu9F5VLv9 (also known as hu9F5VLv8_N60D).

B SEQ ID NO: 132 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54G, L92I, also known as hu9F5VLv8_DIM1).

B SEQ ID NO: 133 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM2).

B SEQ ID NO: 134 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54T, L92I, also known as hu9F5VLv8_DIM3).

B SEQ ID NO: 135 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54R, L92G, also known as hu9F5VLv8_DIM4).

B SEQ ID NO: 136 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM5).

B SEQ ID NO: 137 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cD, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM6).

B SEQ ID NO: 138 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cD, L37Q, M51K, L54R, L92I, also known as hu9F5VLv8_DIM7).

B SEQ ID NO: 139 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37Q, M51K, L54R, L92I, also known as hu9F5VLv8_DIM8).

B SEQ ID NO: 140 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37Q, M51K, L54G, L92I, also known as hu9F5VLv8_DIM9).

B SEQ ID NO: 141 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51K, L54G, L92I, also known as hu9F5VLv8_DIM10).

B SEQ ID NO: 142 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, L92I, also known as hu9F5VLv8_DIM11).

B SEQ ID NO: 143 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, L92G, also known as hu9F5VLv8_DIM12).

B SEQ ID NO: 144 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, also known as

- 24 047155 hu9F5VLv8_DIM13).

B SEQ ID NO: 145 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T, L92I, also known as hu9F5VLv8_DIM14).

B SEQ ID NO: 146 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T, L92G, also known as hu9F5VLv8_DIM15).

B SEQ ID NO: 147 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T, also known as hu9F5VLv8_DIM16).

B SEQ ID NO: 148 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37G, M51G, L54T, L92I, also known as hu9F5VLv8_DIM17).

B SEQ ID NO: 149 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cD, L37G, M51G, L54R, L92I, also known as hu9F5VLv8_DIM18).

B SEQ ID NO: 150 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37I, M51I, L54R, L92I, also known as hu9F5VLv8_DIM19).

B SEQ ID NO: 151 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51I, L54G, L92I, also known as hu9F5VLv8_DIM20).

B SEQ ID NO: 152 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51I, L54G, also known as hu9F5VLv8_DIM21).

B SEQ ID NO: 153 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51E, L54R, L92I, also known as hu9F5VLv8_DIM22).

SEQ ID NO: 154 Light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37Q, M51E, L54G, L92I, also known as hu9F5VLv8_DIM23).

B SEQ ID NO: 155 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37I, M51E, L54R, L92I, also known as hu9F5VLv8_DIM24).

B SEQ ID NO: 156 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cG, L37I, M51E, L54R, L92G, also known as hu9F5VLv8_DIM25).

B SEQ ID NO: 157 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cI, L37I, M51E, L54R, also known as hu9F5VLv8_DIM26).

B SEQ ID NO: 158 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM27).

B SEQ ID NO: 159 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, M51G, L54R, L92I, also known as hu9F5VLv8_DIM28).

B SEQ ID NO: 160 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, L54R, L92I, also known as hu9F5VLv8_DIM29).

B SEQ ID NO: 161 provides the amino acid sequence of the light chain variable region variant hu9F5VLv8 (hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L92I, also known as hu9F5VLv8_DIM30).

B SEQ ID NO: 162 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cS, L37Q, M51G, L54G, L92I, also known as hu9F5VLv9_DIM1).

B SEQ ID NO: 163 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cS, L37Q, M51G, L54R, L92I, also known as hu9F5VLv9_DIM2).

B SEQ ID NO: 164 sets forth the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cS, L37Q, M51G, L54R, L92G, also known as hu9F5VLv9_DIM4).

B SEQ ID NO: 165 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cG, L37Q, M51G, L54R, L92I, also known as

- 25 047155 hu9F5VLv9_DIM5).

B SEQ ID NO: 166 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cG, L37Q, M51K, L54R, L92I, also known as hu9F5VLv9_DIM8).

B SEQ ID NO: 167 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cS, L37Q, M51K, L54G, L92I, also known as hu9F5VLv9_DIM10).

B SEQ ID NO: 168 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cG, L37G, M51G, L54R, L92I, also known as hu9F5VLv9_DIM11).

B SEQ ID NO: 169 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cG, L37G, M51G, L54R, also known as hu9F5VLv9_DIM13).

B SEQ ID NO: 170 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cS, L37I, M51I, L54R, L92I, also known as hu9F5VLv9_DIM19).

B SEQ ID NO: 171 provides the amino acid sequence of the light chain variable region variant hu9F5VLv9 (hu9F5VLv9_V3Q, L27cS, L37Q, M51I, L54G, L92I, also known as hu9F5VLv9_DIM20).

B SEQ ID NO: 172 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27bD).

B SEQ ID NO: 173 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27bT).

B SEQ ID NO: 174 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27bQ).

B SEQ ID NO: 175 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cD, hu9F5VLv8_DIM6, hu9F5VLv8_DIM7 and hu9F5VLv8_DIM18).

SEQ ID NO: 176 represents the amino acid sequence of the Kabat alternative CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cG, hu9F5VLv8_DIM5, hu9F5VLv8_DIM8, hu9F5VLv8_DIM9, in hu9F5VLv8_DIM11, hu9F5VLv8 _DIM12, hu9F5VLv8_DIM13, hu9F5VLv8_DIM14, hu9F5VLv8_DIM15, hu9F5VLv8_DIM16, hu9F5VLv8_DIM23, hu9F5VLv8_DIM24, hu9F5VLv8_DIM25, hu9F5VLv9_DIM5 hu9F5VLv9_DIM8 , hu9F5VLv9_DIM11 and hu9F5VLv9_DIM13).

SEQ ID NO: 177 provides the amino acid sequence of the Kabat alternative CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cS, hu9F5VLv8_DIMl, hu9F5VLv8_DIM2, hu9F5VLv8_DIM19, hu9F5VLv8_DIM20, 8_DIM21, hu9F5VLv8_DIM22, hu9F5VLv8_DIM28, hu9F5VLv8_DIM29, hu9F5VLv8_DIM30, hu9F5VLv9_DIM1, hu9F5VLv9_DIM2, hu9F5VLv9_DIM4, hu9F5VLv9_DIM10, in hu9F5VLv9_DIM19 and hu9F5VLv9_DIM20).

B SEQ ID NO: 178 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cE).

B SEQ ID NO: 179 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cT).

B SEQ ID NO: 180 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cN).

B SEQ ID NO: 181 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cA).

B SEQ ID NO: 182 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L27cP).

B SEQ ID NO: 183 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM26).

B SEQ ID NO: 184 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_I30E).

B SEQ ID NO: 185 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_I30K).

B SEQ ID NO: 186 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_I30G).

B SEQ ID NO: 187 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_I30Q).

B SEQ ID NO: 188 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_T31G).

B SEQ ID NO: 189 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L33N).

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B SEQ ID NO: 190 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L33T).

B SEQ ID NO: 191 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L33S).

B SEQ ID NO: 192 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L33R).

B SEQ ID NO: 193 provides the amino acid sequence of the alternative Kabat CDR-L1 of the humanized antibody 9F5 (present in hu9F5VLv2_L33G).

B SEQ ID NO: 194 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv2_M51E).

B SEQ ID NO: 195 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv2_M51D).

B SEQ ID NO: 196 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM30).

B SEQ ID NO: 197 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM29).

B SEQ ID NO: 198 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM1 and hu9F5VLv9_DIM1).

B SEQ ID NO: 199 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM3, hu9F5VLv8_DIM14, hu9F5VLv8_DIM15, hu9F5VLv8_DIM16 and hu9F5VLv8_DIM17).

SEQ ID NO: 200 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM7, hu9F5VLv8_DIM8 and hu9F5VLv9_DIM8).

SEQ ID NO: 201 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM9, hu9F5VLv8_DIM10 and hu9F5VLv9_DIM10).

B SEQ ID NO: 202 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM19 and hu9F5VLv9_DIM19).

SEQ ID NO: 203 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM20, hu9F5VLv8_DIM21 and hu9F5VLv9_DIM20).

B SEQ ID NO: 204 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM22, hu9F5VLv8_DIM24, hu9F5VLv8_DIM25 and hu9F5VLv8_DIM26).

B SEQ ID NO: 205 provides the amino acid sequence of the alternative Kabat CDR-L2 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM23).

B SEQ ID NO: 206 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv2_A89G).

B SEQ ID NO: 207 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv2_L92D).

B SEQ ID NO: 208 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv2_L92E).

B SEQ ID NO: 209 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM4, hu9F5VLv8_DIM12, hu9F5VLv8_DIM15, hu9F5VLv8_DIM25 and hu9F5VLv9_DIM4).

B SEQ ID NO: 210 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv2_L92Q).

B SEQ ID NO: 211 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv2_L92T).

B SEQ ID NO: 212 represents the amino acid sequence of the Kabat alternative CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv8_DIM1, hu9F5VLv8_DIM2, hu9F5VLv8_DIM3, hu9F5VLv8_DIM5, hu9F5VLv8_DIM6, hu9F5VLv8_DIM 7, hu9F5VLv8_DIM8, hu9F5VLv8_DIM9, hu9F5VLv8_DIM10, hu9F5VLv8_DIM11, hu9F5VLv8_DIM14, hu9F5VLv8_DIM17, hu9F5VLv8_DIM18, hu9F5VLv8_DIM19, 5VLv8_DIM20 , hu9F5VLv8_DIM22, hu9F5VLv8_DIM23, hu9F5VLv8_DIM24, hu9F5VLv8_DIM27, hu9F5VLv8_DIM28, hu9F5VLv8_DIM29, hu9F5VLv8_DIM30, hu9F5VLv9_DIM1, hu 9F5VLv9_DIM2, hu9F5VLv9_DIM5, hu9F5VLv9_DIM8, hu9F5VLv9_DIM10, hu9F5VLv9_DIM11, hu9F5VLv9_DIM19 and u9F5VLv9_DIM20).

B SEQ ID NO: 213 provides the amino acid sequence of the alternative Kabat CDR-L3 of the humanized antibody 9F5 (present in hu9F5VLv2_L93G).

B SEQ ID NO: 214 sets forth the amino acid sequence of the humanized heavy chain variable region of hu10C12VHv1.

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B SEQ ID NO: 215 sets forth the amino acid sequence of the humanized heavy chain variable region of hu10C12VHv2.

B SEQ ID NO: 216 sets forth the amino acid sequence of the humanized light chain variable region of hu10C12VLv1.

B SEQ ID NO: 217 sets forth the amino acid sequence of the humanized light chain variable region of hu10C12VLv2.

B SEQ ID NO: 218 sets forth the amino acid sequence of the heavy chain variable region acceptor CAC20421-VH_huFrwk.

B SEQ ID NO: 219 provides the amino acid sequence of the heavy chain variable region of the murine antibody 12C4.

B SEQ ID NO: 220 sets forth the Kabat amino acid sequence CDR-H2 of mouse antibody 12C4.

B SEQ ID NO: 221 sets forth the amino acid sequence of the humanized heavy chain variable region of hu12C4VHv1.

B SEQ ID NO: 222 sets forth the amino acid sequence of the humanized heavy chain variable region of hu12C4VHv2.

B SEQ ID NO: 223 sets forth the amino acid sequence of the humanized light chain variable region of hu12C4VLv1.

B SEQ ID NO: 224 sets forth the amino acid sequence of the humanized light chain variable region of hu12C4VLv2.

B SEQ ID NO: 225 provides the amino acid sequence of the heavy chain variable region of the murine antibody 17C12.

B SEQ ID NO: 226 provides the amino acid sequence of CDR H1 as determined by the Kabat-Chothia composite determination of mouse antibody 17C12.

B SEQ ID NO: 227 shows the Kabat CDR H2 amino acid sequence of the mouse antibody 17C12.

B SEQ ID NO: 228 provides the amino acid sequence of the light chain variable region of the murine antibody 17C12.

SEQ ID NO: 229 provides the amino acid sequence of Kabat CDR-L1 of mouse antibody 17C12.

B SEQ ID NO: 230 shows the amino acid sequence of Kabat CDR-L2 of mouse antibody 17C12.

B SEQ ID NO: 231 shows the amino acid sequence of Kabat CDR-L3 of mouse antibody 17C12.

B SEQ ID NO: 232 sets forth the amino acid sequence of the humanized heavy chain variable region of hu17C12VHv1.

B SEQ ID NO: 233 sets forth the amino acid sequence of the humanized heavy chain variable region of hu17C12VHv2.

B SEQ ID NO: 234 sets forth the amino acid sequence of the humanized light chain variable region of hu17C12VLv1.

B SEQ ID NO: 235 sets forth the amino acid sequence of the humanized light chain variable region of hu17C12VLv2.

B SEQ ID NO: 236 shows the amino acid sequence of the structural model of the heavy chain variable region 3PP3-VH_mSt.

B SEQ ID NO: 237 provides the amino acid sequence of the structural model of the light chain variable region of 3PP3-VL_mSt.

B SEQ ID NO: 238 sets forth the amino acid sequence of the light chain variable region acceptor QDO16713-VL_huFrwk.

B SEQ ID NO: 239 provides the amino acid sequence of the germline light chain variable region of IGKV2-29*02 and IGKJ4*01.

JB SEQ ID NO: 240 provides the amino acid sequence of the variable region of the heavy chain of the mouse antibody 14H3.

B SEQ ID NO: 241 provides the amino acid sequence of CDR H1 as determined by the Kabat-Chothia composite determination of mouse antibody 14H3.

B SEQ ID NO: 242 sets forth the Kabat CDR H2 amino acid sequence of mouse antibody 14H3.

B SEQ ID NO: 243 sets forth the Kabat CDR H3 amino acid sequence of mouse antibody 14H3.

B SEQ ID NO: 244 provides the amino acid sequence of the light chain variable region of the mouse antibody 14H3.

B SEQ ID NO: 245 shows the amino acid sequence of Kabat CDR L1 of mouse antibody 14H3

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B SEQ ID NO: 246 sets forth the Kabat CDR L2 amino acid sequence of mouse antibody 14H3.

B SEQ ID NO: 247 sets forth the Kabat CDR L3 amino acid sequence of mouse antibody 14H3.

B SEQ ID NO: 248 sets forth the amino acid sequence of the humanized heavy chain variable region of hu14H3VHv1.

B SEQ ID NO: 249 sets forth the amino acid sequence of the humanized heavy chain variable region of hu14H3VHv2.

B SEQ ID NO: 250 sets forth the amino acid sequence of the humanized light chain variable region of hu14H3VLv1.

B SEQ ID NO: 251 sets forth the amino acid sequence of the humanized light chain variable region of hu14H3VLv2.

B SEQ ID NO: 252 provides the amino acid sequence of the structural model of the heavy chain variable region 2VQl-VH_mSt.

B SEQ ID NO: 253 sets forth the amino acid sequence of the heavy chain variable region acceptor QDJ57937-VH_huFrwk.

B SEQ ID NO: 254 provides the amino acid sequence of the germline heavy chain variable region of IGHV1-70*04 and IGHJ4*01.

B SEQ ID NO: 255 provides the amino acid sequence of the structural model of the light chain variable region 2VQ1-VL_mSt.

B SEQ ID NO: 256 sets forth the amino acid sequence of the light chain variable region acceptor ABC66914-VL_huFrwk.

B SEQ ID NO: 257 shows the amino acid sequence of CDR-H2 from AbM of mouse antibody 12C4.

B SEQ ID NO: 258 shows the amino acid sequence of Contact CDR-H2 of mouse antibody 12C4.

B SEQ ID NO: 259 sets forth the amino acid sequence of the Chothia CDR-H1 of the murine antibody 17C12.

B SEQ ID NO: 260 shows the amino acid sequence of CDR-H2 from AbM of mouse antibody 17C12

B SEQ ID NO: 261 shows the amino acid sequence of Contact CDR-H2 of mouse antibody 17C12.

B SEQ ID NO: 262 shows the amino acid sequence of CDR-L1 from Contact of mouse antibody 17C12.

B SEQ ID NO: 263 shows the amino acid sequence of Contact CDR-L2 of mouse antibody 17C12.

B SEQ ID NO: 264 shows the amino acid sequence of Contact CDR-L3 of mouse antibody 17C12.

B SEQ ID NO: 265 sets forth the Kabat amino acid sequence CDR-H1 of mouse antibody 14H3.

B SEQ ID NO: 266 sets forth the amino acid sequence of the Chothia CDR-H1 of the murine antibody 14H3.

B SEQ ID NO: 267 sets forth the amino acid sequence of the Chothia CDR-H2 of the murine antibody 14H3.

B SEQ ID NO: 268 shows the amino acid sequence of CDR-H2 from AbM of mouse antibody 14H3.

B SEQ ID NO: 269 provides the amino acid sequence of Contact CDR-H1 of mouse antibody 14H3.

B SEQ ID NO: 270 shows the amino acid sequence of Contact CDR-H2 of mouse antibody 14H3.

B SEQ ID NO: 271 shows the amino acid sequence of Contact CDR-H3 of mouse antibody 14H3.

B SEQ ID NO: 272 shows the amino acid sequence of Contact CDR-L1 of mouse antibody 14H3.

B SEQ ID NO: 273 shows the amino acid sequence of Contact CDR-L2 of mouse antibody 14H3.

B SEQ ID NO: 274 provides the amino acid sequence of Contact CDR-L3 of mouse antibody 14H3.

B SEQ ID NO: 275 provides the amino acid sequence of the alternative H1 CDR as determined by the Kabat-Chothia composite definition of the humanized antibody 14H3 (present in hu14H3VHv1 and hu14H3VHv2).

B SEQ ID NO: 276 shows the amino acid sequence of the pepti consensus motif

- 29 047155 yes, bound by antibody 9F5, 10C12, 2D11, 12C4, 17C12 and 14H3.

B SEQ ID NO: 277 provides the amino acid sequence of the consensus peptide motif bound by antibody 2D11.

Definitions

Monoclonal antibodies or other biological compounds are usually provided in isolated form. This means that the antibody or other biological compound is generally at least 50% w/w pure, free from interfering proteins and other contaminants resulting from its production or purification, but does not exclude the possibility that the monoclonal antibody is combined with excess a pharmaceutically acceptable carrier or other carrier medium intended to facilitate its use. Sometimes the monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/w free from interfering proteins and contaminants generated during manufacturing or purification. Often the isolated monoclonal antibody or other biological compound is the predominant macromolecule species remaining after its purification.

Specific binding of an antibody to its target antigen means an affinity and/or avidity of at least 10 ⁶ , 10 ⁷ , 108 , 109 , 10 ¹⁰ , 10 ¹¹ or 10 ¹² M ^-1 . Specific binding is markedly higher in magnitude and is distinct from nonspecific binding, which occurs to at least one unrelated target. Specific binding may result from the formation of bonds between specific functional groups or a specific spatial correspondence (eg, a lock and key type), whereas nonspecific binding is usually the result of 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 a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 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 called 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 are classified as gamma, mu, alpha, delta, or epsilon and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a J region of about 12 or more amino acids, while the heavy chain also includes a D region of about 10 or more amino acids. See generally, Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7 (incorporated herein in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region (also referred to herein as a light chain variable domain (VL domain) or a heavy chain variable domain (VH domain), respectively) consists of a framework region interrupted by three complementarity determining regions, or CDRs. Framework regions serve to align CDRs for specific binding to an antigen epitope. CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework regions (FRs) and CDRs: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. VL domain CDRs 1, 2 and 3 are also referred to herein as CDRL1, CDR-L2 and CDR-L3, respectively; CDRs 1, 2 and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2 and CDR-H3, respectively. When the application discloses a VL sequence with R as the C-terminal residue, R can alternatively be considered the N-terminal residue of the light chain constant region. Thus, the application should also be understood as disclosing the VL sequence without the C-terminal R.

The assignment of amino acids to each VL and VH domain is consistent with any traditional CDR definition. Traditional definitions include Kabat (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), Chothia (Chothia & Lesk, J. Mol. Biol. 196:901-917 , 1987; Chothia et al, Nature 342:878-883, 1989); a CDR no Chothia Kabat composite, in which CDR-H1 is a composite of CDR no Chothia and Kabat; ABM determination used by the Oxford Molecular antibody modeling program; and contact definition according to Martin et al (bioinfo.org.uk/abs) (see Table 1). Kabat offers a widely used numbering (Kabat numbering), in which corresponding residues between different heavy chains or between different light chains are assigned the same number. When an antibody is said to contain a CDR according to a particular definition of a CDR (eg, Kabat), that definition indicates the minimum number of CDR residues present in the antibody (ie, a Kabat CDR). This does not exclude that other residues that fall under a different traditional definition of CDR are present, but

- 30 047155 not included in the specified definition. For example, an antibody containing CDRs defined by Kabat includes, among other possibilities, an antibody in which the CDRs contain Kabat CDR residues and no other CDR residues, and an antibody in which the H1 CDR corresponds to the composite definition of the Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and do not contain additional CDR residues based on other definitions.

Table 1. Traditional CDR definitions using Kabat numbering

A loop Kabat Chothia Composite no Chothia definition And Kabat AbM Contact LI 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 Hl H31-H35B H26—H32..H34* H26-H35B* H26-H35B H30-H35B H2 H50-H65 H52-H56 H50-H65 H50-H58 H47-H58 H3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101

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

The term antibody includes intact antibodies and their binding fragments. Typically, the fragments compete with the intact antibody from which they were derived for specific binding to the target, 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 methods 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 having two different heavy/light chain pairs and two different binding sites (see, for example, Songsivilai and Lachmann, Clin. Exp. Immunol, 79:315-321 (1990); Kostelny et al. al., J. Immunol, 148:1547-53 (1992)). In some bispecific antibodies, two different heavy/light chain pairs include humanized heavy chain/light chain pairs 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3 and a heavy chain/light chain pair specific for a different epitope on tau than that bound by 9F5. 10С12, 2D11, 12С4, 17С12 or 14Н3.

In some bispecific antibodies, one heavy chain/light chain pair is a humanized antibody 9F5, a humanized antibody 10C12, a humanized antibody 2D11, a humanized antibody 12C4, a humanized antibody 17C12, or a humanized antibody 14H3, as further disclosed below, and the other heavy chain/light chain pair is derived from an antibody that binds to a receptor expressed on the blood-brain barrier, such as the insulin receptor, insulin-like growth factor (IGF) receptor, leptin receptor, 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 via receptor-mediated transcytosis. Brain uptake of a bispecific antibody can be further enhanced by engineering the bispecific antibody to reduce its affinity for the blood-brain barrier receptor. Reduced affinity for the receptor resulted in greater distribution in the brain (see, e.g., Atwal et al., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3, 84ra44, 2011).

Examples of bispecific antibodies also include: (1) dual variable domain antibody (DVD-Ig), where each light and heavy chain contains two variable domains in tandem through a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) tandem diabody, which is the fusion of two single-chain diabodies, resulting in

- 31 047155 tetravalent bispecific antibody having two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody, resulting in a polyvalent molecule; (4) a so-called dock and lock molecule based on the dimerization and docking domain in protein kinase A, which when applied to a Fab can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to another Fab fragment; or (5) a so-called Scorpio molecule comprising, for example, two scFvs fused to both ends of a human Fc region. Examples of platforms useful for generating bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (Fstar), Fc-engineered IgG1 (Xencor), or DuoBody (Fab arm exchange-based, Genmab).

The term epitope refers to the site of an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or non-contiguous amino acids superimposed on each other by stacking the tertiary structure of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are usually retained when exposed to denaturing solvents, whereas epitopes formed from tertiary folding (also known as conformational epitopes) are usually lost when exposed to denaturing solvents. An epitope typically comprises at least 3 or more, usually at least 5 or 8-10 amino acids, in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and 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 by a simple immunoassay that shows the ability of one antibody to compete with the binding of another antibody to the target antigen. An antibody epitope can also be determined by x-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

Antibody competition is determined by an assay in which a test antibody inhibits the specific binding of a reference antibody to a common antigen (see, eg, Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of test antibody (eg, at least 2x, 5x, 10x, 20x, or 100x) inhibits binding of the reference antibody by at least 50%, as measured in a competitive binding assay. Some test antibodies inhibit the binding of reference antibodies by at least 75%, 90% or 99%. Antibodies identified in a competition assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope located close enough to the epitope bound by the reference antibody to cause steric hindrance.

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

The term patient includes humans and other mammalian subjects who are receiving either prophylactic or therapeutic treatment.

An individual is at increased risk for a disease if they have at least one known risk factor (eg, genetic, biochemical, family history, and situational exposure), such that individuals with that risk factor are at a statistically significantly greater risk of developing the disease than individuals without this risk factor.

The term biological sample refers to a sample of biological material contained within or obtained from a biological source, such as a human or mammal. Such samples may be organs, organelles, tissues, tissue parts, body fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also cover any material resulting from processing of the sample.

The derived material may include cells or their progeny. Processing of the biological sample may include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.

The term control sample refers to a biological sample that is not known or suspected to include lesions of a tau-associated disease, or at least not known or suspected to include lesions of a particular type. Control samples can be obtained from people who do not have tau-associated disease. Alternatively, control samples can be obtained from patients suffering from

- 32 047155 tau-associated disease. Such samples may be obtained at the same time as the biological sample suspected of containing the tau-associated disease or at a different time. The biological sample and the control sample may be obtained from the same tissue. Preferably, control samples consist mainly or entirely of normal, healthy areas and can be used in comparison with a biological sample that is considered to include areas affected by tau-associated disease. Preferably, the tissue in the control sample is the same type as the tissue in the biological sample. Preferably, the tau-associated disease cells believed to be present in the biological sample are from the same cell type (eg, neurons or glia) as the cell type in the control sample.

The term disease refers to any abnormal condition that impairs physiological function. The term is widely used to refer to any disorder, disease, abnormality, pathology, ailment, condition or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

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

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

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, gln, his, lys, arg; group V (residues affecting chain orientation): gly, pro; and group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions include substitutions between amino acids of the same class. Non-conservative substitutions are the replacement of a member of one of these classes by a member of another class.

Percent sequence identity is determined using antibody sequences that are maximally aligned according to the Kabat numbering system. After alignment, if the subject region of an antibody (eg, the entire mature heavy or light chain variable region) is compared to the same region of a reference antibody, the percentage of sequence identity between the target and reference regions of the antibody is the number of positions occupied by the same amino acid as in the target region. , and in the antibody reference region, divided by the total number of aligned positions of the two regions, excluding gaps, multiplied by 100 to convert to a percentage.

Compositions or methods containing or incorporating one or more of the listed elements may include other elements not specifically listed. For example, a composition that contains or includes an antibody may contain the antibody alone or in combination with other ingredients.

The value range designation includes all integers within or defining the range, and all subranges defined by integers within the range.

Unless otherwise obvious from the context, the term about covers non-significant variations, such as values within the standard error of measurement (eg SEM) of a specified value.

Statistical significance means p<0.05.

Singular forms include references to plural forms unless the context clearly indicates 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 information

The invention provides antibodies that bind to tau. Some antibodies specifically bind to an epitope within (Q/E)IVYK(S/P) (SEQ ID NO: 56). Some antibodies specifically bind to a peptide containing the amino acid sequence QIVYKP (SEQ ID NO: 57, corresponding to residues 307-312 of the tau isoform SEQ ID NO: 1). Some antibodies specifically bind to a peptide containing the amino acid sequence EIVYKSP (SEQ ID NO: 58, corresponding to residues 391-397 of the tau isoform SEQ ID NO: 1). These antibodies differ from 3D6 and other antibodies characterized by binding to the microtubule binding region (MTBR) of human tau by the presence of an additional epitope near the C terminus of tau. The additional C-terminal specificity of the epitope provides the basis for the antibody to bind an increased number of conformational forms of tau associated with pathology. Some antibodies specifically bind to a peptide containing the amino acid sequence EIVYKS (SEQ ID NO: 277, corresponding to residues 391-396 of the tau isoform SEQ ID NO: 1). Examples of antibodies according to the invention are 9F5,

- 33 047155

10С12, 2D11, 12С4, 17С12 and 14Н3. Certain antibodies of the invention serve to inhibit or delay the development of tau-associated pathologies and associated worsening of symptoms. Although an understanding of the mechanism is not required to practice the invention, the reduction in toxicity may result from the antibody inducing phagocytosis of tau, inhibiting inter- or intramolecular aggregation of tau or binding to other molecules, stabilization of a non-toxic conformation, inhibition of intercellular or intracellular transmission of pathogenic forms of tau, blockade phosphorylation of tau, preventing tau from binding to cells, or inducing proteolytic degradation of tau, among other mechanisms. Certain antibodies of the invention are useful for increasing tau aggregation by increasing the molecular weight of certain aggregated tau species to reduce toxicity/cellular uptake and/or increase clearance. Large aggregates of tau molecules may exhibit reduced uptake into neuronal cells. Some antibodies of the invention, by divalently binding to tau, bring individual tau molecules closer together, promoting aggregation into a tau aggregate that is too large for uptake by neuronal cells. In addition, Fc-mediated phagocytosis requires that multiple tau-binding antibodies be in close proximity.

A large aggregate of tau molecules may have many anti-tau antibodies associated with it and provide the cluster required for Fc-mediated phagocytosis by macrophages. The antibodies of the 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 otherwise obvious from the context, reference to tau means the naturally occurring human form of tau, including all isoforms, regardless of whether post-translational modification (eg, phosphorylation, glycation, or acetylation) is present. There are six major isoforms (splice variants) of tau found in the human brain. The longest of these variants contains 441 amino acids from which the original met residue is cleaved. Residues are numbered according to isoform 441. Thus, for example, a reference to phosphorylation at position 404 means position 404 of isoform 441 or the corresponding position of any other isoform in a maximum alignment with isoform 441. The amino acid sequences of the isoforms and Swiss-Prot accession numbers are listed below.

Р10636-8 (SEQIDNO:1)

20 30 40 5060

MAEPRQEFEV MEDHAGIYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG

80 90 100 PO120

SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 130 140 150 160 170180

HVTQARMVSK SKDGTGSDDK KAKGADGKTKIATPRGAAPP GQKGQANATRΙΡΑΚΊΡΡΑΡΚ

190 200 210 220 230240

TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAWR TPPKSPSSAK 250 260 270 280 290300

SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV

310 320 330 340 350360

PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 370 380 390 400 410420

THVPGGGNKKIETHKLTFRE NAKAKTDHGA EIVYKSPWS GDTSPRHLSN VSSTGSIDMV 430440

- 34 047155

DSPQLATLAD EVSASLAKQG L

P10636-7 (SEQ ID NO:2) 10 20 30 40 5060

MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 100 110120

SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT 130 140 150 160 170180

KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 190 200 210 220 230240

SRTPSLPTPP TREPKKVAW RTPPKSPSSA KSRLQTAPVP MPDLKNVKSKIGSTENLKHQ 250 260 270 280 290300

PGGGKVQIIN KKLDLSNVQS KCGSKDNIKH VPGGGSVQIV YKPVDLSKVT SKCGSLGNIH 310 320 330 340 350360

HKPGGGQVEV KSEKLDFKDR VQSKIGSLDNITHVPGGGNK KIETHKLTFR ENAKAKTDHG 370 380 390 400410

AEIVYKSPW SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

P10636-6 (human tau 4R0N) (SEQ Ш NO:3)

20 30 40 5060

MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA 70 80 90 100 110120

AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA 130 140 150 160 170180

PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS 190 200 210 220 230240

AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIINKKLDLSNVQ SKCGSKDNIK 250 260 270 280 290300

HVPGGGSVQI VYKPVDLSKV TSKCGSLGNIHHKPGGGQVE VKSEKLDFKD RVQSKIGSLD 310 320 330 340 350360

NITHVPGGGN KKIETHKLTF RENAKAKTDH GAEIVYKSPV VSGDTSPRHL SNVSSTGSID 370380

MVDSPQLATL ADEVSASLAK QGL

P10636-5 (SEQIDNO:4)

20 30 40 5060

MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 100 110120

- 35 047155

SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG 130 140 150 160 170180

HVTQARMVSK SKDGTGSDDK KAKGADGKTKIATPRGAAPP GQKGQANATRIPAKTPPAPK 190 200 210 220 230240

TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAWR TPPKSPSSAK 250 260 270 280 290300

SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIVYK PVDLSKVTSK CGSLGNIHHK 310 320 330 340 350360

PGGGQVEVKS EKLDFKDRVQ SKIGSLDNIT HVPGGGNKKIETHKLTFREN AKAKTDHGAE 370 380 390 400410

IVYKSPWSG DTSPRHLSNV SSTGSIDMVD SPQLATLADE VSASLAKQGL

Pl0636-4 (SEQ ID NO:5)

20 30 40 5060

MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG 70 80 90 100 110120

SETSDAKSTP TAEAEEAGIG DTPSLEDEAA GHVTQARMVS KSKDGTGSDD KKAKGADGKT 130 140 150 160 170180

KIATPRGAAP PGQKGQANAT RIPAKTPPAP KTPPSSGEPP KSGDRSGYSS PGSPGTPGSR 190 200 210 220 230240

SRTPSLPTPP TREPKKVAW RTPPKSPSSA KSRLQTAPVP MPDLKNVKSKIGSTENLKHQ 250 260 270 280 290300

PGGGKVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI 310 320 330 340 350360

THVPGGGNKKIETHKLTFRE NAKAKTDHGA EIVYKSPWS GDTSPRHLSN VSSTGSIDMV 370380

DSPQLATLAD EVSASLAKQG L

Pl0636-2 (SEQ ID NO:6)

20 30 40 5060

MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKAEEAGI GDTPSLEDEA

80 90 100 110120

AGHVTQARMV SKSKDGTGSD DKKAKGADGK TKIATPRGAA PPGQKGQANA TRIPAKTPPA

130 140 150 160 170180

PKTPPSSGEP PKSGDRSGYS SPGSPGTPGS RSRTPSLPTP PTREPKKVAV VRTPPKSPSS

190 200 210 220 230240

AKSRLQTAPV PMPDLKNVKS KIGSTENLKH QPGGGKVQIV YKPVDLSKVT SKCGSLGNIH

250 260 270 280 290300

HKPGGGQVEV KSEKLDFKDR VQSKIGSLDN ITHVPGGGNK KIETHKLTFR ENAKAKTDHG

310 320 330 340350

AEIVYKSPW SGDTSPRHLS NVSSTGSIDM VDSPQLATLA DEVSASLAKQ GL

The reference to tau includes known natural variations, about 30 of which 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 by isoform

- 36 047155

441 are a mutation of lysine to threonine at amino acid residue 257 (K257T), a mutation of isoleucine to valine 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); mutation of glycine to serine 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 can be phosphorylated at one or more amino acid residues, including tyrosine at amino acid positions 18, 29, 97, 310 and 394, serine at amino acid positions 184, 185, 198, 199, 202, 208, 214, 235, 237, 238, 262, 293, 324, 356, 396, 400, 404, 409, 412, 413 and 422; and threonine at amino acid positions 175, 181, 205, 212, 217, 231 and 403.

Unless otherwise obvious from the context, reference to tau or fragments thereof includes naturally occurring human amino acid sequences, including isoforms, mutants and allelic variants thereof.

III. Antibodies

A. Binding Specificity and Functional Properties

The invention provides antibodies that specifically bind to tau. Some antibodies specifically bind to tau at an epitope formed by the amino acids of one or both of two regions of tau that share the IVYK core motif (SEQ ID NO: 276). These regions are defined by residues 307-312 and 391-397 or 391-396 of SEQ ID NO: 1, respectively. Thus, an antibody with a single binding site for tau, such as scFv, can specifically bind to tau on an epitope formed from amino acids within either of these regions individually, or to a hybrid epitope formed from amino acids from both of these regions. An antibody with two binding sites for tau can also bind epitopes within 307-312 and 391-397 or within 391-396 simultaneously from its two binding sites. The epitope can be on the same or different tau molecules. Some antibodies of the invention specifically bind to a peptide consisting of residues 307-312 of tau, namely residues QIVYKP (SEQ ID NO: 57). Some antibodies of the invention specifically bind to a peptide consisting of residues 391-397 of tau, namely EIVYKSP (SEQ ID NO: 58). Some antibodies of the invention specifically bind to a peptide consisting of tau residues 391396, namely EIVYKS (SEQ ID NO: 277). Certain antibodies of the invention specifically bind to a peptide consisting of the (Q/E)IVYK(S/P) consensus motif (SEQ ID NO: 56). 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 tau in unphosphorylated form, as well as in a form in which one or more phosphorylation-sensitive residues are phosphorylated. The invention also provides antibodies that bind to the same epitope as any of the above antibodies, for example, the 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3 epitope. Also included are antibodies that compete for binding to tau with any of the above antibodies, such as, for example, those that compete with 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. In one embodiment, antibodies that bind to the same epitope as a reference antibody, such as 9F5, or compete with a reference antibody, have one or more functional properties, such as the ability to inhibit the internalization of tau in neuronal cells. Optionally, such a property is possessed to the same extent, within the limits of experimental error, or greater than the reference antibody.

The invention provides an antibody that competes with 9F5 for binding to tau and reduces tau-induced toxicity in neurons. The invention provides an antibody that competes with 9F5 for binding to tau and has increased resistance to agitation stress. Examples of humanized 9F5 antibodies that compete with 9F5 for binding to tau and have increased resistance to agitation stress are L27cS/L37Q/M51G/L54R (DIM2), also known as hu9F5VHv9/hu9F5VLv8_DIM2 SEQ ID NO: 127/ SEQ ID NO: 133, L27cG/L37G/M51G/L54T (DIM14), also known as hu9F5VHv9/hu9F5VLv8_DIM14 SEQ ID NO: 127/ SEQ ID NO: 145 and L27cG/L37G/M51G/L54R (DIM13), also known as hu9F5VHv9/hu9F5V Lv8_DIM13 SEQ ID NO: 127/ SEQ ID NO: 144. The invention provides an antibody that competes with 9F5 for binding to tau and has increased resistance to low pH stress. Examples of humanized 9F5 antibodies that compete with 9F5 for binding to tau and have increased resistance to low pH stress are L27cS/L37Q/M51G/L54R (DIM2), also known as hu9F5VHv9/hu9F5VLv8_DIM2 SEQ ID NO: 127/SEQ ID NO : 133, L27cD/L37Q/M51G/L54R (DIM6), also known as hu9F5VHv9/hu9F5VLv8_DIM6 SEQ ID NO: 127/ SEQ ID NO: 137 and L27cG/L37G/M51G/L54R (DIM13), also known as hu9F5VHv9/hu9F5V Lv8_DIM13 SEQ ID NO: 127 / SEQ ID NO: 144. Examples of humanized 9F5 antibodies that compete with 9F5 for binding

- 37 047155 with tau and have increased resistance to stirring stress and increased resistance to low pH stress are DIM 2 [hu9F5VHv9/hu9F5VLv8_DIM2 SEQ ID NO: 127/SEQ ID NO: 133], DIM6 [hu9F5VHv9/hu9F5VLv8_DIM6 SEQ ID NO : 127/SEQ ID NO: 137], DIM7 [hu9F5VHv9/hu9F5VLv8_DIM7 SEQ ID NO: 127/ SEQ ID NO: 138], DIM8 [hu9F5VHv9/hu9F5VLv8_DIM8 SEQ ID NO: 127/ SEQ ID NO: 139], DIM13 [hu9F5VHv9/ hu9F5VLv8_DIM13 SEQ ID NO: 127/ SEQ ID NO: 144], DIM18 [hu9F5VHv9/hu9F5VLv8_DIM18 SEQ ID NO: 127/SEQ ID NO: 149], DIM28

[hu9F5VHv9/hu9F5VLv8_DIM28 SEQ ID NO: 127/SEQ ID NO: 159 and DIM30

[hu9F5VHv9/hu9F5VLv8_DIM30 SEQ ID NO: 127/ SEQ ID NO: 161].

The invention provides an antibody that competes with 9F5 for binding to tau and has a reduced tendency to aggregate under high concentration conditions. An example of an antibody that competes with 9F5 for binding to tau and has a reduced tendency to aggregate under high concentration conditions is L37Q/M51G/L54R in combination with the parent leucine at position L27c (DIM27), also known as hu9F5VHv9/hu9F5VLv8_DIM27 SEQ ID NO: 127/SEQ ID NO: 158.

The above antibodies can be produced de novo by immunization with a tau peptide containing or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), containing the amino acid sequence EIVYKSP (SEQ ID NO: 58), containing the amino acid sequence EIVYKS (SEQ ID NO: 277 ), or containing or consisting of the amino acid sequence (Q/E)IVYK(S/P) (SEQ ID NO: 56), or by immunization with a full-length tau polypeptide or a fragment thereof containing such residues and screening for specific binding to a peptide comprising such leftovers. Such tau peptides are preferably attached to a heterologous conjugate molecule that helps elicit an antibody response to the peptide. The attachment can be direct or through a spacer peptide or amino acid. Cysteine is used as a spacer amino acid because its free SH group facilitates the attachment of the 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 serves to provide a T cell epitope that helps induce an antibody response to the peptide. Several carriers are commonly used, notably keyhole limpet hemocyanin (KLH), egg albumin, and bovine serum albumin (BSA). Peptide spacers can be added to a peptide immunogen as part of solid phase peptide synthesis. Carriers are usually added by chemical crosslinking. Some examples of chemical crosslinkers that can be used include cross-linking N-maleimido-6-aminocaproyl ester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see, for example, Harlow, E. et al., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988; Sinigaglia et al., Nature, 336:778-780 (1988); ; Hammer et al., Cell 74:197-203 (1993); Falk K. et al., Immunogenetics, 39:230-242 (1994); -3373 (1998)). The carrier and spacer, if present, can 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. Hybridoma supernatants can be tested for the ability to bind tau peptide containing or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), a peptide containing or consisting of the amino acid sequence EIVYKSP (SEQ ID NO: 58), a peptide containing or consisting of the amino acid sequence EIVYKS (SEQ ID NO: 277), or a peptide containing or consisting of the amino acid sequence (Q/E)IVYK(S/P) (SEQ ID NO: 56) and/or phosphorylated and non-phosphorylated forms of tau, such as, for example, the full-length tau isoform at position 404 in its phosphorylated form. The peptide can be attached to a carrier or other label to facilitate screening analysis. In this case, the carrier or tag is preferably different from the combination of spacer and carrier molecule used for immunization to eliminate antibodies specific for the spacer or carrier rather than the tau peptide. Any of the tau isoforms can be used.

The antibody designated 9F5 is an example of an antibody that specifically binds to tau. Unless otherwise obvious from the context, reference to 9F5 should be understood to refer to any of the murine, chimeric, venous or humanized forms of this antibody. The antibody was deposited as [DEPOSIT NUMBER]. This antibody specifically binds a peptide containing or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), a peptide containing or consisting of the amino acid sequence EIVYKSP (SEQ ID NO: 58), or a peptide containing or consisting of the amino acid sequence (Q/ E)IVYK(S/P) (SEQ ID NO: 56). The Kabat/Chothia composite CDRs of the 9F5 heavy chain are designated SEQ4 ID NOs: 8, 9 and 10, respectively, and the Kabat light chain CDRs of 9F5 are designated SEQ ID NOs: 12, 13 and 1, respectively.

Additional antibodies that compete with 9F5 for binding to tau and/or bind the same or overlapping epitope as 9F5 have been isolated, designated 10C12, 2D11, 12C4, 17C12, and 14H3, and produced by hybridomas with the same names. 10C12 has mature heavy and light chain variable regions (after signal peptide cleavage) characterized by SEQ ID NO: 7 and SEQ ID NO: 11, respectively. Unless otherwise obvious from the context, reference to 10C12 should be understood

- 38 047155 as referring to any of the murine, chimeric, venous and humanized forms of this antibody. 10C12 deposited as [DEPOSIT NUMBER]. 10C12 is further characterized by its ability to bind both non-pathological and pathological forms and conformations of tau, as well as misfolded/aggregated forms of tau. 10C12 binds structural features such as tau tangles and dystrophic neurites in Alzheimer's disease tissue and precipitates both monomeric and aggregated tau from Alzheimer's disease extracts.

2D 11 has mature heavy and light chain variable regions (after signal peptide cleavage) characterized by SEQ ID NO: 7 and SEQ ID NO: 11, respectively. Unless otherwise obvious from the context, reference to 2D11 should be understood to refer to any of the murine, chimeric, venous and humanized forms of this antibody. 2D11 is deposited as [DEPOSIT NUMBER]. 2D11 is further characterized by its ability to bind both non-pathological and pathological forms and conformations of tau, as well as misfolded/aggregated forms of tau. 2D11 binds structural features such as tau tangles and dystrophic neurites in Alzheimer's disease tissue and precipitates both monomeric and aggregated tau from Alzheimer's disease extracts.

12C4 has mature heavy and light chain variable regions (after signal peptide cleavage) characterized by SEQ ID NO: 219 and SEQ ID NO: 11, respectively. Unless otherwise obvious from the context, reference to 12C4 should be understood to refer to any of the murine, chimeric, venous and humanized forms of this antibody. 12C4 deposited as [DEPOSIT NUMBER]. 12C4 is further characterized by its ability to bind both non-pathological and pathological forms and conformations of tau, as well as misfolded/aggregated forms of tau. 12C4 binds structural features such as tau tangles and dystrophic neurites in Alzheimer's disease tissue and precipitates both monomeric and aggregated tau from Alzheimer's disease extracts.

17C12 has mature heavy and light chain variable regions (after signal peptide cleavage) characterized by SEQ ID NO: 225 and SEQ ID NO: 228, respectively. Unless otherwise obvious from the context, reference to 17C12 should be understood to refer to any of the murine, chimeric, venous and humanized forms of this antibody. 17C12 deposited as [DEPOSIT NUMBER]. 17C12 is further characterized by its ability to bind both non-pathological and pathological forms and conformations of tau, as well as misfolded/aggregated forms of tau.

14H3 has mature heavy and light chain variable regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 240 and SEQ ID NO: 244, respectively. Unless otherwise obvious from the context, reference to 14H3 should be understood to refer to any of the murine, chimeric, venous and humanized forms of this antibody. 14H3 is deposited as [DEPOSIT NUMBER]. 14H3 is further characterized by its ability to bind both non-pathological and pathological forms and conformations of tau, as well as misfolded/aggregated forms of tau. 14H3 binds structural features such as tau tangles and dystrophic neurites in Alzheimer's disease tissue.

Alignments of the mature heavy chain variable regions of murine antibodies 9F5, 10C12, 2D11, 12C4, 14H3 and 17C12 are depicted in FIG. 24, and alignments of the mature light chain variable regions of murine antibodies 9F5, 10C12, 2D11, 12C4, 14H3 and 17C12 are depicted in FIG. 25. The amino acid sequence of the mature heavy chain variable region of the mouse antibody 10C12 has 100% sequence identity with the sequence of the mouse antibody 9F5, and the mature light chain variable region of the mouse antibody 10C12 has 100% sequence identity with the sequence of the mouse antibody 9F5. The amino acid sequence of the mature heavy chain variable region of mouse antibody 2D11 has 100% sequence identity with that of mouse antibody 9F5, and the mature light chain variable region of mouse antibody 2D11 has 100% sequence identity with that of mouse antibody 9F5. The amino acid sequence of the mature heavy chain variable region of mouse antibody 12C4 has 96.6% sequence identity with that of mouse antibody 9F5, and the mature light chain variable region of mouse antibody 12C4 has 100% sequence identity with that of mouse antibody 9F5. The amino acid sequence of the mature heavy chain variable region of mouse antibody 17C12 has 95.9% sequence identity with that of mouse antibody 9F5, and the mature light chain variable region of mouse antibody 17C12 has 70.5% sequence identity with that of mouse antibody 9F5. The amino acid sequence of the mature heavy chain variable region of mouse antibody 14H3 has 35.0% sequence identity with that of mouse antibody 9F5, and the mature light chain variable region of mouse antibody 14H3 has 73.2% sequence identity with that of mouse antibody 9F5.

Optionally, the antibodies of the invention do not include the 10C12 antibody. Optionally, the antibodies of the invention do not include the 2D11 antibody. Optionally, the antibodies of the invention do not include the 12C4 antibody. Optionally, the antibodies of the invention do not include the 17C12 antibody. Optional antibodies

- 39 047155 according to the invention do not include the 14H3 antibody.

Some antibodies of the invention bind to the same or overlapping epitope as the antibody designated 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. The sequences of the mature heavy and light chain variable regions of this 9F5 are designated SEQ ID NO: 7 and 11, respectively. The sequences of the mature heavy and light chain variable regions of 10C12 are designated SEQ ID NO: 7 and 11, respectively. The sequences of the mature heavy and light chain variable regions of 2D11 are designated SEQ ID NO: 7 and 11, respectively. The sequences of the mature 12C4 heavy and light chain variable regions are designated SEQ ID NO: 219 and 11, respectively. The sequences of the mature variable regions of the heavy and light chains of 17C12 are designated SEQ ID NO: 225 and 228, respectively. The sequences of the mature variable regions of the heavy and light chains of 14H3 are designated SEQ ID NO: 240 and 244, respectively. Other antibodies having such binding specificity can be produced by immunizing mice with tau or a portion thereof, including the desired epitope (e.g., a tau peptide containing or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), a tau peptide containing or consisting of the amino acid sequence EIVYKSP (SEQ ID NO: 58), a tau peptide containing or consisting of the amino acid sequence EIVYKS (SEQ ID NO: 277), or a tau peptide containing or consisting of the amino acid sequence (Q/E)IVYK (S/P) (SEQ ID NO: 56)) and screening the resulting antibodies for binding to tau, optionally in competition with an antibody having the mouse variable regions 9F5 (IgG1/kappa), 10C12 (IgG2a/kanpa), 2D11 (IgG2a /kappa), 12C4 (IgG2a/kappa), 17C12 (IgG2a/kanpa) or 14H3 (IgG2a/kappa). The tau fragments, including the desired epitope, can be bound to a carrier that helps elicit an antibody response to the fragment and/or combined with an adjuvant that helps elicit such a response. Such antibodies can be screened for differential binding to tau or a fragment thereof compared to mutants at specified residues. Screening for such mutants more accurately determines binding specificity, allowing the identification of antibodies whose binding is inhibited by mutagenesis of specific residues and which are likely to have the functional properties of other exemplified antibodies. Mutations can be systematic substitutions of alanine (or serine if alanine is already present) one residue at a time or at wider intervals throughout the target or throughout the region where the epitope is known to reside. If the same set of mutations significantly reduces the binding of two antibodies, the two antibodies bind the same epitope.

Antibodies having binding specificity to the selected murine antibody (eg, 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3) can also be produced using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for the production of human antibodies. This method uses the variable region of the heavy or light chain of a selected mouse antibody as starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is created whose members display the same light chain variable region (ie, the mouse starting material) and a different heavy chain variable region. 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 10 ⁹ M ^-1 ). The heavy chain variable region of this phage then serves as starting material for generating additional phage libraries. In this library, each phage displays the same heavy chain variable region (ie, the region identified from the first library displayed) and a different light chain variable region. Light chain variable regions can be obtained, for example, from a library of rearranged human light chain variable regions. Again, a phage that exhibits strong specific binding to tau or a fragment thereof is selected. The resulting antibodies generally have the same or similar epitope specificity as the mouse starting material.

The Kabat/Chothia composite CDRs of the 9F5 heavy chain are designated SEQ ID NO: 8-10, respectively, and the Kabat light chain CDRs of 9F5 are designated SEQ ID NO: 12-14, respectively.

The Kabat/Chothia composite CDRs for heavy chain 10C12 are designated SEQ ID NO: 8-10, respectively, and the Kabat light chain CDRs for 10C12 are designated SEQ ID NO: 12-14, respectively.

The Kabat/Chothia composite CDRs of the 2D11 heavy chain are designated SEQ ID NOs: 8-10, respectively, and the Kabat light chain 2D11 CDRs are designated SEQ ID NOs: 12-14, respectively.

Table 2 refers to CDRs 9F5, 10C12 and 2D11 as defined by Kabat, Chothia, Chothia and Kabat composite definition (also referred to herein as Kabat/Chothia composite definition), AbM and Contact definitions.

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Table 2. CDR 9F5, 10C12 and 2D11, as defined by Kabat, Chothia, composite definition by Chothia and Kabat, definitions by AbM and Contact

A loop Kabat Chothia Composite no Chothia definition and Kabat AbM Contact L1 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L30-L36 SEQ ID NO:47 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:48 L3 L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L96 SEQ ID NO:49 H1 H31-H35B SEQ ID NO:40 H26-H32 SEQIDNO:41 H26-H35B SEQIDNO:8 H26-H35B SEQ ID NO:8 H30-H35B SEQ ID NO:44 H2 H50-H65 SEQ ID NO:9 H52-H56 SEQ ID NO:42 H50-H65 SEQ ID NO:9 H50-H58 SEQIDNO:43 H47-H58 SEQ ID NO:45 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:46

The Kabat/Chothia composite 12C4 heavy chain CDRs are designated SEQ ID NOs: 8, 220 and 10, respectively, and the Kabat 12C4 light chain CDRs are designated SEQ ID NOs: 12-14, respectively.

Table 3 refers to CDR 12C4 as defined by Kabat, Chothia, Chothia and Kabat composite definition (also referred to herein as Kabat/Chothia composite definition), AbM and Contact definitions.

- 41 047155

Table 3. CDR 12C4, as defined by Kabat, Chothia, composite definition

Chothia and Kabat, definitions by AbM and Contact

A loop Kabat Chothia Composite no Chothia definition and Kabat AbM Contact L1 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L24-L34 SEQ ID NO: 12 L30-L36 SEQ ID NO:47 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:48 L3 L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L97 SEQ ID NO: 14 L89-L96 SEQ ID NO:49 H1 H31-H35B SEQ ID NO:40 H26-H32 SEQIDNO:41 H26-H35B SEQ ID NO:8 H26-H35B SEQ ID NO:8 H30-H35B SEQ ID NO:44 H2 H50--H65 SEQ ID NO:220 H52-H56 SEQ ID NO:42 H50--H65 SEQ ID NO:220 H50--H58 SEQ ID NO:257 H47-H58 SEQ ID NO:258 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:46

The 17C12 heavy chain Kabat/Chothia composite CDRs are designated SEQ ID NO: 226, 227 and 10, respectively, and the 17C12 light chain Kabat CDRs are designated SEQ ID NO: 229231, respectively.

Table 4 indicates CDR 17C12 as defined by Kabat, Chothia, Chothia and Kabat composite definition (also referred to herein as Kabat/Chothia composite definition), AbM and Contact definitions.

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Table 4. CDR 17C12, as defined by Kabat, Chothia, composite definition

Chothia and Kabat, definitions by AbM and Contact

A loop Kabat Chothia Composite no Chothia definition and Kabat AbM Contact L1 L24-L34 SEQ ID NO:229 L24-L34 SEQ ID NO:229 L24-L34 SEQ ID NO:229 L24-L34 SEQ ID NO:229 L30-L36 SEQ ID NO:262 L2 L50-L56 SEQ ID NO:230 L50-L56 SEQ ID NO:230 L50-L56 SEQ ID NO:230 L50-L56 SEQ ID NO:230 L46-L55 SEQ ID NO:263 L3 L89-L97 SEQ ID NO:231 L89-L97 SEQIDNO:231 L89-L97 SEQ ID NO:231 L89-L97 SEQ ID NO:231 L89-L96 SEQ ID NO:264 H1 H31-H35B SEQ ID NO:40 H26-H32 SEQ ID NO:259 H26-H35B SEQ ID NO:226 H26-H35B SEQ ID NO:226 H30-H35B SEQ ID NO:44 H2 H50-H65 SEQ ID NO:227 H52-H56 SEQ ID NO:42 H50-H65 SEQ ID NO:227 H50-H58 SEQ ID NO:260 H47-H58 SEQ ID NO:261 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:46

The Kabat/Chothia composite 14H3 heavy chain CDRs are designated SEQ ID NOs: 241-243, respectively, and the Kabat 14H3 light chain CDRs are designated SEQ ID NOs: 245-247, respectively.

Table 5 indicates CDR 14H3 as defined by Kabat, Chothia, Chothia and Kabat composite definition (also referred to herein as Kabat/Chothia composite definition), AbM and Contact definitions.

- 43 047155

Table 5. CDR 14H3, as defined by Kabat, Chothia, composite definition

Chothia and Kabat, definitions by AbM and Contact

A loop Kabat Chothia Composite no Chothia definition and Kabat AbM Contact L1 L24-L34 SEQ ID NO:245 L24-L34 SEQ ID NO:245 L24-L34 SEQ ID NO:245 L24-L34 SEQ ID NO:245 L30-L36 SEQ ID NO:272 L2 L50-L56 SEQ ID NO:246 L50-L56 SEQ ID NO:246 L50-L56 SEQ ID NO:246 L50-L56 SEQ ID NO:246 L46-L55 SEQ ID NO:273 L3 L89-L97 SEQ ID NO:247 L89-L97 SEQ ID NO:247 L89-L97 SEQ ID NO:247 L89-L97 SEQ ID NO:247 L89-L96 SEQ ID NO:274 H1 H31-H35B SEQ ID NO:265 H26-H32 SEQ ID NO:266 H26-H35B SEQ ID NO:241 H26-H35B SEQ ID NO:241 H30-H35B SEQ ID NO:269 H2 H50-H65 SEQ ID NO:242 H52-H56 SEQ ID NO:267 H50-H65 SEQ ID NO:242 H50-H58 SEQ ID NO:268 H47-H58 SEQ ID NO:270 NZ H95-H102 SEQ ID NO:243 H95-H102 SEQ ID NO:243 H95-H102 SEQ ID NO:243 H95-H102 SEQ ID NO:243 H93-H101 SEQ ID NO:271

Other antibodies can be produced by mutagenesis of cDNA encoding the heavy and light chains of an exemplified antibody, such as 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3 in the amino acid sequence of the mature severe variable regions and/or light chain and retaining their functional properties, and/or which differ from the corresponding antibody by a small number of functionally non-essential amino acid substitutions (eg, conservative substitutions), deletions or insertions, are also included in the invention. Also included are monoclonal antibodies having at least one or all six CDRs, as defined by any conventional definition, but preferably by Kabat, that are 90%, 95%, 99% or 100% identical to the corresponding CDRs 9F5, 10C12, 2D11, 12C4 , 17С12 or 14Н3.

The invention also provides antibodies having some or all (eg, 3, 4, 5 and 6) of the CDRs, all or substantially 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. Such antibodies may include a heavy chain variable region that has at least two, and usually all three, CDRs in whole or substantially from a heavy chain variable region 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3 and/or a light chain variable region , which has at least two, and usually all three, CDRs wholly or substantially from the light chain variable region 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. Antibodies can include both heavy and light chains. A CDR is essentially derived from the corresponding 9F5 CDR if it contains no more than 4, 3, 2, or 1 substitution, insertion, or deletion, except that a CDR-H2 (if defined by Kabat) may have no more than 6, 5, 4, 3, 2 or 1 substitution, insertion or deletion. Such antibodies may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to 9F5, 10C12, 2D11, 12C4, 17C12, or 14H3 in the amino acid sequence of the mature severe and severe variable regions. /or light chain and retain its functional properties, and/or differ from 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3 by a small number of functionally non-essential amino acid substitutions (for example, conservative substitutions), deletions or insertions.

Some antibodies identified by such assays may bind to monomeric, misfolded, aggregated, phosphorylated or unphosphorylated forms of tau or other forms. Likewise, some antibodies are immune

- 44 047155 noreactive against non-pathological and pathological forms and conformations of tau.

The invention additionally provides means for specific binding to a peptide consisting of (Q/E)IVYK(S/P) residues (SEQ ID NO: 56), QIVYKP residues (SEQ ID NO: 57), EIVYKSP residues (SEQ ID NO: 58) or EIVYKS residues (SEQ ID NO: 277). An example of an agent is an antibody comprising a heavy chain CDR of SEQ ID NO: 8-10 and a light chain CDR of SEQ ID NO: 12-14. An example of an agent is an antibody comprising heavy chain CDRs SEQ ID NOs: 8, 220 and 10 and light chain CDRs SEQ ID NOs: 12-14. An example of an agent is an antibody comprising the heavy chain CDRs SEQ ID NOs: 226, 227 and 10, and the light chain CDRs SEQ ID NOs: 229-231. An example of an agent is an antibody comprising a heavy chain CDR of SEQ ID NO: 241-243 and a light chain CDR of SEQ ID NO: 245-247.

B. Non-human antibodies

Preparation of other non-human antibodies, for example, mouse, guinea pig, primate, rabbit or rat antibodies to tau or a fragment thereof (for example, a peptide comprising the amino acid sequence QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or (Q/E)IVYK(S/P) (SEQ ID NO: 56)) can be accomplished, for example, by immunizing an animal with tau or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated here for all purposes). Optionally, the immunogen may be 383 amino acid human tau (4R0N). Optionally, the immunogen may be human tau containing the P301S mutation. Optionally, the immunogen may be human tau, wherein the human tau is recombinant His-tagged at the N-terminus. Optionally, the animal is immunized with a tau fragment containing the peptide represented by (Q/E)IVYK(S/P) (SEQ ID NO: 56) linked to a vehicle. Optionally, the peptide is QIVYKP (SEQ ID NO: 57) or EIVYKSP (SEQ ID NO: 58) or EIVYKS (SEQ ID NO: 277). Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or otherwise combined with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvant can be used, as described below. Freund's complete adjuvant followed by incomplete adjuvant can be used to immunize laboratory animals. To obtain polyclonal antibodies, rabbits or guinea pigs are usually used. Mice are commonly used to produce monoclonal antibodies. Antibodies are screened for specific binding to tau or an epitope within tau (eg, QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or (Q/E)IVYK( S/P) (SEQ ID NO: 56)). Optionally, screening can be performed against 15 amino acid peptides containing QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or any other consensus motif provided by (Q/E) IVYK(S/P) (SEQ ID NO: 56). Optionally, the peptides contain QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277). Such screening can be accomplished by determining the binding of the antibody to a set of tau variants, such as tau variants containing or consisting of amino acid residues 307-312, 391-397, or 391-396 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 grafted onto human acceptor antibody sequences (see, for example, Queen, US 5530101 and 5585089; Winter, US 5225539; Carter, US 6407213; US Adair, 5859205 and Foote , US 6881557). The acceptor antibody sequences may be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having at least three, four, five or all of the CDRs, wholly or substantially from the donor antibody and variable region and constant region framework sequences, if present, wholly or substantially from human antibody sequences . Likewise, the humanized heavy chain has at least one, two, and typically all three CDRs derived entirely or substantially from the donor antibody heavy chain, and the heavy chain variable region framework sequence and the heavy chain constant region, if present, are derived essentially from the framework sequence human heavy chain variable region and human constant region. Likewise, the humanized light chain has at least one, two, and typically all three CDRs derived entirely or substantially from the light chain of the donor antibody, and the light chain variable region framework sequence and the light chain constant region, if present, are derived substantially from the framework sequence human light chain variable region and human constant region. Unlike nanobodies and dAbs, a humanized antibody contains a humanized heavy chain and a humanized light chain. The CDR in a humanized antibody is substantially similar to the corresponding CDR in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (as determined according to any conventional definition)

- 45 047155 division, but preferably defined by Kabat) are identical between the corresponding CDRs. The antibody variable chain framework or antibody constant region framework sequences are essentially derived from a human variable region framework sequence or a human constant region framework, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical . To be classified as humanized according to the 2014 World Health Organization (WHO) International Nonproprietary Name (INN) definition of humanized antibody, the antibody must have at least 85% identity with human germline antibody sequences (i.e. to somatic hypermutation). Mixed antibodies are antibodies for which one chain of the antibody (eg, the heavy chain) meets the threshold value, but the other chain (eg, the light chain) does not meet the threshold value. An antibody is classified as chimeric if neither chain meets the threshold, even if the framework variable regions for both chains were essentially human with some back mutations in mice. See Jones et al. (2016) The INNs and outs of antibody nonproprietary names, mAbs 8:1, 1-9, DOI: 10.1080/19420862.2015.1114320. See also WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: https://www.who.int/medicines/services/inn/BioRev2014.pdf ), is incorporated herein by reference. For the avoidance of doubt, the term humanized as used herein is not intended to be limited to the 2014 WHO INN definition of humanized antibodies. Some of the humanized antibodies proposed herein have at least 85% sequence identity with human germline sequences. and some of the humanized antibodies provided herein have less than 85% sequence identity to human germline sequences. Some of the heavy chains of the humanized antibodies provided herein have about 60 to 100% sequence identity to human germline sequences, for example, in the range of about 60 to 69%, 70 to 79%, 80 to 84%, or from 85 to 89%. Some heavy chains do not meet the 2014 WHO INN definition and have, for example, about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% 75%, 76%, 77%, 78%, 79%, 80%, 81% or 82%, 83 or 84% sequence identity to human germline sequences, while other heavy chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or more sequence identity with human germline sequences. Some of the light chains of the humanized antibodies provided herein have from about 60 to 100% sequence identity with human germline sequences, for example, in the range from about 80 to 84% or from 85 to 89%. Some light chains do not meet the 2014 WHO INN definition and have, for example, about 81%, 82%, 83% or 84% sequence identity with human germline sequences, while other light chains meet the 2014 WHO INN definition and have about 85%, 86%, 87%, 88%, 89% or more sequence identity with human germline sequences. Certain humanized antibodies provided herein that are chimeric as defined by the 2014 WHO INN have heavy chains with less than 85% identity to human germline sequences paired with light chains having less than 85% identity to germline sequences. human lines. Certain humanized antibodies provided herein are mixed as defined by the 2014 WHO INN, for example, having a heavy chain with at least 85% sequence identity to human germline sequences paired with a light chain having less than 85% sequence identity with human germline sequences, or vice versa. Certain humanized antibodies provided herein meet the 2014 WHO INN definition of humanized and have a heavy chain with at least 85% sequence identity to human germline sequences paired with a light chain with at least 85% sequence identity. sequence identity with human germline sequences. Examples of 12C4 antibodies that meet the 2014 WHO INN definition of humanized include antibodies having a mature heavy chain having the amino acid sequence of SEQ ID NO: 221 or SEQ ID NO: 222 paired with a mature light chain sequence having the amino acid sequence of SEQ ID NO: 223 or SEQ ID NO: 224. Examples of 14H3 antibodies that meet the 2014 WHO INN definition of humanized include antibodies having a mature heavy chain with the amino acid sequence SEQ ID NO: 248 or SEQ ID NO: 249 paired with a mature light chain sequence having the amino acid sequence of SEQ ID NO: 251 or SEQ ID NO: 252. Certain humanized antibodies provided herein meet the 2014 WHO INN definition of mixed. Examples of 9F5 antibodies that meet the 2014 WHO INN definition as mixed include antibodies having a mature heavy chain with the amino acid sequence SEQ ID NO: 15-22 and SEQ ID NO: 127-128 paired with

- 46 047155 mature light chain sequence having the amino acid sequence of SEQ ID NO: 26-29 or SEQ ID NO: 130-131. Examples of 10C12 antibodies that meet the 2014 WHO INN definition as mixed include antibodies having a mature heavy chain amino acid sequence of SEQ ID NO: 214 or SEQ ID NO: 215 paired with a mature light chain sequence having the amino acid sequence SEQ ID NO: 216 or SEQ ID NO: 217. Examples of 17C12 antibodies that meet the 2014 WHO INN definition as mixed include antibodies having a mature heavy chain with the amino acid sequence SEQ ID NO: 232 or SEQ ID NO: 233 paired with a mature light chain sequence having the amino acid sequence of SEQ ID NO: 235. Additional humanized 9F5 antibodies of the invention include antibodies having a mature heavy chain having any of the amino acid sequence of SEQ ID NO: 15-22 and SEQ ID NO: 127-128 paired with a mature light chain having any amino acid sequence from SEQ ID NO: 23-25. Additional humanized 17C12 antibodies of the invention include antibodies having a mature heavy chain of the amino acid sequence SEQ ID NO: 232 or SEQ ID NO: 233 paired with a mature light chain of the amino acid sequence SEQ ID NO: 234.

Although humanized antibodies often include all six CDRs (defined by any conventional definition, but preferably defined by Kabat) from a murine antibody, they can also be made using fewer than all CDRs (e.g., at least 3, 4, or 5 CDRs) from mouse antibody (eg, 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 a subset of CDR residues required for binding, called SDRs, are required to maintain binding in the humanized antibody. CDR residues that are not antigen-contacting and not included in the SDR can be identified based on previous studies (e.g., residues H60-H65 in the H2 CDR are often not required) from Kabat CDR regions located outside the Chothia hypervariable loops (Chothia, J Mol. Biol. 196:901, 1987) by molecular modeling and/or empirically, or as described by Gonzales et al., Mol. Immunol. 41: 863, 2004. In such humanized antibodies, at positions in which one or more donor CDR residues are missing or in which entire donor CDRs are omitted, the amino acid occupying that position may be the amino acid occupying the corresponding position (Kabat numbering) in acceptor antibody sequences. The number of such acceptor to donor amino acid substitutions in the CDR that must be included reflects a balance between competing considerations. Such substitutions are potentially useful in reducing the number of murine amino acids in a humanized antibody and therefore reducing potential immunogenicity and/or compliance with the WHO INN definition of humanized. However, substitutions can also cause changes in affinity, so it is preferable to avoid significant reductions in affinity. The positions to be replaced in the CDR and the amino acids to be replaced can also be selected empirically.

The human acceptor antibody sequences may be selected from a variety of known human antibody sequences to provide a high degree of sequence identity (eg, 65-85% identity) between the human acceptor sequence variable region frameworks and the corresponding antibody donor chain variable region frameworks.

Some humanized and chimeric antibodies have the same (within experimental error) or improved functional properties, for example, binding affinity to human tau, inhibition of tau internalization into neurons, as described in the examples, as the murine antibodies from which they were derived. For example, some humanized and chimeric antibodies have a binding affinity that is a factor of 3, 2, or 1 from the murine antibody from which they were derived, or an affinity that is indistinguishable within experimental error. Some humanized and chimeric antibodies inhibit the internalization of tau into neurons, as described in the Examples, by a factor of 3, 2, or 1 from the mouse antibody from which they were derived, or inhibit the same thing, within experimental error, as the mouse antibody from which they were received.

An example of a 9F5 heavy chain acceptor sequence is the mature human heavy chain variable region of humanized Fab 48G7 with PDB accession code 2RCS-VH_huFrwk (SEQ ID NO: 32). An example of a 9F5 heavy chain acceptor sequence is the mature human heavy chain, GenBank AAN16432-VH_huFrwk (SEQ ID NO: 31). The variable domains of Fab 9F5 and 48G7 also have the same length with respect to the CDR-H1, H2 loops. An example of a 9F5 heavy chain acceptor sequence is the mature human heavy chain variable region, IMGT No. IGHV169-2*01 (SEQ ID NO: 33). Chothia CDR-H1 in IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33) is in canonical class 1 and Chothia CDR-H2 is in canonical class 2. IMGT No. IGHV1-692*01 (SEQ ID NO: 33) belongs to human heavy chain subgroup 1. An example of an acceptor

- 47047155 of the 9F5 light chain sequence is the mature human light chain variable region 1911357B-VL_huFrwk (SEQ ID NO: 36). An example of a 9F5 light chain acceptor sequence is the mature human light chain variable region, CAB51297-VL_huFrwk (SEQ ID NO: 35). The light chain variable domain of antibodies 9F5, CAB51297 and 1911357B also has the same length with respect to the CDR-L1, L2 and L3 loops. An example of a 9F5 light chain acceptor sequence is the mature human light chain variable region with IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). CDR-L1 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 4. CDR-L2 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. Chothia CDR-L3 IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to the subgroup 2 kappa people.

An example of a 10C12 heavy chain acceptor sequence is mature human CAC20421 (SEQ ID NO: 218). The variable domains VH 10C12 and CAC20421 also have the same length with respect to the CDR-H1, H2 loops. An example of a 10C12 heavy chain acceptor sequence is the mature human heavy chain variable region, IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33). Chothia CDR-H1 in IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33) is in canonical class 1 and Chothia CDR-H2 is in canonical class 2. IMGT No. IGHV1-69-2*01 ( SEQ ID NO: 33) belongs to human heavy chain subgroup 1. An example of a 10C12 light chain acceptor sequence is the mature human light chain variable region CAB51297-VL_huFrwk (SEQ ID NO: 35). The light chain variable domain of antibodies 10C12 and CAB51297 also has the same length with respect to the CDR-L1, L2 and L3 loops. An example of a 10C12 light chain acceptor sequence is the mature human light chain variable region with IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). CDR-L1 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 4. CDR-L2 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. Chothia CDR-L3 IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to the subgroup 2 kappa people.

An example of a 12C4 heavy chain acceptor sequence is the mature human heavy chain variable region CAC20421-VH_huFrwk (SEQ ID NO: 218). The variable domains VH 12C4 and CAC20421 also have the same length with respect to the CDR-H1, H2 loops. An example of a 12C4 heavy chain acceptor sequence is the mature human heavy chain variable region, IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33). Chothia CDR-H1 in IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33) is in canonical class 1 and Chothia CDR-H2 is in canonical class 2. IMGT No. IGHV1-69-2*01 ( SEQ ID NO: 33) belongs to human heavy chain subgroup 1. An example of a 12C4 light chain acceptor sequence is the mature human light chain variable region CAB51297-VL_huFrwk (SEQ ID NO: 35). The light chain variable domain of antibodies 12C4 and CAB51297 also has the same length with respect to the CDR-L1, L2 and L3 loops. An example of a 12C4 light chain acceptor sequence is the mature human light chain variable region with IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). CDR-L1 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 4. CDR-L2 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. Chothia CDR-L3 IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to the subgroup 2 kappa people.

An example of a 17C12 heavy chain acceptor sequence is the mature human heavy chain variable region CAC20421-VH_huFrwk (SEQ ID NO: 218). The variable domains of the heavy chains 17C12 and CAC20421 also have the same length with respect to the CDR-H1, H2 loops. An example of a 17C12 heavy chain acceptor sequence is the mature human heavy chain variable region, IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33). Chothia CDR-H1 in IMGT No. IGHV1-69-2*01 (SEQ ID NO: 33) is in canonical class 1 and Chothia CDR-H2 is in canonical class 2. IMGT No. IGHV1-69-2*01 ( SEQ ID NO: 33) belongs to human heavy chain subgroup 1. An example of a 17C12 light chain acceptor sequence is the mature human light chain variable region QDO16713-VL_huFrwk (SEQ ID NO: 238). The light chain variable domain of antibodies 17C12 and QDO16713 also has the same length with respect to the CDR-L1, L2 and L3 loops. An example of a 17C12 light chain acceptor sequence is the mature human light chain variable region with IMGT No. IGKV2-29*02 and IGKJ4*01 (SEQ ID NO: 239).

An example of a 14H3 heavy chain acceptor sequence is the mature human heavy chain variable region QDJ57937-VH_huFrwk (SEQ ID NO: 253). The VH variable domains 14H3 and QDJ57937 also have the same length with respect to the CDR-H1, H2 loops. An example of a 14H3 heavy chain acceptor sequence is the mature human heavy chain variable region, IGHV 1-70*04 and IGHJ4*01 (SEQ ID NO: 254). An example of a 14H3 light chain acceptor sequence is the mature human light chain variable region, ABC66914-VL_huFrwk (SEQ ID NO: 256). The light chain variable domain of antibodies 14H3 and ABC66914 also has the same length with respect to the CDR-L1, L2 and L3 loops. An example of a 14H3 light chain acceptor sequence is the mature human light chain variable region with IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:

- 48 047155

37). CDR-L1 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 4. CDR-L2 by Chothia IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. Chothia CDR-L3 IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to canonical class 1. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) belongs to the subgroup 2 kappa people.

If more than one human acceptor antibody sequence is selected, a composite or hybrid of these acceptors can be used, and the amino acids used at the various positions of the humanized light chain and heavy chain variable regions can be taken from any human acceptor antibody sequence used. For example, human mature heavy chain variable regions AAN16432-VH_huFrwk (SEQ ID NO: 31) and humanized 48G7 Fab with PDB accession code 2RCS-VH_huFrwk (SEQ ID NO: 32) were used as hybrid acceptor sequences to humanize the mature heavy chain variable region 9F5. Examples of positions where the two acceptors differ are position H1 (E or Q), H5 (V or Q), H1 1 (V or L), H12 (K or V), H20 (V or L), H23 ( K or T), H28 (T or N), H38 (R or K), H40 (A or R), H42 (G or E), H43 (K or Q), H48 (M or I), H54 (D or N), H66 (R or K), H69 (M or I), H75 (T or S), H76 (D or N), H80 (M or L), H81 (E or Q), H83 (R or T), H108 (L or T) or H109 (V or L). Humanized versions of the 9F5 heavy chain variable region may include any amino acid at any of these positions. The human germline sequence IMGT No. IGHV1-69-2*01 (SEQ ID NO: 25) was also used as an acceptor sequence to humanize the mature heavy chain variable region of 9F5. For example, the human mature light chain variable regions CAB51297-VL_huFrwk (SEQ ID NO: 35) and 1911357B-VL_huFrwk (SEQ ID NO: 36) were used as hybrid acceptor sequences to humanize the 9F5 mature light chain variable region. Examples of positions where the two acceptors differ are positions L7 (S or A), L8 (P or A), L9 (L or F), L11 (L or N), L15 (P or L), L17 (E or T), L18 (P or S), L30 (Y or I), L31 (N or T), L54 (R or L), L60 (D or N), L66 (G or E) or L74 (K or R)). Humanized versions of the 9F5 light chain variable region may include any amino acid at any of these positions. The human germline sequence IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) was also used as an acceptor sequence to humanize the mature 9F5 light chain variable region.

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

For example, when an amino acid differs between a mouse variable framework residue and a selected human variable framework residue, the human framework amino acid may be replaced by an equivalent murine antibody framework amino acid if the amino acid is reasonably expected to:

(1) non-covalently binds antigen directly;

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

(3) otherwise interacts with the CDR region (e.g., is within about 6 A of the CDR region) (e.g., identified by modeling a light or heavy chain on the solved structure of a known homologous immunoglobulin chain) or (4) is a residue involved in VL-VH interface.

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

Framework residues from classes (1)-(3) as defined by Queen, US 5530101, are sometimes alternately called canonical and vernier residues. Framework residues that help determine the conformation of the CDR loop are sometimes called canonical residues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton & Martin, J. Mol. Biol. 263:800-815 ( 1996)). Scaffolding residues that maintain the conformations of the antigen-binding loop and play a role in fine-tuning the matching of antibody to antigen are sometimes called vernier residues (Foote & Winter, J. Mol. Biol 224:487-499 (1992)).

Other scaffold residues that are candidates for replacement are residues that create a potential glycosylation site. Still other candidates for replacement are human acceptor framework amino acids, which are unusual for human immunoglobulin at this position. These amino acids can be replaced by amino acids from the equivalent position of the murine donor antibody or from the equivalent positions of more typical human immunoglobulins.

Other framework residues that are candidates for replacement are N

- 49 047155 terminal glutamine residues (Q), which can be replaced with glutamic acid (E) to minimize the potential for pyroglutamate conversion [Y. Diana Liu, et al., 2011, J. Biol. Chem., 286: 1121111217]. The conversion of glutamic acid (E) to pyroglutamate (pE) occurs more slowly than from glutamine (Q). Due to the loss of primary amine when glutamine is converted to pE, antibodies become more acidic. Incomplete conversion causes heterogeneity in the antibody, which can be observed as multiple peaks using charge-based analytical methods. Differences in heterogeneity may indicate insufficient process control.

Examples of humanized 9F5 heavy chain variable regions with N-terminal glutamine to glutamate substitutions are SEQ ID NO: 16 (hu9F5VHv2), SEQ ID NO: 17 (hu9F5VHv3), SEQ ID NO: 18 (hu9F5VHv4), SEQ ID NO: 19 (hu9F5VHv5 ), SEQ ID NO: 20 (hu9F5VHv6), SEQ ID NO: 21 (hu9F5VHv7), SEQ ID NO: 22 (hu9F5VHv8), SEQ ID NO: 109 (hu9F5VHv4_L80P), SEQ ID NO: 110 (hu9F5VHv4_L80D), SEQ ID NO : 111 (hu9F5VHv4_L82cG), SEQ ID NO: 112 (hu9F5VHv4_L82cD), SEQ ID NO: 113 (hu9F5VHv4_L82P), SEQ ID NO: 114 (hu9F5VHv4_L80G), SEQ ID NO: 115 (hu9F5VHv4_L82K), SEQ ID NO : 116 (hu9F5VHv4_L82R) , SEQ ID NO: 117 (hu9F5VHv4_L82E), SEQ ID NO: 118 (hu9F5VHv4_L82N), SEQ ID NO: 119 (hu9F5VHv4_Y79D), SEQ ID NO: 120 (hu9F5VHv4_Y79N), SEQ ID NO: 121 (hu9F5VHv4_Y79G), SE QID NO: 122 (hu9F5VHv5_M80E), SEQ ID NO: 123 (hu9F5VHv5_M80G), SEQ ID NO: 124 (hu9F5VHv4_L82cS), SEQ ID NO: 125 (hu9F5VHv4_Y79Q), SEQ ID NO: 126 (hu9F5VHv4_S82aG), SEQ ID NO: 127 (hu9F5VHv9), SEQ ID NO: 128 (hu9F5VHv10), SEQ ID NO: 129 (hu9F5VHv10_L82cG). An example of a humanized 10C12 heavy chain variable region with an N-terminal glutamine to glutamate substitution is SEQ ID NO: 215 (hu10C12VHv2). An example of a humanized 12C4 heavy chain variable region with an N-terminal glutamine to glutamate substitution is SEQ ID NO: 222 (hu12C4VHv2). An example of a humanized 17C12 heavy chain variable region with an N-terminal glutamine to glutamate substitution is SEQ ID NO: 233 (hu17C12VHv2).

Examples of humanized antibodies include humanized forms of murine 9F5, designated Hu9F5.

Murine antibody 9F5 comprises mature heavy and light chain variable regions having the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11, respectively. The invention provides 29 examples of humanized mature heavy chain variable regions: hu9F5VHv1 (SEQ ID NO: 15), hu9F5VHv2 (SEQ ID NO: 16), hu9F5VHv3 (SEQ ID NO: 17), hu9F5VHv4 (SEQ ID NO: 18), hu9F5VHv5 ( SEQ ID NO: 19), hu9F5VHv6 (SEQ ID NO: 20 hu9F5VHv7 (SEQ ID NO: 21), hu9F5VHv8 (SEQ ID NO: 22), hu9F5VHv4_L80P (SEQ ID NO: 109), hu9F5VHv4_L80D (SEQ ID NO: 110), hu9F5VHv4_L82cG (SEQ ID NO: 111), hu9F5VHv4_L82cD (SEQ ID NO: 112), hu9F5VHv4_L82P (SEQ ID NO: 113), hu9F5VHv4_L80G (SEQ ID NO: 114), hu9F5VHv4_L82K (SEQ ID NO 1 15:), hu9F5VHv4_L82R (SEQ ID NO : 116), hu9F5VHv4_L82E (SEQ ID NO: 117), hu9F5VHv4_L82N (SEQ ID NO: 118), hu9F5VHv4_Y79D (SEQ ID NO: 119), hu9F5VHv4_Y79N (SEQ ID NO: 120), hu9F5VHv4_Y79G (SE Q ID NO: 121), hu9F5VHv5_M80E (SEQ ID NO: 122), hu9F5VHv5_M80G (SEQ ID NO: 123), hu9F5VHv4_L82cS (SEQ ID NO: 124), hu9F5VHv4_Y79Q (SEQ ID NO: 125), hu9F5VHv4_S82aG (SEQ ID NO: 126), hu9F5VHv9 ( SEQ ID NO: 127), hu9F5VHv10 (SEQ ID NO: 128) and hu9F5VHv10_L82cG (SEQ ID NO: 129). The invention further provides 95 examples of mature light chain variable regions hu9F5VLv1 (SEQ ID NO: 23), hu9F5VLv2 (SEQ ID NO: 24), hu9F5VLv3 (SEQ ID NO: 25), hu9F5VLv4 (SEQ ID NO: 26), hu9F5VLv5 (SEQ ID NO: 27), hu9F5VLv6 (SEQ ID NO: 28:), hu9F5VLv7 (SEQ ID NO: 29), hu9F5VLv8 (SEQ ID NO: 130), hu9F5VLv9 (SEQ ID NO: 131), hu9F5VLv2_M51E (SEQ ID NO: 61) , hu9F5VLv2_M51D (SEQ ID NO: 62), hu9F5VLv2_L27cD (SEQ ID NO: 63), hu9F5VLv2_L27cG (SEQ ID NO: 64), hu9F5VLv2_L27cS (SEQ ID NO: 65), hu9F5VLv2_L27cE (SEQ ID NO: 66), hu9F5VLv2_I30E (SEQ ID NO: 67), hu9F5VLv2_I30K (SEQ ID NO: 68), hu9F5VLv2_L27cT (SEQ ID NO: 69), hu9F5VLv2_L27cN (SEQ ID NO: 70, hu9F5VLv2_L27bD (SEQ ID NO: 71), hu9F5VLv2_I30G (SEQ ID NO: 72), hu9F5VLv2_L33N (SEQ ID NO: 73), hu9F5VLv2_L27cA (SEQ ID NO: 74), hu9F5VLv2_L33T (SEQ ID NO: 75), hu9F5VLv2_L33S (SEQ ID NO: 76), hu9F5VLv2_L33R (SEQ ID NO: 77), hu9F5VLv2_I30Q (SE QID NO: 78), hu9F5VLv2_L27bT (SEQ ID NO: 79, hu9F5VLv2_T31G (SEQ ID NO: 80), hu9F5VLv2_L27bQ (SEQ ID NO: 81), hu9F5VLv2_L33G (SEQ ID NO: 82), hu9F5VLv2_L27cP (SEQ ID NO: 83), hu9F5VLv2_V78R (SEQ ID NO: 84), hu9F5VLv2_I75D (SEQ ID NO: 85), hu9F5VLv2_V78D (SEQ ID NO: 86), hu9F5VLv2_V78E (SEQ ID NO: 87), hu9F5VLv2_V78P (SEQ ID NO: 88), hu9F5VLv2_V78K (SEQ ID NO: 89) , hu9F5VLv2_R77D (SEQ ID NO: 90), hu9F5VLv2_V78G (SEQ ID NO: 91), hu9F5VLv2_S76P (SEQ ID NO: 92), hu9F5VLv2_I75P (SEQ ID NO: 93), hu9F5VLv2_I75Q (SEQ ID NO: 94), hu9F5VLv2_I75G (SEQ ID NO: 95), hu9F5VLv2_L73P (SEQ ID NO: 96), hu9F5VLv2_L73G (SEQ ID NO: 97), hu9F5VLv2_V78Q (SEQ ID NO: 98), hu9F5VLv2_S76G (SEQ ID NO: 99), hu9F5VLv2_L92D (SEQ ID NO: 100), hu9F5VLv2_Y86T (SEQ ID NO: 101), hu9F5VLv2_L92E (SEQ ID NO: 102), hu9F5VLv2_L92G (SEQ ID NO: 103), hu9F5VLv2_L92Q (SEQ ID NO: 104), hu9F5VLv2_L93G (SEQ ID NO: 10 5), hu9F5VLv2_V85G (SEQ ID NO : 106), hu9F5VLv2_L92T (SEQ ID NO: 107), hu9F5VLv2_A89G (SEQ ID NO: 108), hu9F5VLv8_DIM1 (SEQ ID NO: 132), hu9F5VLv8_DIM2 (SEQ ID NO: 133), hu9F5VLv8_DIM3 (SEQ ID NO : 134), hu9F5VLv8_DIM4 (SEQ ID NO: 135), hu9F5VLv8_DIM5 (SEQ ID NO: 136), hu9F5VLv8_DIM6 (SEQ ID NO: 137), hu9F5VLv8_DIM7 (SEQ ID NO: 138, hu9F5VLv8_DIM8 (SEQ ID

- 50 047155

NO: 139), hu9F5VLv8_DIM9 (SEQ ID NO: 140), hu9F5VLv8_DIM10 (SEQ ID NO: 141), hu9F5VLv8_DIM11 (SEQ ID NO: 142), hu9F5VLv8_DIM12 (SEQ ID NO: 143), hu9F5VLv8_DIM13 ID NO: 144), hu9F5VLv8_DIM14 (SEQ ID NO: 145), hu9F5VLv8_DIM15 (SEQ ID NO: 146), hu9F5VLv8_DIM16 (SEQ ID NO: 147), hu9F5VLv8_DIM17 (SEQ ID NO: 148), hu9F5VLv8_DIM18 (SEQ ID NO: 14 9), hu9F5VLv8_DIM19 (SEQ ID NO : 150), hu9F5VLv8_DIM20 (SEQ ID NO: 151), hu9F5VLv8_DIM21 (SEQ ID NO: 152), hu9F5VLv8_DIM22 (SEQ ID NO: 153), hu9F5VLv8_DIM23 (SEQ ID NO: 154), hu9F5VLv8_DIM24 (SE Q ID NO: 155), hu9F5VLv8_DIM25 (SEQ ID NO: 156), hu9F5VLv8_DIM26 (SEQ ID NO: 157), hu9F5VLv8_DIM27 (SEQ ID NO: 158), hu9F5VLv8_DIM28 (SEQ ID NO: 159), hu9F5VLv8_DIM29 (SEQ ID NO: 160), hu9F5VLv8_DIM 30 (SEQ ID NO: 161), hu9F5VLv9_DIMl (SEQ ID NO: 162), hu9F5VLv9_DIM2 (SEQ ID NO: 163), hu9F5VLv9_DIM4 (SEQ ID NO: 164), hu9F5VLv9_DIM5 (SEQ ID NO: 165), hu9F5VLv9_DIM8) (SEQ ID NO: 1 66), hu9F5VLv9_DIM10 (SEQ ID NO: 167), hu9F5VLv9_DIM11 (SEQ ID NO: 168), (hu9F5VLv9_DIM13 (SEQ ID NO: 169), hu9F5VLv9_DIM19 (SEQ ID NO: 170) and hu9F5VLv9_DIM20 (SEQ ID NO: 171). In fig. 1A-1B and 4A-4B show an alignment of the murine 9F5 heavy chain variable region and various humanized antibodies. In fig. 2A-2B and 5A-5B show an alignment of the murine 9F5 light chain variable region and various humanized antibodies. In fig. 6A-6C show an alignment of the light chain variable region of the humanized variant hu9F5VLv8 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/or antigen binding, mediation of interaction between the heavy and light chains, interaction with the constant region, site for desirable or undesired post-translational modification, a residue unusual for its position in the human variable region sequence and due to of this potential immunogenicity, decreased aggregation potential, and for other reasons, the following 48 variable region framework positions were considered as candidates for substitutions in the 95 examples of mature human light chain variable regions and 29 examples of mature human heavy chain variable regions, as further indicated in the examples: L3 (V3Q), L7 (A7S), L8 (A8P), L9 (F9L), L11 (N11L), L15 (L15P), L17 (T17E), L18 (S18P), L37 (L37Q, L37G, L37I), L39 ( R39K), L60 (N60D), L64 (G64S), L66 (E66G), L73 (L73P, L73G), L74 (R74K), L75 (I75D, I75P, I75Q, I75G), L76 (S76P, S76G), L77 ( R77D), L78 (V78R, V78D, V78E, V78P, V78K, V78G, V78Q), L85 (V85G), L86 (Y86T), L100 (G100Q), H1 (Q1E), H5 (Q5V), H11 (L11V), Н12 (V12K), Н17 (S17T), Н20 (L20I), Н23 (TOK), Н38 (K38R, K38Q), Н40 (R40A), Н42 (E42G), Н43 (Q43K), Н48 (I48M), Н66 (K66R) ), H69 (I69M), H75 (S75T), H76 (N76D), H79 (Y79Q, Y79D, Y79N, Y79G), H80 (L80M, L80P, L80D, L80G, L80E), H81 (Q81E), H82 (L82P, L82K, L82R, L82R, L82E, L82N), H82a (S82aG), H82c (L82cG, L82cD, L82cS), H83 (T83R), H93 (A93T), H94 (S94T), H108 (T108L) and H109 (L109V). The following 14 variable region CDR positions were considered as candidates for replacement in 95 examples of mature human light chain variable regions and 29 examples of mature human heavy chain variable regions, as follows in the examples: L27b (L27bD, L27bT, L27bQ), L27c (L27cD , L27cG, L27cS, L27cE, L27cT, L27cN, L27cA, L27cP, L27cI), L30 (I30Y, I30E, I30K, I30G, DOQ), L31 (T31N, T31G), L33 (L33N, L33T, L33S, L33R, L33G) , L51 (M51G, M51E, M51D, M51K, M51I), L54 (L54R, L54G, L54T), L89 (A89G), L92 (L92D, L92E, L92G, L92Q, L92T, L92I), L93 (E93G), H28 ( N28T), H51 (I48M), H54 (N54D) and H56 (D56E). In certain humanized antibodies, the 9F5 CDR-H1, as defined by the Kabat-Chothia composite definition, has an amino acid sequence comprising SEQ ID NO: 50. In certain humanized antibodies, the 9F5 CDRH1, as defined by the Kabat-Chothia composite definition, has an amino acid sequence comprising SEQ ID NO: 50. a Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51. In certain humanized antibodies, 9F5 Kabat CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51. In certain humanized antibodies, 9F5 Kabat CDR-H2 has an amino acid sequence sequence comprising SEQ ID NO: 52. In certain humanized antibodies, Kabat's 9F5 CDR-L2 has an amino acid sequence comprising SEQ ID NO: 55. In certain humanized antibodies, Kabat's 9F5 CDR-L1 has an amino acid sequence comprising SEQ ID NO: 53 , and Kabat CDR-L2 has an amino acid sequence comprising SEQ ID NO: 55. In certain humanized 9F5 antibodies, Kabat CDR-L1 has an amino acid sequence comprising SEQ ID NO: 54, and Kabat CDR-L2 has an amino acid sequence comprising SEQ ID NO: 55. In certain humanized antibodies, Kabat's 9F5 CDR-L1 has an amino acid sequence including a sequence selected from the group consisting of SEQ ID NOs: 172-193. In some humanized antibodies, Kabat's 9F5 CDR-L2 has an amino acid sequence including a sequence selected from the group consisting of SEQ ID NOs: 194-205. In certain humanized antibodies, Kabat's 9F5 CDR-L3 has an amino acid sequence including a sequence selected from the group consisting of SEQ ID NOs: 206-213.

Here, as elsewhere, the first-mentioned residue is that of a humanized antibody formed by grafting a Kabat CDR, or a Chothia-Kabat composite definition CDR in the case of CDR-H1, onto a human acceptor framework, and the second-mentioned residue is

- 51 047155 is the balance being considered to replace such balance. Thus, within the variable region frameworks, the first residue mentioned is human, and within the CDRs, the first residue mentioned is mouse.

Exemplary antibodies include any permutations or combinations of the severe variable regions as hu9F5VHv1/hu9F5VLv 1, hu9F5VLv1/hu9F5VLv5, hu9F5VHv2/hu9F5VLv2, hu9F5VHv2/hu9F5VLv6, hu9F5VHv3/hu9F5VLv3 , hu9F5VHv3/hu9F5VLv7, hu9F5VHv4/hu9F5VLv4, hu9F5VHv5/hu9F5VLv1, hu9F5VLv5 /hu9F5VLv5, hu9F5VHv6/hu9F5VLv2, hu9F5VHv6/hu9F5VLv6, hu9F5VHv7/u9F5VLv3, hu9F5VHv7/hu9F5VLv7, examples of mature hu9F5VHv1/hu9F5VLv2, hu9F5VHv1/hu9F5 VLv6, hu9F5VHv2/hu9F5VLv3, hu9F5VHv2/hu9F5VLv7, hu9F5VHv3/hu9F5VLv4, hu9F5VHv4/hu9F5VLv1, hu9F5VLv4/hu9F5VLv5, hu9F5VHv5 /hu9F5VLv2, hu9F5VHv5/hu9F5VLv6, hu9F5VHv6/hu9F5VLv3, hu9F5VHv6/hu9F5VLv7, hu9F5VHv7/hu9F5VLv4, hu9F5VHv8/hu9F5VLv 1, and light chains hu9F5VHv1/hu9 F5VLv4, hu9F5VHv2/hu9F5VLv1, hu9F5VLv2/hu9F5VLv5, hu9F5VHv3/hu9F5VLv2, hu9F5VHv3/hu9F5VLv6, hu9F5VHv4/hu9F5VLv3 , hu9F5VHv4/hu9F5VLv7, hu9F5VHv5/hu9F5VLv4, hu9F5VHv6/hu9F5VLv1, hu9F5VHv6/hu9F5VLv5, hu9F5VHv7/hu9F5VLv2, hu9F5VHv7/hu9F5VLv6, v8/hu9F5VLv3, hu9F5VHv1/hu9F5VLv3, hu9F5VHv1/hu9F5VLv7, hu9F5VHv2/hu9F5VLv4, hu9F5VHv3/hu9F5VLv1, hu9F5VLv3/hu9F5VLv5, hu9F5VHv4 /hu9F5VLv2, hu9F5VHv4/hu9F5VLv6, hu9F5VHv5/hu9F5VLv3, hu9F5VHv5/hu9F5VLv7, hu9F5VHv6/hu9F5VLv4, hu9F5VHv7/hu9F5VLv1, hu9F5VLv7/hu9F5VLv5 , hu9F5VHv8/hu9F5VLv2, hu9F5VHv8/ hu9F5VLv4, hu9F5VLv8/ hu9F5VLv5, hu9F5VHv8/ hu9F5VLv6, hu9F5VHv8/ hu9F5VLv7.

Exemplary antibodies include any permutations or combinations of the exemplary mature heavy and light chain variable regions hu9F5VHv1 (SEQ ID NO: 15), hu9F5VHv2 (SEQ ID NO: 16), hu9F5VHv3 (SEQ ID NO: 17), hu9F5VHv4 ( SEQ ID NO: 18), hu9F5VHv5 (SEQ ID NO: 19), hu9F5VHv6 (SEQ ID NO: 20), hu9F5VHv7 (SEQ ID NO: 21), hu9F5VHv8 (SEQ ID NO: 22), hu9F5VHv9 (SEQ ID NO: 127 ), hu9F5VHv10 (SEQ ID NO: 128), hu9F5VHv10_L82cG (SEQ ID NO: 129), hu9F5VHv4_L80P (SEQ ID NO: 109), hu9F5VHv4_L80D (SEQ ID NO: 110), hu9F5VHv4_L82cG (SEQ ID NO: 11 1), hu9F5VHv4_L82cD (SEQ ID NO: 112), hu9F5VHv4_L82P (SEQ ID NO: 113), hu9F5VHv4_L80G (SEQ ID NO: 114), hu9F5VHv4_L82K (SEQ ID NO: 115), hu9F5VHv4_L82R (SEQ ID NO: 116), hu9F5VHv4_L82E ( SEQ ID NO: 117) , hu9F5VHv4_L82N (SEQ ID NO: 118), hu9F5VHv4_Y79D (SEQ ID NO: 119), hu9F5VHv4_Y79N (SEQ ID NO: 120), hu9F5VHv4_Y79G (SEQ ID NO: 121), hu9F5VHv5_M80E (SEQ ID NO: 122), hu9F5VHv5_M80G (SEQ ID NO: 123), hu9F5VHv4_L82Cs (SEQ ID NO: 124), hu9F5VHv4_Y79Q (SEQ ID NO: 125) and hu9F5VHv4_S82aG (SEQ ID NO: 126), with any of the exemplary mature light chain variable regions of hu9F5VLv1 (SEQ ID NO: 23), hu9F5VLv2 (SEQ ID NO: 24), hu9F5VLv3 (SEQ ID NO: 25), hu9F5VLv4 (SEQ ID NO: 26), hu9F5VLv5 (SEQ ID NO: 27, hu9F5VLv6 (SEQ ID NO: 28), hu9F5VLv7 (SEQ ID NO: 29), hu9F5VLv8 (SEQ ID NO: 130), hu9F5VLv9 (SEQ ID NO: 131), hu9F5VLv2_M51E (SEQ ID NO: 61), hu9F5VLv2_M51D (SEQ ID NO: 62), hu9F5VLv2_L27cD (SEQ ID NO: 63) , hu9F5VLv2_L27cG (SEQ ID NO: 64), hu9F5VLv2_L27cS (SEQ ID NO: 65), hu9F5VLv2_L27cE (SEQ ID NO: 66), hu9F5VLv2_I30E (SEQ ID NO: 67), hu9F5VLv2_I30K (SEQ ID NO: 6 8), hu9F5VLv2_L27cT (SEQ ID NO: 69), hu9F5VLv2_L27cN (SEQ ID NO: 70), hu9F5VLv2_L27bD (SEQ ID NO: 71), hu9F5VLv2_I30G, (SEQ ID NO: 72), hu9F5VLv2_L33N (SEQ ID NO: 73), hu9F5VLv2_L27cA (SEQ ID NO: 74) , hu9F5VLv2_L33T (SEQ ID NO: 75), hu9F5VLv2_L33S (SEQ ID NO: 76), hu9F5VLv2_L33R (SEQ ID NO: 77), hu9F5VLv2_I30Q (SEQ ID NO: 78), hu9F5VLv2_L27bT (SEQ ID NO: 79), hu9F5VLv2_T31G (SEQ ID NO: 80), hu9F5VLv2_L27bQ (SEQ ID NO: 81), hu9F5VLv2_L33G (SEQ ID NO: 82), hu9F5VLv2_L27cP (SEQ ID NO: 83), hu9F5VLv2_V78R (SEQ ID NO: 84), hu9F5VLv2_I75D (SEQ ID NO : 85), hu9F5VLv2_V78D (SEQ ID NO: 86), hu9F5VLv2_V78E (SEQ ID NO: 87), hu9F5VLv2_V78P (SEQ ID NO: 88), hu9F5VLv2_V78K (SEQ ID NO: 89), hu9F5VLv2_R77D (SEQ ID NO: 90), F5VLv2_V78G (SEQ ID NO : 91), hu9F5VLv2_S76P (SEQ ID NO: 92), hu9F5VLv2_I75P (SEQ ID NO: 93), hu9F5VLv2_I75Q (SEQ ID NO: 94), hu9F5VLv2_I75G (SEQ ID NO: 95), hu9F5VLv2_L73P (SEQ ID NO: 96), hu9F5VLv2_L73G (SEQ ID NO: 97), hu9F5VLv2_V78Q (SEQ ID NO: 98), hu9F5VLv2_S76G (SEQ ID NO: 99), hu9F5VLv2_L92D (SEQ ID NO: 100), hu9F5VLv2_Y86T (SEQ ID NO: 101), hu9F5VLv2_L92E ( SEQ ID NO: 102), hu9F5VLv2_L92G (SEQ ID NO: 103), hu9F5VLv2_L92Q (SEQ ID NO: 104), hu9F5VLv2_L93G (SEQ ID NO: 105), hu9F5VLv2_V85G (SEQ ID NO: 106), hu9F5VLv2_L92T (SEQ ID NO: 107), hu9F5VLv2_A89G ( SEQ ID NO: 108), hu9F5VLv8_DIM1 (SEQ ID NO: 132), hu9F5VLv8_DIM2 (SEQ ID NO: 133), hu9F5VLv8_DIM3 (SEQ ID NO: 134), hu9F5VLv8_DIM4 (SEQ ID NO: 135), hu9F5VLv8_DIM5 (SEQ ID NO: 136 ), hu9F5VLv8_DIM6 (SEQ ID NO: 137), hu9F5VLv8_DIM7 (SEQ ID NO: 138), hu9F5VLv8_DIM8 (SEQ ID NO: 139), hu9F5VLv8_DIM9 (SEQ ID NO: 140), hu9F5VLv8_DIM10 (SEQ ID NO: 141), hu9F5VLv8_DIM11 (SEQ ID NO: 142), hu9F5VLv8_DIM12 (SEQ ID NO: 143), hu9F5VLv8_DIM13 (SEQ ID NO: 144), hu9F5VLv8_DIM14 (SEQ ID NO: 145), hu9F5VLv8_DIM15 (SEQ ID NO: 146), hu9F5VLv8_DIM16 ( SEQ ID NO: 147) , hu9F5VLv8_DIM17 (SEQ ID NO: 148), hu9F5VLv8_DIM18 (SEQ ID NO: 149), hu9F5VLv8_DIM19 (SEQ ID NO: 150), hu9F5VLv8_DIM20 (SEQ ID NO: 151), hu9F5VLv8_DIM21 (SEQ ID NO: 152), hu9F5VLv8_DIM22 (SEQ ID NO: 153), hu9F5VLv8_DIM23 (SEQ ID NO: 154), hu9F5VLv8_DIM24 (SEQ ID NO: 155), hu9F5VLv8_DIM25 (SEQ ID NO: 156), hu9F5VLv8_DIM26 (SEQ ID NO: 157),

- 52 047155 hu9F5VLv8_DIM27 (SEQ ID NO: 158), hu9F5VLv8_DIM28 (SEQ ID NO: 159), hu9F5VLv8_DIM29 (SEQ ID NO: 160), hu9F5VLv8_DIM30 (SEQ ID NO: 161), hu9F5VLv9_DIM 1 (SEQ ID NO: 162), hu9F5VLv9_DIM2 ( SEQ ID NO: 163), hu9F5VLv9_DIM4 (SEQ ID NO: 164), hu9F5VLv9_DIM5 (SEQ ID NO: 165), hu9F5VLv9_DIM8 (SEQ ID NO: 166), hu9F5VLv9_DIM10 (SEQ ID NO: 167), hu9F5VLv9_DIM11 (SE Q ID NO: 168 ), hu9F5VLv9_DIM13 (SEQ ID NO: 169), hu9F5VLv9_DIM19 (SEQ ID NO: 170) and hu9F5VLv9_DIM20 (SEQ ID NO: 171).

The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM18 (also known as hu9F5VLv8_V3Q, L27cD, L37G, M51G, L54R, L92I, SEQ ID NO: 149) . The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM11 (also known as hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, L92I, SEQ ID NO: 142) . The invention was offered an antibody in which the humanized variable area of the heavy chain HU9F5VHV9 (SEQ ID NO: 127) is combined with a humanized variable area of the light chain HU9F5VLV8_DIM28 (also known as HU9F5VLV8_V3Q, L27CS, M51G, L54R, L92I, S. EQ ID No: 159). The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM17 (also known as hu9F5VLv8_V3Q, L27cS, L37G, M51G, L54T, L92I, SEQ ID NO: 148) .

The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region /hu9F5VLv8_DIM6 (also known as hu9F5VLv8_V3Q, L27cD, L37Q, M51G, L54R, L92I, SEQ ID NO: 137 ). The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM14 (also known as hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T, L92I, SEQ ID NO: 145) . The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM5 (also known as hu9F5VLv8_V3Q, L27cG, L37Q, M51G, L54R, L92I, SEQ ID NO: 136) . The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM7 (also known as hu9F5VLv8_V3Q, L27cD, L37Q, M51K, L54R, L92I, SEQ ID NO: 138) .

The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM27 (also known as hu9F5VLv8_V3Q, L37Q, M51G, L54R, L92I, SEQ ID NO: 158). The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM12 (also known as hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, L92G, SEQ ID NO: 143) . The invention was offered an antibody in which the humanized variable area of the heavy chain HU9F5VHV9 (SEQ ID NO: 127) is combined with a humanized variable area of the light chain HU9F5VLV8_DIM13 (also known as HU9F5VLV8_V3Q, L27CG, L37G, M51G, L54R, S. EQ ID No: 144). The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM2 (also known as hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54R, L92I, SEQ ID NO: 133) .

In the invention, an antibody was proposed, in which the humanized variable area of the heavy chain HU9F5VHV9 (SEQ ID NO: 127) is combined with a humanized variable area of the light chain HU9F5VLV8_DIM29 (also known as HU9F5VLV8_V3Q, L27CS, L37Q, L54R, L92I, S. EQ ID No: 160). The invention was offered an antibody in which the humanized variable area of the heavy chain HU9F5VHV9 (SEQ ID NO: 127) is combined with a humanized variable area of a light circuit /HU9F5VLV8_DIM30 (also known as HU9F5VLV8_V3Q, L27CS, L37Q, M51G, L92I, S. EQ ID No: 161). The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv9 (SEQ ID NO: 127) is combined with the humanized light chain variable region hu9F5VLv8_DIM8 (also known as hu9F5VLv8_V3Q, L27cG, L37Q, M51K, L54R, L92I, SEQ ID NO: 139) .

The invention provides an antibody in which the humanized heavy chain variable region hu9F5VHv10 (SEQ ID NO: 128) is combined with the humanized light chain variable region hu9F5VLv9_DIM11 (also known as hu9F5VLv9_V3Q, L27cG, L37G, M51G, L54R, L92I, SEQ ID NO: 168).

The invention provides variants of the humanized 9F5 antibody wherein the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu9F5VHv1 (SEQ ID NO: 15), hu9F5VHv2 ( SEQ ID NO: 16), hu9F5VHv3

- 53 047155 (SEQ ID NO: 17), hu9F5VHv4 (SEQ ID NO: 18), hu9F5VHv5 (SEQ ID NO: 19), hu9F5VHv6 (SEQ ID NO: 20), hu9F5VHv7 (SEQ ID NO: 21), hu9F5VHv8 (SEQ ID NO: 22), hu9F5VHv9 (SEQ ID NO: 127), hu9F5VHv10 (SEQ ID NO: 128), hu9F5VHv10_L82cG (SEQ ID NO: 129), hu9F5VHv4_L80P (SEQ ID NO: 109), hu9F5VHv4_L80D (SEQ ID NO: 11 0) , hu9F5VHv4_L82cG (SEQ ID NO: 111), hu9F5VHv4_L82cD (SEQ ID NO: 112), hu9F5VHv4_L82P (SEQ ID NO: 113), hu9F5VHv4_L80G (SEQ ID NO: 114), hu9F5VHv4_L82K (SEQ ID NO : 115), hu9F5VHv4_L82R (SEQ ID NO: 116), hu9F5VHv4_L82E (SEQ ID NO: 117), hu9F5VHv4_L82N (SEQ ID NO: 118), hu9F5VHv4_Y79D (SEQ ID NO: 119), hu9F5VHv4_Y79N (SEQ ID NO: 120), hu9F5VHv4_Y79G (SE Q ID NO: 121), hu9F5VHv5_M80E (SEQ ID NO: 122), hu9F5VHv5_M80G (SEQ ID NO: 123), hu9F5VHv4_L82Cs (SEQ ID NO: 124), hu9F5VHv4_Y79Q (SEQ ID NO: 125) or hu9F5VHv4_S82aG (SEQ ID NO: 126), and humanized mature variable region light chain exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu9F5VLv1 (SEQ ID NO: 23), hu9F5VLv2 (SEQ ID NO: 24), hu9F5VLv3 (SEQ ID NO: 25 ), hu9F5VLv4 (SEQ ID NO: 26), hu9F5VLv5 (SEQ ID NO: 27, hu9F5VLv6 (SEQ ID NO: 28), hu9F5VLv7 (SEQ ID NO: 29), hu9F5VLv8 (SEQ ID NO: 130, hu9F5VLv9 (SEQ ID NO : 131), hu9F5VLv2_M51E (SEQ ID NO: 61), hu9F5VLv2_M51D (SEQ ID NO: 62), hu9F5VLv2_L27cD (SEQ ID NO: 63), hu9F5VLv2_L27cG (SEQ ID NO: 64), hu9F5VLv2_L27cS (SEQ ID NO: 65), hu9F5VLv2_L27cE (SEQ ID NO: 66), hu9F5VLv2_I30E (SEQ ID NO: 67), hu9F5VLv2_I30K (SEQ ID NO: 68), hu9F5VLv2_L27cT (SEQ ID NO: 69), hu9F5VLv2_L27cN (SEQ ID NO: 70), hu9F5VLv2_L27bD (SEQ ID NO: 71), hu9F5VLv2_I30G, (SEQ ID NO: 72), hu9F5VLv2_L33N (SEQ ID NO: 73), hu9F5VLv2_L27cA (SEQ ID NO: 74), hu9F5VLv2_L33T (SEQ ID NO: 75), hu9F5VLv2_L33S (SEQ ID NO: 76), hu9F5VLv2_L33R (SEQ ID NO: 77), hu9F5VLv2_I30Q (SEQ ID NO: 78), hu9F5VLv2_L27bT (SEQ ID NO: 79), hu9F5VLv2_T31G (SEQ ID NO: 80), hu9F5VLv2_L27bQ (SEQ ID NO: 81), hu9F5VLv2_L33G ( SEQ ID NO: 82), hu9F5VLv2_L27cP (SEQ ID NO: 83), hu9F5VLv2_V78R (SEQ ID NO: 84), hu9F5VLv2_I75D (SEQ ID NO: 85), hu9F5VLv2_V78D (SEQ ID NO: 86), hu9F5VLv2_V78E (SEQ ID NO: 8 7), hu9F5VLv2_V78P ( SEQ ID NO: 88), hu9F5VLv2_V78K (SEQ ID NO: 89), hu9F5VLv2_R77D (SEQ ID NO: 90), hu9F5VLv2_V78G (SEQ ID NO: 91), hu9F5VLv2_S76P (SEQ ID NO: 92), hu9F5VLv2_I75P (SEQ ID NO: 93 ), hu9F5VLv2_I75Q (SEQ ID NO: 94), hu9F5VLv2_I75G (SEQ ID NO: 95), hu9F5VLv2_L73P (SEQ ID NO: 96), hu9F5VLv2_L73G (SEQ ID NO: 97), hu9F5VLv2_V78Q (SEQ ID NO: 98), hu 9F5VLv2_S76G (SEQ ID NO: 99), hu9F5VLv2_L92D (SEQ ID NO: 100), hu9F5VLv2_Y86T (SEQ ID NO: 101), hu9F5VLv2_L92E (SEQ ID NO: 102), hu9F5VLv2_L92G (SEQ ID NO: 103), hu9F5VLv2_L92Q (SE Q ID NO: 104) , hu9F5VLv2_L93G (SEQ ID NO: 105), hu9F5VLv2_V85G (SEQ ID NO: 106), hu9F5VLv2_L92T (SEQ ID NO: 107), hu9F5VLv2_A89G (SEQ ID NO: 108), hu9F5VLv8_DIM1 (SEQ ID NO: 1 32), hu9F5VLv8_DIM2 (SEQ ID NO: 133), hu9F5VLv8_DIM3 (SEQ ID NO: 134), hu9F5VLv8_DIM4 (SEQ ID NO: 135), hu9F5VLv8_DIM5 (SEQ ID NO: 136), hu9F5VLv8_DIM6 (SEQ ID NO: 137), hu9F5VLv8_DIM7 (SEQ ID NO: 138), hu9F5VLv8_DIM8 (SEQ ID NO: 139), hu9F5VLv8_DIM9 (SEQ ID NO: 140), hu9F5VLv8_DIM10 (SEQ ID NO: 141), hu9F5VLv8_DIM11 (SEQ ID NO: 142), hu9F5VLv8_DIM12 (SEQ ID NO: 143), hu9F5VLv8_DIM13 (SEQ ID NO : 144), hu9F5VLv8_DIM14 (SEQ ID NO: 145), hu9F5VLv8_DIM15 (SEQ ID NO: 146), hu9F5VLv8_DIM16 (SEQ ID NO: 147), hu9F5VLv8_DIM17 (SEQ ID NO: 148), hu9F5VLv8_DIM18 (SE Q ID NO: 149), hu9F5VLv8_DIM19 (SEQ ID NO: 150), hu9F5VLv8_DIM20 (SEQ ID NO: 151), hu9F5VLv8_DIM21 (SEQ ID NO: 152), hu9F5VLv8_DIM22 (SEQ ID NO: 153), hu9F5VLv8_DIM23 (SEQ ID NO: 154), hu9F5VLv8_DIM 24 (SEQ ID NO: 155), hu9F5VLv8_DIM25 (SEQ ID NO: 156), hu9F5VLv8_DIM26 (SEQ ID NO: 157), hu9F5VLv8_DIM27 (SEQ ID NO: 158), hu9F5VLv8_DIM28 (SEQ ID NO: 159), hu9F5VLv8_DIM29 (SEQ ID NO: 160), hu9F5VLv8_DIM30 ( SEQ ID NO: 161), hu9F5VLv9_DIM1 (SEQ ID NO: 162), hu9F5VLv9_DIM2 (SEQ ID NO: 163), hu9F5VLv9_DIM4 (SEQ ID NO: 164), hu9F5VLv9_DIM5 (SEQ ID NO: 165), hu9F5VLv9_DIM8 (SEQ ID NO: 166 ), hu9F5VLv9_DIM10 (SEQ ID NO: 167), hu9F5VLv9_DIM11 (SEQ ID NO: 168), hu9F5VLv9_DIM13 (SEQ ID NO: 169), hu9F5VLv9_DIM19 (SEQ ID NO: 170) or hu9F5VLv9_DIM20 (SEQ ID NO: 171). In some such antibodies, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or all 62 positions subject to backmutations or other mutations in SEQ ID NO: 15-22, 109-129 , SEQ ID NOs: 23-29, SEQ ID NOs: 61-108 and SEQ ID NOs: 130-171, are likewise subject to back mutation or other mutations. In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by E, H17 is occupied by T, H20 is occupied by I, H69 is occupied by M, H75 is occupied by T, H93 is occupied by T, H94 is occupied by T, and H109 is occupied by V. In some humanized 9F5 antibodies, positions H1, H17, H20, H69, H75, H94 and H109 are occupied by E, T, I, M, T, T, T and V, respectively.

In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H66 is occupied by R and H81 is occupied by E. In some humanized 9F5 antibodies, positions H66 and H81 are occupied by R and E, respectively.

In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H23 is occupied by I, and H83 is occupied by R. In some humanized 9F5 antibodies, positions H23 and H83 are occupied by K and R, respectively.

- 54 047155

In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H43 is occupied by K, H51 is occupied by V, H76 is occupied by D, M80 is occupied by M, and H108 is occupied by L. In some humanized 9F5 antibodies, positions H43, H51, H76, H80 and H108 are occupied by K, V, D, M and L, respectively.

In some humanized 9F5 antibodies, the H28 position in the VH region is occupied by T.

In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H54 is occupied by D and H56 is occupied by E. In some humanized 9F5 antibodies, positions H54 and H56 are occupied by D and E, respectively.

In some humanized 9F5 antibodies, the H40 position in the VH region is occupied by A. In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H5 is occupied by V, H11 is occupied by V, H12 is occupied by K, H38 is occupied by R, and H42 busy G.

In some humanized 9F5 antibodies, positions H5, H11, H12, H38 and H42 are occupied by V, V, K, R and G, respectively.

In some humanized 9F5 antibodies, at least one of the following positions in the VH region is occupied by a specified amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H12 is occupied by V or K, H17 is occupied by S or T, H20 occupied by L or I, H23 occupied by T or K, H28 occupied by N or T, H38 occupied by K, R or Q, H40 occupied by R or A, H42 occupied by E or G, H43 occupied by Q or K, H48 occupied by I or M, H51 occupied I or V, H54 occupied N or D, H56 occupied D or E, H66 occupied K or R, H69 occupied I or M, H75 occupied S or T, H76 occupied N or D, H79 occupied Y, Q, D, N or G, H80 occupied by L, M, P, D, G or E, H81 occupied by Q or E, H82 occupied by L, P, K, R, E or N, H82a occupied by S or G, H82c occupied by L, G, D or S, H83 occupied by T or R, H93 occupied by A or T, H94 occupied by S or T, H108 occupied by T or L, H109 occupied by L or V.

In some humanized 9F5 antibodies, positions H1, H17, H20, H69, H75, H93, H94 and H109 in the VH region are occupied by E, T, I, M, T, T, T and V, respectively, as in hu9F5VHv2. In some humanized 9F5 antibodies, positions H1, H17, H20, H66, H69, H75, H81, H93, H94 and H109 in the VH region are occupied by E, T, I, R, M, T, E, T, T and V, respectively , as in hu9F5VHv3. In some humanized 9F5 antibodies, positions H1, H17, H20, H23, H28, H66, H69, H75, H81, H83, H93, H94 and H109 in the VH region are occupied by E, T, I, K, T, R, M, T , E, R, T, T and V, respectively, as in hu9F5VHv4. In some humanized 9F5 antibodies, positions H1, H17, H20, H23, H28, H43, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E , T, I, K, T, K, V, D, E, R, M, T, D, M, E, R, T, T, L and V, respectively, as in hu9F5VHv5. In some humanized 9F5 antibodies, positions H1, H17, H20, H23, H28, H40, H43, H48, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in areas VH are occupied by E, T, I, K, T, A, K, M, V, D, E, R, M, T, D, M, E, R, T, T, L and V, respectively, as in hu9F5VHv6. In some humanized 9F5 antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H40, H42, H43, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH area are occupied by E, V, V, K, T, I, K, R, A, G, K, V, D, E, R, M, T, D, M, E, R , T, T, L and V, respectively, as in hu9F5VHv7. In some humanized 9F5 antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H40, H42, H43, H51, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108 and H109 in the VH region is occupied by E, V, V, K, T, I, K, R, A, G, K, V, R, M, T, D, M, E, R, T, T, L and V , respectively, as in hu9F5VHv8. In some humanized 9F5 antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E , V, V, K, T, I, K, Q, G, K, R, M, T, M, E, R, T, T, L and V, respectively, as in hu9F5VHv9. In some humanized 9F5 antibodies, the heavy chain variable region contains the amino acid sequence SEQ ID NO: 127.

In some humanized 9F5 antibodies, positions H1, H5, H11, H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H83, H93, H94, H108 and H109 in the VH region are occupied by E , V, V, K, T, I, K, K, E, K, R, M, T, M, E, R, T, T, L and V, respectively, as in hu9F5VHv10. In some humanized 9F5 antibodies, the heavy chain variable region contains the amino acid sequence SEQ ID NO: 128.

In some humanized 9F5 antibodies, positions H1, H5, NI, H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H82c, H83, H93, H94, H108 and H109 in the VH region occupied by E, V, V, K, T, I, K, K, E, K, R, M, T, M, E, G, R, T, T, L and V, respectively, as in hu9F5VHv10_L82cG.

In some humanized 9F5 antibodies, at least one of the following positions in the VL region is occupied by the indicated amino acid: L7 is occupied by S, L8 is occupied by P, L15 is occupied by P, and L100 is occupied by Q. In some humanized 9F5 antibodies, positions L7, L8, L15 and L100 are occupied S, P, P and Q respectively.

In some humanized 9F5 antibodies, the L66 position in the VL region is occupied by a G. In some humanized 9F5 antibodies, the L64 position in the VL region is occupied by an S.

In some humanized 9F5 antibodies, the L17 position in the VL region is occupied by E.

In some humanized 9F5 antibodies, at least one of the following positions in the VL region is occupied by the indicated amino acid: L11 is occupied by L, L51 is occupied by G, and L54 is occupied by R. In some humanized 9F5 antibodies, positions L11, L51, and L54 are occupied by L, G, and R, respectively. .

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In some humanized 9F5 antibodies, the L30 position in the VL region is occupied by Y.

In some humanized 9F5 antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L3 is occupied by V or Q, L7 is occupied by A or S, L8 is occupied by A or P, L9 is occupied by F or L, L11 is occupied by N or L, L15 occupied by L or P, L17 occupied by T or E, L18 occupied by S or P, L27b occupied by L, D, T or Q, L27c occupied by L, D, G, S, E, T, N, A, P or I, L30 occupied by I, Y, E, K, G or Q, L31 occupied by T, N or G, L33 occupied by L, N, T, S, R or G, L37 occupied by L, Q, G or I, L39 occupied by R or K , L51 occupied by M, G, E, D, K or I, L54 occupied by L, R, G or T, L60 occupied by N or D, L64 occupied by G or S, L66 occupied by E or G, L73 occupied by L, P or G , L74 occupied by R or K, L75 occupied by I, D, P, Q or G, L76 occupied by S, P or G, L77 occupied by SEQ ID NO: 146 or D, L78 occupied by V, R, D, E, P, K , G or Q, L85 occupied by V or G, L86 occupied by Y or T, L89 occupied by A or G, L92 occupied by L, D, E, G, Q, T or I, L93 occupied by E or G, L100 occupied by G or Q .

In some humanized 9F5 antibodies, positions L64 and L66 in the VL region are occupied by S and G, respectively, as in hu9F5VLv1. In some humanized 9F5 antibodies, positions L7, L8, L15, L64, L66 and L100 in the VL region are occupied by S, P, P, S, G and Q, respectively, as in hu9F5VLv2. In some humanized 9F5 antibodies, positions L7, L8, L15, L17, L66 and L100 in the VL region are occupied by S, P, P, E, G and Q, respectively, as in hu9F5VLv3.

In some humanized 9F5 antibodies, positions L7, L8, L11, L15, L17, L51, L54, L66 and L100 in the VL region are occupied by S, P, L, P, E, G, R, G and Q, respectively, as in hu9F5VLv4 . In some humanized 9F5 antibodies, the light chain variable region contains any amino acid sequence from SEQ ID NOs: 133, 135-137, 142-144, 149, 158, 159 and 168. In some humanized 9F5 antibodies, the light chain variable region contains the amino acid sequence of SEQ ID NO: 133. In certain humanized 9F5 antibodies, the light chain variable region contains the amino acid sequence SEQ ID NO: 137. In certain humanized 9F5 antibodies, the light chain variable region contains the amino acid sequence SEQ ID NO: 149. In certain humanized 9F5 antibodies, the light chain variable region contains amino acid sequence SEQ ID NO: 159.

In some humanized 9F5 antibodies, positions L7, L8, L11, L15, L17, L30, L51, L54, L66 and L100 in the VL region are occupied by S, P, L, P, E, Y, G, R, G and Q, respectively , as in hu9F5VLv5. In some humanized 9F5 antibodies, positions L7, L8, L11, L15, L17, L30, L51, L54 and L100 in the VL region are occupied by S, P, L, P, E, Y, G, R and Q, respectively, as in hu9F5VLv6 . In some humanized 9F5 antibodies, positions L7, L8, L9, L11, L15, L17, L18, L31, L39, L51, L54, L60, L66, L74 and L100 in the VL region are occupied by S, P, L, L, P, E , P, N, K, G, R, D, G, K and Q, respectively, as in hu9F5VLv7.

In some humanized 9F5 antibodies, positions L7, L8, L11, L15, L17, L39, L64, L66, L74 and L100 in the VL region are occupied by S, P, L, P, E, K, S, G, K and Q, respectively , as in hu9F5VLv8. In some humanized 9F5 antibodies, the L3 position in the VL region is occupied by Q. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by D, G, I, L or S, the L37 position in the VL region is occupied by G, I, L or Q, the L51 position in the VL region is occupied by E, G, I, K or M, position L54 in the VL region is occupied by G, L, R or T, and position L92 in the VL region is occupied by G, I or L. In some humanized 9F5 antibodies, position L27c in the VL region is occupied by VL is occupied by D or S, position L37 in the VL region is occupied by G, L or Q, position L51 in the VL region is occupied by G or K, position L54 in the VL region is occupied by R, and position L92 in the VL region is occupied by I.

In some humanized 9F5 antibodies, position L27c in the VL region is occupied by D, position L37 in the VL region is occupied by G, and position L51 in the VL region is occupied by G. In some humanized 9F5 antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127. and the light chain variable region has the amino acid sequence comprising SEQ ID NO: 149.

In some humanized 9F5 antibodies, position L27c in the VL region is occupied by D, position L37 in the VL region is occupied by Q, and position L51 in the VL region is occupied by G. In some humanized 9F5 antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127. and the light chain variable region has the amino acid sequence comprising SEQ ID NO: 137.

In some humanized 9F5 antibodies, position L27c in the VL region is occupied by S, position L37 in the VL region is occupied by L, and position L51 in the VL region is occupied by G. In some humanized 9F5 antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127. and the light chain variable region has the amino acid sequence comprising SEQ ID NO: 159.

In some humanized 9F5 antibodies, position L27c in the VL region is occupied by D, position L37 in the VL region is occupied by Q, and position L51 in the VL region is occupied by K.

In some humanized 9F5 antibodies, the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 127 and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 138.

In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by S, the L37 position in the VL region is occupied by Q, and the L51 position in the VL region is occupied by G. In some humanized

- 56 047155 9F5 antibodies, the heavy chain variable region has the amino acid sequence comprising SEQ ID NO: 127, and the light chain variable region has the amino acid sequence comprising SEQ ID NO: 133.

In some humanized 9F5 antibodies, positions L7, L8, L11, L15, L17, L39, L60, L64, L66, L74 and L100 in the VL region are occupied by S, P, L, P, E, K, D, S, G, K and Q, respectively, as in hu9F5VLv9. In some humanized 9F5 antibodies, the L3 position in the VL region is occupied by Q.

In some humanized 9F5 antibodies, position L27c in the VL region is occupied by G or S, position L37 in the VL region is occupied by G, I, or Q, position L51 in the VL region is occupied by G, I, or K, position L54 in the VL region is occupied by G or R, and position L92 in the VL region is occupied by G, I, or L. In some humanized 9F5 antibodies, position L27c in the VL region is occupied by G, position L37 in the VL region is occupied by G, position L51 in the VL region is occupied by G, and position L54 in the VL region is occupied by R. In some humanized 9F5 antibodies, the L92 position in the VL region is occupied by I. In some humanized 9F5 antibodies, the heavy chain variable region has an amino acid sequence comprising SEQ ID NO: 129, and the light chain variable region has an amino acid sequence comprising SEQ ID NO: 168.

The light chain variable region of any of the above antibodies may be modified to further reduce immunogenicity. For example, in some humanized antibodies, the L27b position in the VL region is occupied by D, T or Q; position L27c in area VL is occupied by D, G, S, E, T, N, A, I or P; position L30 in area VL is occupied by E, K, G or Q; position L31 in area VL is occupied by G; position L33 in the VL region is occupied by N, T, S, R or G; position L37 in the VL region is occupied by Q, G or I, position L51 in the VL region is occupied by E, D, G, K or I; position L54 in the VL region is occupied by G, R or T, position L60 in the VL region is occupied by D, position L73 in the VL region is occupied by P or G; position L75 in area VL is occupied by D, P, Q or G; position L76 in the VL region is occupied by P or G; position L77 in area VL is occupied by D; position L78 in region VL is occupied by R, D, E, P, K, G or Q; position L85 in area VL is occupied by G; position L86 in the VL region is occupied by T; position L89 in area VL is occupied by G; position L92 in area VL is occupied by D, E, G, Q, I or T; and/or position L93 in the VL region is occupied by G (Kabat numbering).

In some humanized 9F5 antibodies, the L51 position in the VL region is occupied by E, as in hu9F5VLv2_M51E. In some humanized 9F5 antibodies, the L51 position in the VL region is occupied by D, as in hu9F5VLv2_M51D. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by D, as in hu9F5VLv2_L27cD. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by G, as in hu9F5VLv2_L27cG. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by S, as in hu9F5VLv2_L27cS. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by E, as in hu9F5VLv2_L27cE. In some humanized 9F5 antibodies, the L30 position in the VL region is occupied by E, as in hu9F5VLv2_I30E. In some humanized 9F5 antibodies, the L30 position in the VL region is occupied by K, as in hu9F5VLv2_I30K. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by a T, as in hu9F5VLv2_L27cT. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by N, as in hu9F5VLv2_L27cN)

In some humanized 9F5 antibodies, the L27b position in the VL region is occupied by D, as in hu9F5VLv2_L27bD. In some humanized 9F5 antibodies, the L30 position in the VL region is occupied by G, as in hu9F5VLv2_I30G. In some humanized 9F5 antibodies, the L33 position in the VL region is occupied by N, as in hu9F5VLv2_L33N. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by A, as in hu9F5VLv2_L27cA. In some humanized 9F5 antibodies, the L33 position in the VL region is occupied by a T, as in hu9F5VLv2_L33T. In some humanized 9F5 antibodies, the L33 position in the VL region is occupied by S, as in hu9F5VLv2_L33S. In some humanized 9F5 antibodies, the L33 position in the VL region is occupied by R, as in hu9F5VLv2_L33R. In some humanized 9F5 antibodies, the L30 position in the VL region is occupied by Q, as in hu9F5VLv2_I30Q. In some humanized 9F5 antibodies, the L27b position in the VL region is occupied by a T, as in hu9F5VLv2_L27bT. In some humanized 9F5 antibodies, the L31 position in the VL region is occupied by G, as in hu9F5VLv2_T31G.

In some humanized 9F5 antibodies, the L27b position in the VL region is occupied by a Q, as in hu9F5VLv2_L27bQ) In some humanized 9F5 antibodies, the L33 position in the VL region is occupied by a G, as in hu9F5VLv2_L33G. In some humanized 9F5 antibodies, the L27c position in the VL region is occupied by P, as in hu9F5VLv2_L27cP. In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by an R, as in hu9F5VLv2_V78R. In some humanized 9F5 antibodies, the L75 position in the VL region is occupied by D, as in hu9F5VLv2_I75D. In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by D, as in hu9F5VLv2_V78D. In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by E, as in hu9F5VLv2_V78E. In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by P, as in hu9F5VLv2_V78P. In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by K, as in hu9F5VLv2_V78K. In some humanized 9F5 antibodies, the L77 position in the VL region is occupied by D, as in hu9F5VLv2_R77D.

In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by G, as in hu9F5VLv2_V78G. In some humanized 9F5 antibodies, position L76 in the VL region is occupied by P,

- 57 047155 as in hu9F5VLv2_S76P. In some humanized 9F5 antibodies, the L75 position in the VL region is occupied by P, as in hu9F5VLv2_I75P. In some humanized 9F5 antibodies, the L75 position in the VL region is occupied by Q, as in hu9F5VLv2_I75Q. In some humanized 9F5 antibodies, the L75 position in the VL region is occupied by G, as in hu9F5VLv2_I75G. In some humanized 9F5 antibodies, the L73 position in the VL region is occupied by P, as in hu9F5VLv2_L73P. In some humanized 9F5 antibodies, the L73 position in the VL region is occupied by G, as in hu9F5VLv2_L73G. In some humanized 9F5 antibodies, the L78 position in the VL region is occupied by Q, as in hu9F5VLv2_V78Q. In some humanized 9F5 antibodies, the L76 position in the VL region is occupied by G, as in hu9F5VLv2_S76G. In some humanized 9F5 antibodies, the L92 position in the VL region is occupied by D, as in hu9F5VLv2_L92D.

In some humanized 9F5 antibodies, the L86 position in the VL region is occupied by a T, as in hu9F5VLv2_Y86T. In some humanized 9F5 antibodies, the L92 position in the VL region is occupied by E, as in hu9F5VLv2_L92E. In some humanized 9F5 antibodies, the L92 position in the VL region is occupied by G, as in hu9F5VLv2_L92G. In some humanized 9F5 antibodies, the L92 position in the VL region is occupied by Q, as in hu9F5VLv2_L92Q. In some humanized 9F5 antibodies, the L93 position in the VL region is occupied by G, as in hu9F5VLv2_L93G). In some humanized 9F5 antibodies, the L85 position in the VL region is occupied by G, as in hu9F5VLv2_V85G. In some humanized 9F5 antibodies, the L92 position in the VL region is occupied by a T, as in hu9F5VLv2_L92T. In some humanized 9F5 antibodies, the L89 position in the VL region is occupied by G, as in hu9F5VLv2_A89G.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, G and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54G, L92I, also known as hu9F5VLv8_DIM1. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, R and I, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM2. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, G and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54T, L92I, also known as hu9F5VLv8_DIM3. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, R and G, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L54R, L92G, also known as hu9F5VLv8_DIM4. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, G, R and I, as in hu9F5VLv8_V3Q, L27cG, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM5.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, Q, G, R and I, respectively, as in hu9F5VLv8_V3Q, L27cD, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM6. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, Q, K, R and I, respectively, as in hu9F5VLv8_V3Q, L27cD, L37Q, M51K, L54R, L92I, also known as hu9F5VLv8_DIM7. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, K, R and I, respectively, as in hu9F5VLv8_V3Q, L27cG, L37Q, M51K, L54R, L92I, also known as hu9F5VLv8_DIM8. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, K, G and I, respectively, as in hu9F5VLv8_V3Q, L27cG, L37Q, M51K, L54G, L92I, also known as hu9F5VLv8_DIM9. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, K, G and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51LY, L54G, L92I, also known as hu9F5VLv8_DIM10.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, Q, G, R and I, respectively, as in hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, L92I, also known as hu9F5VLv8_DIM11 In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, R and G, respectively, as in hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, L92G, also known as hu9F5VLv8_DIM12. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, G, G, G and R, respectively, as in hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54R, also known as hu9F5VLv8_DIM13. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, T and I, respectively, as in hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T, L92I, also known like hu9F5VLv8_DIM14. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, T and G, respectively, as in hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T, L92G, hu9F5VLv8_DIM15 .

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, G, G, G, T, respectively, as in hu9F5VLv8_V3Q, L27cG, L37G, M51G, L54T also known as hu9F5VLv8_DIM16. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, G, G, T and I, respectively, as hu9F5VLv8_V3Q, L27cS, L37G, M51G, L54T, L92I, also known like hu9F5VLv8_DIM17. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, D, G, G, R and I, respectively, as in hu9F5VLv8_V3Q, L27cD, L37G, M51G, L54R, L92I, also known as hu9F5VLv8_DIM18. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q,

- 58 047155

S, I, I, R and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37I, M51I, L54R, L92I, also known as hu9F5VLv8_DIM19. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, I, G and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51I, L54G, L92I, also known as hu9F5VLv8_DIM20.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, S, Q, I and G, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51I, L54G, also known as hu9F5VLv8_DIM21. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, E, R and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51E, L54R, L92I, also known as hu9F5VLv8_DIM22. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, E, G and I, respectively, as in hu9F5VLv8_V3Q, L27cG, L37Q, M51E, L54G, L92I, also known as hu9F5VLv8_DIM23. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, I, E, R and I, respectively, as in hu9F5VLv8_V3Q, L27cG, L37I, M51E, L54R, L92I, also known as hu9F5VLv8_DIM24. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, I, E, R and G, respectively, as in hu9F5VLv8_V3Q, L27cG, L37I, M51E, L54R, L92G, also known as hu9F5VLv8_DIM25.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, I, I, E and R, respectively, as in hu9F5VLv8_V3Q, L27cI, L37I, M51E, L54R, also known as hu9F5VLv8_DIM26. In some humanized 9F5 antibodies, positions L3, L37, L51, L54 and L92 in the VL region are occupied by Q, Q, G, R and I, respectively, as in hu9F5VLv8_V3Q, L37Q, M51G, L54R, L92I, also known as hu9F5VLv8_DIM27. In some humanized 9F5 antibodies, positions L3, L27c, L51, L54 and L92 in the VL region are occupied by Q, S, G, R and I, respectively, as in hu9F5VLv8_V3Q, L27cS, M51G, L54R, L92I, also known as hu9F5VLv8_DIM28. In some humanized 9F5 antibodies, positions L3, L27c, L37, L54 and L92 in the VL region are occupied by Q, S, Q, R and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, L54R, L92I, also known as hu9F5VLv8_DIM29. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51 and L92 in the VL region are occupied by Q, S, Q, G and I, respectively, as in hu9F5VLv8_V3Q, L27cS, L37Q, M51G, L92I, also known as hu9F5VLv8_DIM30.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, G and I, respectively, as in hu9F5VLv9_V3Q, L27cS, L37Q, M51G, L54G, L92I, also known as hu9F5VLv9_DIM1. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, R and I, respectively, as in hu9F5VLv9_V3Q, L27cS, L37Q, M51G, L54R, L92I, also known as hu9F5VLv9_DIM2. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, G, R and G, respectively, as in hu9F5VLv9_V3Q, L27cS, L37Q, M51G, L54R, L92G, also known as hu9F5VLv9_DIM4). In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, G, R and I, respectively, as in hu9F5VLv9_V3Q, L27cG, L37Q, M51G, L54R, L92I, also known as hu9F5VLv9_DIM5. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, Q, K, R and I, respectively, as in hu9F5VLv9_V3Q, L27cG, L37Q, M51LY, L54R, L92I, also known as hu9F5VLv9_DIM8.

In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, K, G and I, respectively, as in hu9F5VLv9_V3Q, L27cS, L37Q, M51K, L54G, L92I, also known as hu9F5VLv9_DIM10. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, G, G, G, R and I, respectively, as in hu9F5VLv9_V3Q, L27cG, L37G, M51G, L54R, L92I, also known as hu9F5VLv9_DIM11. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51 and L54 in the VL region are occupied by Q, G, G, G and R, respectively, as in hu9F5VLv9_V3Q, L27cG, L37G, M51G, L54R, also known as hu9F5VLv9_DIM13. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, I, I, R and I, respectively, as in hu9F5VLv9_V3Q, L27cS, L37I, M51I, L54R, L92I, also known as hu9F5VLv9_DIM19. In some humanized 9F5 antibodies, positions L3, L27c, L37, L51, L54 and L92 in the VL region are occupied by Q, S, Q, I, G and I, respectively, as in hu9F5VLv9_V3Q, L27cS, L37Q, M51I, L54G, L92I, also known as hu9F5VLv9_DIM20.

The heavy chain variable region of any of the above antibodies may also be modified to further reduce immunogenicity. For example, in some humanized antibodies, the H79 position is occupied by D, N, G or Q; position H80 is occupied by P, D, E or G; position H82 is occupied by P, K, R, E or N; position H82a is occupied by G; and/or position H82c is occupied by G, D or S.

In some humanized 9F5 antibodies, the H80 position is occupied by P, as in hu9F5VHv4_L80P. In some humanized 9F5 antibodies, the H80 position is occupied by D, as in hu9F5VHv4_L80D. In some humanized 9F5 antibodies, the H82c position is occupied by G, as in hu9F5VHv4_L82cG and hu9F5VHv10_L82cG. In some humanized 9F5 antibodies, position H82c is occupied by D, as in hu9F5VHv4_L82cD. In some humanized 9F5 antibodies, the H82 position is occupied by P, as in hu9F5VHv4_L82P. In some humanized 9F5 antibodies, the H80 position is occupied by G, as in

- 59 047155 hu9F5VHv4_L80G. In some humanized 9F5 antibodies, the H82 position is occupied by K, as in hu9F5VHv4_L82K. In some humanized 9F5 antibodies, the H82 position is occupied by R, as in hu9F5VHv4_L82R. In some humanized 9F5 antibodies, the H82 position is occupied by E, as in hu9F5VHv4_L82E. In some humanized 9F5 antibodies, the H82 position is occupied by N, as in hu9F5VHv4_L82N.

In some humanized 9F5 antibodies, position H79 is occupied by D, as in hu9F5VHv4_Y79D. In some humanized 9F5 antibodies, the H79 position is occupied by N, as in hu9F5VHv4_Y79N. In some humanized 9F5 antibodies, the H79 position is occupied by G, as in hu9F5VHv4_Y79G. In some humanized 9F5 antibodies, the H80 position is occupied by E, as in hu9F5VHv5_M80E. In some humanized 9F5 antibodies, the H80 position is occupied by G, as in hu9F5VHv5_M80G. In some humanized 9F5 antibodies, the H82c position is occupied by S, as in hu9F5VHv4_L82cS. In some humanized 9F5 antibodies, the H79 position is occupied by Q, as in hu9F5VHv4_Y79Q. In some humanized 9F5 antibodies, the H82a position is occupied by G, as in hu9F5VHv4_S82aG.

In some humanized 9F5 antibodies, the heavy chain variable region has >85% identity with the human sequence. In some humanized 9F5 antibodies, the light chain variable region has >85% identity with the human sequence. In some humanized 9F5 antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. In some humanized 9F5 antibodies, three heavy chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 8, 9 and 10) and three light chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 12, 13 and 14 ); provided that position H28 is occupied by N or T, position H51 is occupied by I or V, position H54 is occupied by N or D, position H56 is occupied by D or E, position L27b is occupied by L, D, T or Q, position L27c is occupied by L, D, G, S, E, T, N, A, P or I, position L30 occupied by I, Y, E, K, G or Q, position L31 occupied by T, N or G, position L33 occupied by L N, T, S, R or G, position L51 occupied by M, G, E, D, K or I, position L54 occupied by L, R, G or T, position L89 occupied by A or G, position L92 occupied by L, D, E, G, Q, T or I, and position L93 is occupied by E or G.

In certain humanized antibodies, 9F5 CDR-H1, as defined by the Kabat-Chothia composite definition, has an amino acid sequence comprising SEQ ID NO: 50. In certain humanized antibodies, Kabat 9F5 CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51 or SEQ ID NO : 52. In certain humanized antibodies, Kabat's 9F5 CDR-L1 has an amino acid sequence comprising SEQ ID NO: 53 or SEQ ID NO: 54. In certain humanized antibodies, Kabat's 9F5 CDR-L2 has an amino acid sequence comprising SEQ ID NO: 55 In certain humanized antibodies, Kabat's 9F5 CDR-L1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 172-193. In certain humanized antibodies, Kabat's 9F5 CDR-L2 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 194-205. In certain humanized antibodies, Kabat's 9F5 CDR-L3 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 206-213.

Examples of humanized antibodies include humanized forms of murine 10C12, designated Hu10C12.

Murine antibody 10C12 includes mature heavy and light chain variable regions having the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11, respectively. The invention provides 2 examples of humanized mature heavy chain variable regions: hu10C12VHv1 and hu10C12VHv2. The invention additionally provides 2 examples of mature light chain variable regions hu10C12VLv1 and hu10C12VLv2. In fig. 7 and 8 show the alignment of the heavy chain variable region and the light chain variable region, respectively, of murine 10C12 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/or antigen binding, mediation of interaction between the heavy and light chains, interaction with the constant region, site for desirable or undesired post-translational modification, a residue unusual for its position in the human variable region sequence and due to of this potential immunogenicity, decreased aggregation potential, and for other reasons, the following 9 variable region framework positions were considered as candidates for substitutions in 2 examples of mature human light chain variable regions and 2 examples of mature human heavy chain variable regions, as further indicated in the examples: L64 (G64S), L104 (V104L), H1 (Q1E), H24 (V24A), H48 (M48I), H67 (V67A), H69 (I69M), H93 (A93T) and H94 (R94T).

Here, as elsewhere, the first mentioned residue is that of a humanized antibody formed by grafting a Kabat CDR, or a Chothia-Kabat composite definition CDR in the case of CDR-H1, onto a human acceptor framework, and the second mentioned residue is a residue considered for replacement such a remainder. Thus, within the variable region frameworks, the first residue mentioned is human, and within the CDRs, the first residue mentioned is

- 60 047155 the crumpled remainder is mouse.

Examples of antibodies include any permutations or combinations of the exemplary mature heavy and light chain variable regions hu10C12VHv1/ hu10C12VLv1, hu10C12VHv1/ hu10C12VLv2, hu10C12VHv2/ hu10C12VLv1, hu10C12VHv2/ hu10C12VL v2.

Examples of antibodies include any permutation or combination of the exemplary mature heavy chain variable regions hu10C12VHv1 (SEQ ID NO: 214) and hu10C12VHv2 (SEQ ID NO: 215) with any of the exemplary mature light chain variable regions hu10C12VLvl (SEQ ID NO : 216) and hu10C12VLv2 (SEQ ID NO: 217).

The invention provides variants of the humanized antibody 10C12, in which the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu10C12VHv1 (SEQ ID NO: 214) or hu10C12VHv2 ( SEQ ID NO: 215), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu10C12VLv1 (SEQ ID NO: 216) or hul10C12VLv2 (SEQ ID NO: 217). In some such antibodies, at least 1, 2, 3, 4, 5, 6, 7, 8, or all 9 backmutations or other mutations in SEQ ID NO: 214-215 and SEQ ID no: 216-217) are retained.

In some humanized 10C12 antibodies, at least one of the following positions in the VH region is occupied by the indicated amino acid: H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, and H94 is occupied by T. In some humanized antibodies, 10C12 positions H24, H48, H67, H69, H93 and H94 are occupied by A, I, A, M, T and T, respectively.

In some humanized 10C12 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, H94 is occupied by T.

In some humanized 10C12 antibodies, positions H24, H48, H67, H69, H93 and H94 are occupied by A, I, A, M, T and T, respectively, as in hu10C12VHv1. In some humanized 10C12 antibodies, positions H1, H24, H48, H67, H69, H93 and H94 are occupied by E, A, I, A, M, T and T, respectively, as in hu10C12VHv2.

In some humanized 10C12 antibodies, the L64 position in the VL region is occupied by S.

In some humanized 10C12 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L64 is occupied by S, L104 is occupied by V or L.

In some humanized 10C12 antibodies, positions L64 and L104 in the VL region are occupied by S, as in hu10C12VLv1. In some humanized 10C12 antibodies, positions L64 and L104 in the VL region are occupied by S and L, respectively, as in hu10C12VLv2.

In some humanized 10C12 antibodies, the heavy chain variable region has >85% identity with the human sequence. In some humanized 10C12 antibodies, the light chain variable region has >85% identity with the human sequence. In some humanized 10C12 antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. In some humanized 10C12 antibodies, three heavy chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 8, 9 and 10) and three light chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 12, 13 and 14 ).

Examples of humanized antibodies include humanized forms of murine 12C4, designated Hu12C4.

Murine antibody 12C4 includes mature heavy and light chain variable regions having the amino acid sequences of SEQ ID NO: 219 and SEQ ID NO: 11, respectively. The invention provides 2 examples of humanized mature heavy chain variable regions: hu12C4VHv1 and hu12C4VHv2. The invention additionally provides 2 examples of mature light chain variable regions hu12C4VLv1 and hu12C4VLv2. In fig. 9 and 10 show the alignment of the heavy chain variable region and the light chain variable region, respectively, of murine 12C4 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/or antigen binding, mediation of interaction between the heavy and light chains, interaction with the constant region, site for desirable or undesired post-translational modification, a residue unusual for its position in the human variable region sequence and due to of this potential immunogenicity, obtaining aggregation potential, and for other reasons, the following 6 variable region framework positions were considered as candidates for substitutions in 2 examples of mature human light chain variable regions and 2 examples of mature human heavy chain variable regions, as further indicated in the examples: L64 (G64S), L104 (V104L), H1 (Q1E), H48 (M48I), H93 (A93T) and H94 (R94T).

Here as elsewhere, the first-mentioned residue is that of a humanized antibody formed by grafting a Kabat CDR, or a Chothia-Kabat composite definition CDR in the case of CDR-H1, onto a human acceptor framework, and the second-mentioned residue is

- 61 047155 the balance considered to replace such balance. Thus, within the variable region frameworks, the first residue mentioned is human, and within the CDRs, the first residue mentioned is mouse.

Examples of antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions hu12C4VHv1/ hu12C4VLv1, hu12C4VHv1/ hu12C4VLv2, hu12C4VHv2/ hu12C4VLv1, hu12C4VHv2/ hu12C4VLv2.

Examples of antibodies include any permutation or combination of the exemplary mature heavy chain variable regions hu12C4VHv1 (SEQ ID NO: 221) and hu12C4VHv2 (SEQ ID NO: 222) with any of the exemplary mature light chain variable regions hu12C4VLv1 (SEQ ID NO : 223) and hu12C4VLv2 (SEQ ID NO: 224).

The invention provides variants of the humanized 12C4 antibody wherein the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu12C4VHv1 (SEQ ID NO: 221) or hu12C4VHv2 ( SEQ ID NO: 222), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu12C4VLv1 (SEQ ID NO: 223) or hu12C4VLv2 (SEQ ID NO: 224). In some such antibodies, at least 1, 2, 3, 4, 5, or all 6 of the back mutations or other mutations in SEQ ID NOs: 221-222 and SEQ ID NOs: 223-224) are retained.

In some humanized 12C4 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H48 is occupied by M or I, H93 is occupied by A or T, H94 is occupied by R or T.

In some humanized 12C4 antibodies, positions H1, H48, H93 and H94 in the VH region are occupied by E, I, T and T, respectively, as in hu12C4VHv2.

In some humanized 12C4 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L64 is occupied by G or S, L104 is occupied by V or L.

In some humanized 12C4 antibodies, positions L64 and L104 in the VL region are occupied by S and L, respectively, as in hu12C4VLv2.

In some humanized 12C4 antibodies, the heavy chain variable region has >85% identity with the human sequence. In some humanized 12C4 antibodies, the light chain variable region has >85% identity with the human sequence. In some humanized 12C4 antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. In some humanized 12C4 antibodies, three heavy chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 8, 220 and 10) and three light chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 12, 13 and 14 ).

Examples of humanized antibodies are humanized forms of murine 17C12, designated Hu17C12.

Murine antibody 17C12 includes mature heavy and light chain variable regions having the amino acid sequences of SEQ ID NO: 225 and SEQ ID NO: 228, respectively. The invention provides 2 examples of humanized mature heavy chain variable regions: hu17C12VHv and hu17C12VHv2. The invention additionally provides 2 examples of mature light chain variable regions hu17C12VLv1 and hu17C12VLv2. In fig. 11 and 12 show the alignment of the heavy chain variable region and the light chain variable region, respectively, of murine 17C12 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/or antigen binding, mediation of interaction between the heavy and light chains, interaction with the constant region, site for desirable or undesired post-translational modification, a residue unusual for its position in the human variable region sequence and due to of this potential immunogenicity, obtaining aggregation potential, and for other reasons, the following 13 variable region framework positions were considered as candidates for substitutions in 2 examples of mature human light chain variable regions and 2 examples of mature human heavy chain variable regions, as further indicated in the examples: L2 (I2V), L36 (Y36L), L43 (P43S), H1 (Q1E), H2 (V2I), H24 (V24A), H48 (M48I), H67 (V67A), H69 (I69M), H93 (A93T), H94 (R94T), H108 (T108L) and H113 (R113S).

Here, as elsewhere, the first mentioned residue is that of a humanized antibody formed by grafting a Kabat CDR, or a Chothia-Kabat composite definition CDR in the case of CDR-H1, onto a human acceptor framework, and the second mentioned residue is a residue considered for replacement such a remainder. Thus, within the variable region frameworks, the first residue mentioned is human, and within the CDRs, the first residue mentioned is mouse.

Examples of antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions hu17C12VHv1/hu17C12VLv1, hu17C12VHv1/hu17C12VLv2, hu17C12VHv2/hu17C12VLv1, hu17C12VHv2/hu17C12VLv2.

- 62 047155

Examples of antibodies include any permutation or combination of the exemplary mature heavy chain variable regions hu17C12VHv1 (SEQ ID NO: 232) and hu17C12VHv2 (SEQ ID NO: 233) with any of the exemplary mature light chain variable regions hu17C12VLv1 (SEQ ID NO : 234) and hu17C12VLv2 (SEQ ID NO: 235).

The invention provides variants of the humanized antibody 17C12, in which the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98% or 99% identity with hu17C12VHv1 (SEQ ID NO: 232) or hu17C12VHv2 ( SEQ ID NO: 233), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu17C12VLv1 (SEQ ID NO: 234) or hu17C12VLv2 (SEQ ID NO: 235). Some such antibodies have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 back mutations or other mutations in SEQ ID NOs: 232-233 and SEQ ID NOs : 234-235 saved.

In some humanized 17C12 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H2 is occupied by I, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, and H94 is occupied by T. In some In humanized antibodies 17C12, positions H2, H24, H48, H67, H69, H93 and H94 are occupied by E, A, I, A, M, T and T, respectively.

In some humanized 17C12 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H1 is occupied by Q or E, H2 is occupied by I, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, H94 occupied by T, H108 occupied by T or L, H1 13 occupied by R or S.

In some humanized 17C12 antibodies, positions H2, H24, H48, H67, H69, H93 and H94 in the VH region are occupied by E, A, I, A, M, T and T, respectively, as in hu17C12VHv1. In some humanized 17C12 antibodies, positions H1, H2, H24, H48, H67, H69, H93, H94, H108 and H113 in the VH region are occupied by E, I, A, I, A, M, T, T, L, and S, respectively, as in hu17C12VHv2.

In some humanized 17C12 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is occupied by V, and L36 is occupied by L. In some humanized 17C12 antibodies, positions L2 and L36 are occupied by V and L, respectively.

In some humanized 17C12 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is occupied by V, L36 is occupied by L, L43 is occupied by P or S.

In some humanized 17C12 antibodies, positions L2 and L36 in the VL region are occupied by V and L, respectively, as in hu17C12VLv1. In some humanized 17C12 antibodies, positions L2, L36 and L43 in the VL region are occupied by V, L and S, respectively, as in hu17C12VLv2.

In some humanized 17C12 antibodies, the heavy chain variable region has >85% identity with the human sequence. In some humanized 17C12 antibodies, the light chain variable region has >85% identity with the human sequence. In some humanized 17C12 antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. In some humanized 9F5 antibodies, three heavy chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 226, 227 and 10), and three light chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NOs: 229-231).

Examples of humanized antibodies are humanized forms of murine 14H3, designated Hu14H3.

Murine antibody 14H3 includes mature heavy and light chain variable regions having the amino acid sequences of SEQ ID NO: 240 and SEQ ID NO: 244, respectively. The invention provides 2 examples of humanized mature heavy chain variable regions: hu14H3VHv1 and hu14H3VHv2. The invention additionally provides 2 examples of mature light chain variable regions hu14H3VLv1 and hu14H3VLv2. In fig. 13 and 14 show the alignment of the heavy chain variable region and the light chain variable region, respectively, of murine 14H3 and various humanized antibodies.

For reasons such as possible influence on CDR conformation and/or antigen binding, mediation of interaction between the heavy and light chains, interaction with the constant region, site for desirable or undesired post-translational modification, a residue unusual for its position in the human variable region sequence and due to of this potential immunogenicity, obtaining aggregation potential, and for other reasons, the following 8 variable region framework positions were considered as candidates for substitutions in 2 examples of mature human light chain variable regions and 2 examples of mature human heavy chain variable regions, as further indicated in the examples: L2 (I2V), L7 (T7S), L37 (L37Q), L87 (Y87F), L100 (G100Q), L104 (V104L), H108 (M108L) and Refinery (L113S). The following variable region CDR position was considered as a candidate for replacement in 2 examples of mature human heavy chain variable regions, as further specified in the examples: H35B (G35BS). In some humanized antibodies, the 14H3 CDR-H1, as defined by the Kabat-Chothia composite definition, has the amino acid sequence comprising SEQ ID NO: 275.

- 63 047155

Here, as elsewhere, the first mentioned residue is that of a humanized antibody formed by grafting a Kabat CDR, or a Chothia-Kabat composite definition CDR in the case of CDR-H1, onto a human acceptor framework, and the second mentioned residue is a residue considered for replacement such a remainder. Thus, within the variable region frameworks, the first residue mentioned is human, and within the CDRs, the first residue mentioned is mouse.

Examples of antibodies include any permutations or combinations of the exemplified mature heavy and light chain variable regions hu14H3VHv1/hu14H3VLv1, hu14H3VHv1/hu14H3VLv2, hu14H3VHv2/hu14H3VLv1, hu14H3VHv2/hu14H3VLv2.

Examples of antibodies include any permutation or combination of the exemplary mature heavy chain variable regions hu14H3VHv1 (SEQ ID NO: 248) and hu14H3VHv2 (SEQ ID NO: 249) with any of the exemplary mature light chain variable regions hu14H3VLv1 (SEQ ID NO : 250) and hu14H3VLv2 (SEQ ID NO: 251).

The invention provides variants of the humanized 14H3 antibody in which the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu14H3VHv1 (SEQ ID NO: 249) or hu14H3VHv2 ( SEQ ID NO: 250), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity with hu14H3VLv1 (SEQ ID NO: 251) or hu14H3VLv2 (SEQ ID NO: 252). In some such antibodies, at least 1, 2, 3, 4, 5, 6, 7, 8, or all 9 backmutations or other mutations in SEQ ID NOs: 249-250 and SEQ ID NOs: 251-252 are retained.

In some humanized 14H3 antibodies, the H35B position in the VH region is occupied by S.

In some humanized 14H3 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H35B is occupied by S, H108 is occupied by M or L, H113 is occupied by L or S.

In some humanized 14H3 antibodies, the H35B position in the VH region is occupied by S, as in hu14H3VHv1. In some humanized 14H3 antibodies, positions H35B, H108 and H113 in the VH region are occupied by S, L and S, respectively, as in hu14H3VHv2.

In some humanized 14H3 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is occupied by V, and L87 is occupied by F. In some humanized 14H3 antibodies, positions L2 and L87 are occupied by V and F, respectively.

In some humanized 14H3 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is occupied by V, L7 is occupied by T or S, L37 is occupied by L or Q, L87 is occupied by F, L100 is occupied by G or Q, L104 is occupied by V or L .

In some humanized 14H3 antibodies, positions L2 and L87 in the VL region are occupied by V and F, respectively, as in hu14H3VLv1. In some humanized 14H3 antibodies, positions L2, L7, L37, L87, L100 and L104 in the VL region are occupied by V, S, Q, F, Q and L, respectively, as in hu14H3VLv2.

In some humanized 14H3 antibodies, the heavy chain variable region has >85% identity with the human sequence. In some humanized 14H3 antibodies, the light chain variable region has >85% identity with the human sequence. In some humanized 14H3 antibodies, the heavy chain variable region and the light chain variable region each have >85% identity to the human germline sequence. In some humanized 14H3 antibodies, the three heavy chain CDRs correspond to the Kabat/Chothia composite definition (SEQ ID NO: 241-243); provided that the H35B position is occupied by a G or S and the three light chain CDRs meet the Kabat/Chothia composite definition (SEQ ID NO: 245-247).

In certain humanized antibodies, the 14H3 CDR-H1, as defined by the Kabat-Chothia composite definition, has the amino acid sequence comprising SEQ ID NO: 275.

The CDR regions of such humanized antibodies 9F5, 10C12, 2D11, 12C4, 17C12 and 14H3 may be identical or substantially identical to the CDR regions of antibodies 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3. CDR regions may be defined according to any traditional definition (eg, Chothia, or a composite definition of Chothia and Kabat), but preferably they correspond to the Kabat definition.

The positions of the variable region frameworks correspond to Kabat numbering unless otherwise noted. Other such variants typically differ from the sequences of the example heavy and light chains Hu9F5, Hu10C12, Hu12C4, Hu17C12, or Hu14H3 by a small number (e.g., typically no more than 1, 2, 3, 5, 10, or 15) substitutions, deletions, or insertions. Such differences typically occur in the framework but can also occur in the CDR.

Possibility of Additional Variations in Humanized Variants 9F5, 10C12, 2D11, 12C4, 17C12, and 14H3 represent additional back mutations in the variable region frameworks. Many of the framework residues not contacting the CDR in the humanized mAb may accommodate amino acid substitutions from corresponding positions in the donor murine mAb or other murine or human antibodies, and even many potential CDR-contacting residues are also under

- 64 047155 are given to the replacement. Even the amino acids in the CDRs can be changed, for example, residues found at the corresponding position of the human acceptor sequence are used to provide variable region frameworks. In addition, alternative human acceptor sequences may be used, for example for the heavy and/or light chain. When using different acceptor sequences, one or more of the back mutations recommended above can be omitted since the corresponding donor and acceptor residues are already the same without back mutations.

Preferably, substitutions or back mutations in the humanized variants 9F5, 10C12, 2D11, 12C4, 17C12 and 14H3 (conserved or not) do not significantly affect the binding affinity or potency of the humanized mAb, that is, its ability to bind to tau.

Humanized 9F5 antibodies are further characterized by their ability to bind phosphorylated and unphosphorylated tau, as well as misfolded/aggregated forms of tau.

D. Chimeric and venated antibodies

The invention also provides chimeric and venated forms of non-human antibodies, in particular antibodies 9F5, 10C12, 2D11, 12C4, 1702 or 14H3 from the examples.

A chimeric antibody is an antibody in which the mature light and heavy chain variable regions of a non-human antibody (eg, mouse) are combined with human light and heavy chain constant regions. Such antibodies retain substantially or completely the binding specificity of the murine antibody and are approximately two-thirds human sequence.

A venated antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some non-human variable region framework residues of the non-human antibody, replacing other variable region framework residues that may contribute to B or T cell epitopes, such as accessible residues ( Padlan, Mol. Immunol. 28:489, 1991) replace with residues from corresponding positions in the human antibody sequence. The result is an antibody in which the CDRs belong entirely or substantially to the non-human antibody, and the variable region frameworks of the non-human antibody are made more human-like due to the substitutions. The invention includes venated forms of antibodies 9F5, 10C12, 2D11, 12C4, 17C12 and 14H3.

E. Human antibodies

Human antibodies that specifically bind to tau or a fragment thereof (e.g., a peptide comprising or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or (Q /E)IVYK(S/P) (SEQ ID NO: 56)) is obtained using various methods described below. Some human antibodies are selected by competitive binding experiments, the Winter phage display method described above, or otherwise to have the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the Examples. Human antibodies can also be tested for epitope specificity using only a tau fragment, such as a tau fragment containing or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or (Q/E)IVYK(S/P) (SEQ ID NO: 56) as a target antigen, and/or by screening antibodies for a set of tau variants, such as tau variants containing various mutations in the amino acid residues 307-312, 391-397 or 391-396 SEQ ID NO: 1.

Methods for producing human antibodies include the trioma method described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, US Patent No. 4,634,664 and Engleman et al., US Patent No. 4,634,666, use of transgenic mice incorporating human immunoglobulin genes (see, for example, Lonberg et al., WO 93/12227 (1993); US 5877397; US 5874299; US 5814318; US 5770429; US 5633425; US 5545806; 1/10741 (1991)) phage display methods (see, for example, 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 (eg, immortalization of memory B cells isolated from humans, eg by EBV, screening for desired properties, cloning and expression of recombinant forms).

F. Selection of constant region

The variable regions of the heavy and light chains of chimeric, venated or humanized antibodies may be linked to at least a portion of the 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, the human IgG1 and IgG3 isotypes have complement-dependent cytotoxicity, but the human IgG2 and IgG4 isotypes do not. Human IgG1 and IgG3 also induce stronger cell-mediated functions than human IgG2 and IgG4. Light chain constant regions 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

- 65 047155 of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991, 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 exon numbering according to IMGT (Lefranc, see above).

One or more amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be absent or derivatized in part or all of the molecules. Substitutions can be made in constant regions to decrease or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., US Pat. No. 5,624,821; Tso et al., US Pat. No. 5,834,597; and Lazar et al. ., Proc. Natl. Sci. USA 103:4005, 2006) or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Examples of 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. Substitution at any or all of positions 234, 235, 236 and/or 237 reduces affinity for Fcy receptors, especially the FcyRI receptor (see, for example, US 6,624,821). Alanine substitution at positions 234, 235 and 237 of human IgG1 can be used to reduce effector functions. Some antibodies have alanine substitutions at positions 234, 235 and 237 of human IgG1 to reduce effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are replaced with alanine, and position 235 is replaced 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 the EU numbering of human IgG1. Some antibodies use a mutation at one or more positions, 318, 320 and 322 of the EU numbering of human IgG1. In some antibodies, positions 234 and/or 235 are replaced with alanine and/or position 329 is replaced with glycine. In some antibodies, positions 234 and 235 are replaced 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 separate heavy chains, light chains, as Fab, Fab', F(ab')2 and Fv, or as single-chain antibodies in which the heavy chain variable domains and light chain are connected through a spacer.

Human constant regions exhibit allotypic variation and isoallotypic variation among individuals, that is, constant regions may vary among individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of one or more other isotypes. For example, another heavy chain constant region is Glm3 IgG1 with or without a C-terminal lysine. A reference to a human constant region includes a constant region with any naturally occurring allotype or any permutation of residues occupying positions in natural allotypes.

G. Expression of recombinant antibodies

A number of methods are known for producing chimeric and humanized antibodies using cell lines expressing antibodies (eg, hybridomas). For example, immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods. In one method, the VH heavy chain variable region is cloned by RT-PCR using mRNA obtained from hybridoma cells. Consensus primers are used for the VH region leader peptide spanning the translation initiation codon as a 5' primer and a 3' primer specific for the g2b constant regions. Examples of primers are described in US patent publication US 2005/0009150 Schenk et al. (hereinafter Schenk). Sequences from multiple independently obtained clones can be compared to ensure no changes occur during amplification. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained using RT-PCR methodology with 5'-RACE (rapid amplification of cDNA ends) and a 3'g2b-specific primer.

The VL light chain variable region can be cloned in a similar manner. In one approach, a set of consensus primers is designed to amplify VL regions using a 5' primer designed to hybridize to the VL region containing the translation initiation codon and a 3' primer specific for the Ck region located downstream of the V-J junction region. In the second approach, 5′-RACE RT-PCR methodology is used to clone the cDNA encoding VL. Examples of primers are described in Schenk, 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 reconstructed to encode splice donor sequences downstream of the corresponding VDJ or VJ junctions and cloned into a mammalian expression vector such as pCMV-h/1 for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human γ1 and Ck constant regions as exonic fragments downstream of the inserted variable region cassette. After sequence verification, expression vectors

- 66 047155 heavy chain and light chain can be cotransfected into CHO cells to produce chimeric antibodies. Conditioned media are collected 48 h after transfection and analyzed by Western blotting for antibody production or ELISA for antigen binding. Chimeric antibodies are humanized as described above.

Chimeric, venous, humanized and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of the antibody chains, including naturally associated 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 is incorporated into an appropriate host, that host is maintained under conditions that are suitable for high-level expression of the nucleotide sequences and for the collection and purification of cross-reacting antibodies.

These expression vectors are typically reproducible in the host as episomes or as part of the host's chromosomal DNA. Typically, expression vectors contain selection markers, such as ampicillin resistance or hygromycin resistance, to enable detection of those cells transformed with the desired DNA sequences.

E. coli is a prokaryotic host useful for the expression of antibodies, especially antibody fragments. Other microorganisms such as yeast can also be used for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, origin of replication, termination sequences and the like, if desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C and enzymes that are responsible for the use of maltose and galactose.

Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed and these include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells and non-antibody producing myeloma cells including Sp2/0 and NS0 . The cells may be non-human. Expression vectors for these cells may include expression control sequences such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary informational processing sites such as ribosome binding sites , RNA splicing sites, polyadenylation sites, and transcription termination sequences. Expression control sequences may include 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 included in transgenes for insertion into the genome of the transgenic animal and subsequent expression in the milk of the transgenic animal (see, for example, US Pat. No. 5,741,957; US Pat. No. 5,304,489; and US Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked to a promoter and enhancer from a mammary gland-specific gene such as casein or beta-lactoglobulin.

Vectors containing DNA segments of interest can be transferred into a host cell in a manner dependent on the type of host cell. For example, calcium chloride transfection is typically used for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or virus-based transfection may be used for other host cells. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection. To produce transgenic animals, transgenes can be microinjected into fertilized oocytes or they can be incorporated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs), and the nuclei of such cells are transferred into enucleated oocytes.

Once the vector(s) encoding the antibody heavy and light chains are introduced into cell culture, pools of cells can be tested for growth performance and product quality in serum-free media. Pools of high-producing cells can then be subjected to FACS-based single cell cloning to create monoclonal lines. Specific productivity above 50 or 100 pg per cell per day can be used, which corresponds to product titers above 7.5 g/l of culture. Antibodies derived from single cell clones can also be tested for turbidity, filtration properties by PAGE, IEF, UV scanning, HPEC, carbohydrate-oligosaccharide mapping, mass spectrometry and binding assays such as ELISA or Biacore. The selected clone can then be jarred in several vials and stored frozen

- 67 047155 new form for subsequent use.

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

Commercial antibody production methodology may be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell banking, use of selection markers to enhance copy number, CHO terminator, or improve protein titer (see e.g. US 5786464; US 6063598; WO 2008/012142; WO 2008/012142; O2 009/027471 and US 5888809).

IV. Active immunogens

The agent used for active immunization serves to induce in the patient the same types of antibodies that are described above in connection with passive immunization. The agents used for active immunization may be the same types of immunogens that are used to generate 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 307-312 or 391-397 or 391-396 of SEQ ID NO: 1, such as, for example, a tau peptide comprising or consisting of residues 307-312, 391-397 or 391-396 of SEQ ID NO: 1) or a tau peptide containing or consisting of the amino acid sequence QIVYKP (SEQ ID NO: 57), a tau peptide containing or consisting of the amino acid sequence EIVYKSP (SEQ ID NO: 58), a tau peptide containing or consisting of an acid the sequence EIVYKS (SEQ ID NO: 277) or a tau peptide containing or consisting of the amino acid sequence (Q/E)IVYK(S/P) (SEQ ID NO: 56). To induce binding of antibodies to the same or overlapping epitope as 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3, the epitope specificity of these antibodies can be mapped (eg, by testing binding to a series of overlapping peptides spanning tau). A tau fragment consisting of, including, or overlapping the epitope can then be used as an immunogen. Such fragments are usually used in non-phosphorylated form.

The heterologous carrier and adjuvant, if used, may be the same as those used to produce monoclonal antibodies, but may also be selected for better pharmaceutical suitability for use in humans. Suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid or toxoid of other pathogenic bacteria such as diphtheria (eg CRM197), E. coli, cholera or H. pylori, or an attenuated toxin derivative. T cell epitopes are also suitable carrier molecules. Some conjugates can be formed by linking the agents of the invention with an immunostimulatory polymer molecule (for example, tripalmitoyl-glycerol cysteine (Pam ₃ Cys), mannan (mannose polymer) or glucan (β 1^-2 polymer)), cytokines (for example, IL -1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF) and chemokines (eg, MIP1-a and β and RANTES). Immunogens can be associated with carriers with or without amino acid spacers (eg, gly-gly). Additional carriers include virus-like particles. Virus-like particles (VLPs), also called pseudovirions or viral particles, are subunit structures consisting of multiple copies of the viral capsid and/or envelope protein that are capable of self-assembly into VLPs with defined spherical symmetry in vivo. (Powilleit, et al., (2007) PLoS ONE 2(5):e415.) Alternatively, peptide immunogens can be bound to at least one artificial T cell epitope capable of binding most MHC class II molecules, such as epitope pan DR (PADRE). PADRE is described in US 5,736,142, WO 95/07707 and in 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 induced antibodies and/or the binding affinity of induced antibodies compared to the situation if the peptide is used alone. Various adjuvants can be used in combination with an immunogenic tau fragment to induce an immune response. Some adjuvants enhance the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Some 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, Framingham, MA; now Antigenics, Inc., New York, NY). Other adjuvants are oil-in-water emulsions (eg, squalene or peanut oil), optionally in combination with immunostimulants. like this

- 68 047155 as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers and killed mycobacteria. Ribi adjuvants are oil-in-water emulsions. Ribi contains a metabolizable oil (squalene) emulsified with a saline solution 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 a component of a therapeutic composition with the active agent, or they may be administered separately, before, simultaneously with, or after administration of the therapeutic agent.

Analogs of natural tau fragments that induce anti-tau antibodies can also be used. For example, one or more or all of the L-amino acids may be replaced by D-amino acids in such peptides. Also, the order of amino acids can be reversed (retropeptide). Optionally, the peptide includes all D-amino acids in reverse order (retroinverse peptide). Peptides and other compounds that do not necessarily have significant amino acid sequence similarity to tau peptides, but nevertheless serve as tau peptide mimics and induce a similar immune response. Anti-idiotypic antibodies against tau monoclonal antibodies can also be used as described above. Such anti-I antibodies mimic an antigen and induce an immune response to it (see Essential Immunology, Roit ed., Blackwell Scientific Publications, Palo Alto, CA 6th ed., p. 181).

Peptides (and optionally a carrier fused to the peptide) can also be administered in the form of a nucleic acid encoding the peptide and expressed in situ in the patient. The nucleic acid segment encoding the immunogen is typically associated with regulatory elements, such as a promoter and enhancer, that cause the DNA segment to be expressed 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 the immunoglobulin light or heavy chain genes or the CMV major intermediate early promoter and enhancer are suitable for directing 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, then the chains are preferably bound as a single chain antibody. Antibodies for passive administration can also be obtained, for example, using affinity chromatography from the serum of patients treated with peptide immunogens.

The DNA can be delivered in an unprotected form (i.e., without colloidal or encapsulating materials). Alternatively, a number of viral vector systems can be used, including retroviral systems (see, for example, Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)), including retrovirus-derived vectors such as MMLV, HIV-1 and ALV; adenoviral vectors {see, for example, Bett et al. J. Virol. 67, 5911 (1993)); adeno-associated viral vectors {see, for example, Zhou et al., J. Exp. Med. 179, 1867 (1994)), lentiviral vectors, such as vectors based on HIV or FIV gag sequences, viral vectors from the pox family, including vaccinia virus and fowlpox viruses, viral vectors from the alpha virus family, such as vectors derived from from Sindbis virus 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)).

DNA encoding an immunogen or encoding the heavy and/or light chains of an antibody, or a vector containing them, can be packaged in liposomes. Suitable lipids and related analogues are described in US 5,208,036, US 5,264,618, US 5,279,833 and US 5,283,185. Vectors and DNA encoding an immunogen or encoding the heavy and/or light chains of an antibody can also be adsorbed or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-coglycolides) (see, for example, McGee et al., J. Micro Encap. 1996).

Vectors or segments thereof encoding antibody heavy and/or light chains can be incorporated into cells ex vivo, for example, into cells explanted from an individual patient (eg, lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells with subsequent re-implantation of the cells into the patient, usually after selecting cells that contain the transgenes. (see for example WO 2017/091512). Examples of patient-derived cells include patient-derived induced pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural or mesenchymal).

The vector or segment thereof encoding the heavy and/or light chains of the antibody can be introduced into any region of interest in cells ex vivo, such as an albumin gene or other safe haven gene. Cells comprising the vector can be implanted with or without prior differentiation. Cells can be implanted into a specific tissue, such as secretory tissue or a site of pathology, or systemically, such as by infusion into the blood. For example, the cells can be implanted into a patient's secretory tissue, such as the liver, without necessarily first differentiating into cells present in that tissue, such as hepatocytes in the case of the liver. Expression of the antibody in the liver leads to secretion of the antibody into the blood.

- 69 047155

H. Antibody Screening Tests

Initially, antibodies can be screened for putative binding specificity as described above. Active immunogens can also be tested 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 sera are tested for appropriate binding specificity.

Antibodies that have the desired binding specificity can then be tested in cellular and animal models. The cells 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 quadruple 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 neuroblastoma cell line N2a by adding doxycycline. Cellular models allow us to study the toxicity of tau to cells in a soluble or aggregated state, the appearance of tau aggregates after turning on tau gene expression, the dissolution of tau aggregates after turning off gene expression again, and the effectiveness of antibodies in inhibiting the formation of tau aggregates or their disaggregation.

Antibodies or active immunogens can also be screened in transgenic animal models of tau-associated diseases. Such transgenic animals may include a tau transgene (eg, any human isoform) and, optionally, a human APP transgene, among others, such as tau phosphorylating kinase, ApoE, presenilin, or alpha-synuclein. Such transgenic animals are prone to develop at least one sign or symptom of a disease associated with tau.

An example of a transgenic animal is the K3 mouse line (Itner et al., Proc. Natl. Acad. Sci. USA 105(41): 15997-6002 (2008)). These mice have a human tau transgene with the K 369 I mutation (a mutation associated with Pick's disease) and a Thy 1.2 promoter. This model shows a rapid progression of neurodegeneration, motor impairment, and degeneration of cerebellar afferent fibers and granule cells. Another animal example is the JNPL3 mouse line. These mice have a human tau transgene with the P301L mutation (a mutation associated with frontotemporal dementia) and the Thy 1.2 promoter (Taconic, Germantown, N.Y., Lewis, et al., Nat Genet. 25:402-405 (2000)). In these mice, neurodegeneration occurs gradually. Mice develop neurofibrillary tangles in several regions of the brain and spinal cord, which are incorporated herein by reference in their entirety). This is an excellent model for studying the consequences of tangle development and for screening treatments that can inhibit the formation of these aggregates. Another advantage of these animals is the relatively early onset of pathology. In the homozygous line, behavioral abnormalities associated with tau pathology can be observed after at least 3 months, 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 effect of treatment. Active immunization of these mice for 6–13 months with AI with KLH-PHF-1 produced titers of approximately 1000 and showed fewer neurofibrillary tangles, less pSer422, and less body weight loss compared to untreated control mice.

The activity of antibodies or active agents can be assessed by various criteria, including reduction in the amount of total tau or phosphorylated tau, reduction in other pathological characteristics such as amyloid Ab deposits, and inhibition or delay, or behavioral disturbances. Active immunogens can also be tested for antibody induction in sera. Both passive and active immunogens can be tested for the passage of antibodies across the blood-brain barrier into the brain of the transgenic animal. Antibodies or antibody-inducing fragments can also be tested in non-human primates that develop symptoms of diseases characterized by tau either naturally or by induction. Antibody or active agent tests are typically performed in combination with a control in which a parallel experiment is performed except that the antibody or active agent is missing (eg, replaced by vehicle). Reduction, delay or inhibition of signs or symptoms of disease associated with the test antibody or active agent can then be assessed relative to the control.

V. Patients amenable to treatment

The presence of neurofibrillary tangles has been found in a number of diseases, including Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic or boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia , frontotemporal lobar degeneration, disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP). Real schemes

- 70 047155 we can also be used to treat or prevent any of these diseases. Because of the widespread association between neurological diseases and conditions, as well as tau, the present regimens can be used for the treatment or prevention of any subject who exhibits elevated levels of tau or phosphorylated tau (eg, in CSF) compared to the average value in individuals without neurological diseases. The present regimens can also be used to treat or prevent a neurological disease in individuals who have a tau mutation associated with a neurological disease. The present methods are particularly suitable for the treatment or prevention of Alzheimer's disease, especially in patients.

Treatable patients include individuals at risk of developing the disease but not showing symptoms, as well as patients who are currently showing symptoms. Patients at risk for developing the disease include those with a known genetic risk for developing the disease. These individuals include those who have relatives who have had the disease and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk include tau mutations, such as those discussed above, as well as mutations in other genes associated with neurological disease. For example, the ApoE4 allele in heterozygous and, especially, 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, particularly mutations at position 717 and positions 670 and 671, called the Hardy mutation and the Swedish mutation, respectively, mutations in the presenilin, PS1 and PS2 genes, AD, hypercholesterolemia or atherosclerosis in a family history medical history. Individuals currently suffering from Alzheimer's disease can be recognized using PET imaging by the characteristic dementia as well as the presence of the risk factors described above. In addition, a number of diagnostic tests are available to identify individuals with AD. These include measuring tau or phospho-tau levels in the CSF and Av42. Elevated levels of tau or phospho-tau and decreased levels of Av42 indicate the presence of AD. Some mutations associated with Parkinson's disease. Ala30Pro or A1a53 or mutations in other genes associated with Parkinson's disease, such as leucine-rich kinase, PARK8. Individuals may also be diagnosed with any of the neurological disorders mentioned above using DSM IV TR (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Revised) criteria.

In asymptomatic patients, treatment can begin at any age (eg, 10, 20, 30). However, it is usually not necessary to begin treatment until the patient is 40, 50, 60, or 70 years old. Treatment usually involves taking several doses over a period of time. Treatment can be monitored by analyzing antibody levels over time. If the response decreases, a booster dose is prescribed. For potential Down syndrome patients, treatment may begin prenatally with maternal administration of a therapeutic agent or shortly after birth.

I. Nucleic acids

The invention further provides nucleic acids encoding any of the heavy and light chains described above (for example, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15-22, SEQ ID NO: 23-29, 61- 108, 109-129, 130-171, 214-217, 219, 221-224, 225, 228, 232-235, 240, 244 and 248-251). Optionally, such nucleic acids further encode a signal peptide and can be expressed by the signal peptide linked to the variable region. Nucleic acid coding sequences may be operably linked to regulatory sequences to provide expression of coding sequences such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The regulatory sequences may include a promoter, for example a prokaryotic promoter or a eukaryotic promoter. Nucleic acids encoding heavy or light chains can be codon optimized for expression in the host cell. The nucleic acids encoding the heavy and light chains may encode the gene of choice. The nucleic acids encoding the heavy and light chains may be in isolated form or may be cloned into one or more vectors. Nucleic acids can be synthesized, for example, by solid phase synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be joined as one contiguous nucleic acid, for example in an expression vector, or may be separate, for example each cloned into its own expression vector.

J. Conjugated antibodies

Conjugated antibodies that specifically bind to antigens such as tau are useful in detecting the presence of tau; monitoring and evaluating the effectiveness of therapeutic agents used to treat patients 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 disease type C, supranuclear palsy , frontotemporal dementia, frontotemporal lobar degeneration, disease,

- 71 047155 characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GTT), Parkinson's disease or progressive supranuclear palsy (PSP); inhibiting or reducing tau aggregation; inhibiting or reducing the formation of tau fibrils; reducing or clearing tau deposits; stabilization of non-toxic conformations of tau; treatment or prevention of 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 disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GTT), Parkinson's disease or progressive supranuclear palsy (PSP). For example, such antibodies can be conjugated to other therapeutic compounds, other proteins, other antibodies and/or detectable labels. See WO 03/057838; US 8455622. Such therapeutic compounds can be any agent that can be used to treat, control, ameliorate, prevent or improve an undesirable condition or disease in a patient, such as Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism , post-traumatic dementia or boxer dementia, Pick disease, Niemann-Pick disease 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, variant Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP).

Conjugate therapeutic compounds may include cytotoxic agents, cytotoxic agents, neurotrophic agents, neuroprotective agents, radiotherapeutics, radioactive (radiopharmaceutical), fluorescent, paramagnetic tracers, ultrasound contrast agents, immunomodulators, or any biologically active agents that promote or enhance antibody activity or alter bioavailability and distribution in the body or within organs. A cytotoxic agent can be any agent that is toxic to a cell. The cytostatic agent may be any agent that inhibits cell proliferation. A neurotrophic agent can be any agent, including chemical or protein agents, that promotes the maintenance, growth or differentiation of neurons. A neuroprotective agent can be any agent, including chemical or protein agents, that protects neurons from acute injury or degenerative processes. An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunological response. A radiotherapy agent can be any molecule or compound that emits radiation. If such therapeutic moieties are coupled to a tau-specific antibody, such as the antibodies described herein, the bound therapeutic moieties will have a specific affinity for cells affected by the tau-associated disease compared to normal cells. Therefore, administration of conjugated antibodies directly targets cancer cells with minimal damage to surrounding normal healthy tissue. This may be particularly useful for therapeutic compounds that are too toxic when administered alone. In addition, smaller amounts of therapeutic compounds can be used.

Some such antibodies can be modified to act as immunotoxins. See, for example, US Pat. No. 5,194,594. For example, ricin, a plant-derived cell toxin, can be coupled to antibodies using the bifunctional reagents Sacetyl mercaptosuccinic anhydride for antibody and succinimidyl 3-(2-pyridyldithio)propionate for ricin. See Pietersz et al., Cancer Res. 48(16):4469–4476 (1998). Binding results in loss of ricin's B-chain binding activity without impairing either the toxic potential of the ricin A-chain or the activity of the antibody. Likewise, saporin, an inhibitor of ribosomal assembly, can be associated with antibodies through a disulfide bond between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

Some such antibodies may be associated with radioisotopes. Examples of radioisotopes include, for example, yttrium ⁹⁰ (90Y), indium ¹¹¹ (111In), ¹³¹ I, ⁹⁹ mTc, radioactive silver-111, radioactive silver-199 and bismuth ²¹³ . The binding of radioisotopes to antibodies can be accomplished using conventional bifunctional chelates. Sulfur-based linkers can be used to link radioactive silver-111 and radioactive silver 199. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Connections

- 72 047155 reduction of silver radioisotopes may include reduction of immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used, which will react with such isotopes to form 11Πn-ibritumomab tiuxetan and 90Yibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

Some such antibodies may be associated with other therapeutic compounds. Such therapeutic compounds may be, for example, cytotoxic, cytostatic, neurotrophic or neuroprotective. For example, antibodies can be conjugated to toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binders (eg, auristatins), or minor groove binders such as calicheamicin. Other representative therapeutic compounds include agents known to be used for the treatment, control or amelioration of 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 disease type C, supranuclear paralysis, frontotemporal dementia, frontotemporal lobar degeneration, argyrophil granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant Alzheimer's disease with bodies Lewy disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease, or progressive supranuclear palsy (PSP).

Antibodies can also be associated with other proteins. For example, antibodies can be associated with finomers. Finomers are small binding proteins (eg, 7 kDa) derived from the human Fyn SH3 domain. They 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 creating multispecific antibody-based fusion proteins. For example, finomers can be fused to the N-terminus and/or C-terminus of antibodies to create bi- and trispecific FynomAbs with different architectures. Finomers can be selected using finomer libraries using screening technologies using FACS, Biacore and cell-based assays, which 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).

Antibodies described herein may also be linked or conjugated to one or more other antibodies (eg, to form antibody heteroconjugates). Such other antibodies may bind to different epitopes within tau or may bind to a different target antigen.

Antibodies may also be associated 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, post-encephalitic parkinsonism, post-traumatic or boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontal -temporal lobar degeneration, disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam Parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP), and/or to evaluate the effectiveness of treatment. Such antibodies are particularly useful in making such determinations in subjects suffering from or predisposed to developing diseases such as Alzheimer's disease, Down 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/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, disease variant Alzheimer's with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP), or in appropriate biological samples obtained from such subjects. Representative detectable labels that may be fused or linked to the 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;

- 73 047155 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, ¹¹¹ In), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn and ¹¹⁷ Tin; positron-active metals using various positron emission tomographs; non-radioactive paramagnetic metal ions; and molecules that are labeled with radioactive isotopes or conjugated to specific radioisotopes.

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

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

VI. Pharmaceutical compositions and methods of use

For prophylactic purposes, an antibody or antibody inducing agent or pharmaceutical composition is administered to a patient susceptible to or otherwise at risk of developing a disease (eg, Alzheimer's disease) at a schedule (dose, frequency, and route of administration) effective to reduce the risk, reduce the severity, or delaying the onset of at least one sign or symptom of the disease. In particular, the regimen is preferably effective in inhibiting or delaying tau or phospho-tau and paired filaments formed therefrom in the brain and/or inhibiting or delaying its toxic effects and/or inhibiting/or delaying the development of behavioral disorders. For therapeutic purposes, an antibody or antibody inducing agent is administered to a patient suspected of having or already suffering from a disease (eg, Alzheimer's disease) in a schedule (dose, frequency and route of administration) effective to alleviate or at least prevent further deterioration of 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 strands derived therefrom, associated toxicities and/or behavioral disturbances.

A regimen is considered therapeutically or prophylactically effective if an individual treated patient achieves a more favorable outcome than the average outcome of a control population of matched patients not treated with the methods of the invention, or if a more favorable outcome is demonstrated in treated patients compared to control patients in controlled clinical trial (eg, phase II, phase II/III, or phase III) at the p < 0.05, 0.01, or even 0.001 level.

Effective dosages vary depending on many different factors, such as the route of administration, the target site, the physiological state of the patient, whether the patient is a carrier of ApoE, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic.

Examples of antibody dosage ranges 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 weight patient's body. The antibody may be administered at such dosages daily, every other day, weekly, biweekly, monthly, quarterly, or any other schedule determined by empirical analysis. An example treatment involves administering multiple dosage forms over an extended period of time, such as at least six months. Additional example treatment regimens suggest administration once every two weeks, or once a month, or once every 3-6 months.

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The amount of agent for active administration ranges from 0.1-500 μΓ per patient and more typically from 1-100 or 1-10 μg per injection for administration to a person. The timing of injections can vary widely, from once a day to once a year to once every ten years. A typical regimen consists of immunization followed by booster injections at specified intervals, such as 6 weeks or two months. Another regimen consists of immunization followed by booster injections at 1, 2, and 12 months. Another regimen involves administration every two months for life. Alternatively, booster injections may be intermittent, as indicated by monitoring the immune response.

Antibodies or antibody-inducing agents are preferably administered via a peripheral route (i.e., one in which the administered or induced antibody 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, intradermal or intramuscular. Some routes of active immunization are subcutaneous and intramuscular. This type of injection is most often performed in the muscles of the arm or leg. tissue where deposits have accumulated, for example through intracranial injection.

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and are manufactured under GMP conditions. Pharmaceutical compositions may be presented in unit dosage form (ie, single dose dosage form). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or excipients. The drug 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 saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain constituents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies can be lyophilized for mixing with a suitable vehicle, such as sterile pyrogen-free water, before use.

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

Antibodies are administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or alleviates at least one sign or symptom of the disease being treated. If the patient is already suffering from a disease, the regimen may be said to be a therapeutically effective regimen. If the patient is at increased risk of developing the disease compared to the general population, but is not yet experiencing symptoms, the regimen may be considered preventatively effective. In some cases, therapeutic or prophylactic efficacy may be observed in an individual patient relative to control of the medical history or past use experience in the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated in preclinical or clinical trials in a population of treated patients compared with a control population of untreated patients.

Examples of antibody dosages are 0.1-60 mg/kg (for example, 0.5, 3, 10, 30 or 60 mg/kg) or 0.5-5 mg/kg body weight (for example, 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-4000 mg or 10-1500 mg as a fixed dose. Among other factors, dosage depends on the patient's condition and response to previous treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disease is acute or chronic.

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

The frequency of administration depends on the half-life of the antibody in the bloodstream, the condition of the patient, and the route of administration, among other factors. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or progression of the disease being treated. An example of intravenous administration frequency is the range from weekly to quarterly depending on the ongoing cause

- 75 047155 treatment, although more or less frequent dosing is also possible. For subcutaneous administration, the approximate dosing frequency is daily to monthly, although more or less frequent dosing is also possible.

The number of doses administered depends on whether the disease is acute or chronic and the disease's response to treatment. For acute diseases or exacerbations of a chronic disease, 1 to 10 doses are often sufficient. Sometimes a single bolus dose, not necessarily in divided form, is sufficient to treat an acute disease or an exacerbation of a chronic disease. Treatment can be repeated in case of relapse of acute disease or in case of exacerbation. For chronic diseases, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or for the lifetime of the patient.

A. Diagnostic and monitoring methods

In vivo imaging, diagnostic modalities and optimization of immunotherapy

The invention provides methods for imaging tau deposits in vivo (eg, neurofibrillary tangles and tau inclusions) in a patient. These methods work by administering a reagent, such as an antibody that binds tau (eg, murine, humanized, chimeric, or venated antibody 9F5, 10C12, 2D11, 12C4, 17C12, or 14H3), to the patient and then detecting the reagent after binding. Antibodies that specifically bind to tau at an epitope within amino acid residues QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or (Q/E)IVYK(S) may be used /P) (SEQ ID NO: 56). In some methods, an antibody binds to a peptide consisting of an epitope within amino acid residues QIVYKP (SEQ ID NO: 57), EIVYKSP (SEQ ID NO: 58), EIVYKS (SEQ ID NO: 277) or (Q/E)IVYK(S /P) (SEQ ID NO: 56). The clearance response to administered antibodies can be avoided or reduced by using antibody fragments that do not have a full-length constant region, such as Fab. In some methods, the same antibody can serve as both a drug and a diagnostic reagent.

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 dosage of the reagent should be within the same limits as for methods of use as treatment. Typically, the reagent is labeled, although in some methods the primary reagent with affinity for tau is unlabeled and a secondary agent is used to bind to the primary reagent as a label. The choice of label depends on the detection means. For example, a fluorescent tag is suitable for optical detection. The use of paramagnetic tags is suitable for tomographic detection without surgical intervention. Radioactive tracers can also be detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

Methods for imaging tau deposits in vivo are useful for diagnosing or confirming the diagnosis of tauopathy, such as Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy and Pick's disease, or a predisposition to such disease. For example, these methods can be used for patients with symptoms of dementia. If a patient has abnormal neurofibrillary tangles, he or she most likely has Alzheimer's disease. Alternatively, if a patient has abnormal tau inclusions, then, depending on the location of the inclusions, the patient may suffer from frontotemporal lobar degeneration. These methods can also be used in asymptomatic patients. The presence of abnormal tau protein deposits indicates a predisposition to symptomatic disease in the future. The methods are also useful for monitoring disease progression and/or response to treatment in patients who have previously been diagnosed with tau-associated disease.

Diagnosis can be made by comparing the number, size and/or intensity of tagged loci with corresponding baseline values. Baseline values may represent average levels in a population of disease-free individuals. Baseline values may also represent previous levels determined in the same patient. For example, baseline values may be determined in a patient prior to initiation of tau immunotherapy treatment, and the measured values thereafter may be compared to baseline values. A decrease in values relative to the initial level indicates a positive response to treatment.

In some patients, a PET scan may help diagnose tauopathy. PET scanning can be performed, for example, using a conventional PET scanner and ancillary equipment. The scan usually includes one or more areas of the brain that are typically associated with tau protein deposits, and, as a control, one or more areas that typically show small amounts of deposits, if present.

The signal detected in a PET scan can be represented as a multidimensional image. Multidimensional imaging can be performed in two planes representing a cross-section of the brain, in three planes representing a three-dimensional image of the brain

- 76 047155 brain, or in four planes, representing changes in a three-dimensional image of the brain over time. A color scale can be used, with different colors representing different amounts of label and presumably the tau protein deposit detected. Scan results can also be presented numerically, with numbers relating to the amount of tag detected and therefore the amount of tau protein deposits. Label present in a brain region known to be associated with deposits in a particular tauopathy (eg, Alzheimer's disease) can be compared with label present in a region known not to be associated with deposits to obtain a ratio , indicating the extent of deposits in the first area. For the same radiolabeled ligand, such ratios provide a comparable measure of tau deposition and its variation across patients.

In some methods, a PET scan is performed at the same time as an MRI or CAT scan or at the same patient visit. MRI or CAT scans provide more detailed images of the anatomical structures of the brain than PET scans. However, the image obtained from a PET scan can be superimposed on an MRI or CAT scan image, which more accurately indicates the location of the PET ligand and thus the tau deposits relative to anatomical structures in the brain. Some machines can perform both a PET scan and an MRI or CAT scan without changing the patient's position between scans, making image superimposition easier.

Suitable PET ligands include radiolabeled antibodies of the invention (eg, murine, humanized, chimeric or venous antibodies 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3). The radioisotope used may be, for example, C ¹¹ , N ¹³ , O ¹⁵ , F ¹⁸ or I ¹²³ . The interval between PET ligand administration and scanning may depend on the PET ligand and, in particular, on the rate of its uptake and clearance into the brain, as well as the half-life of its radioactive tracer.

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

Maintenance scans can be carried out at intervals of, for example, six months to ten years, preferably 1 to 5 years. Some patients have preventative scans every year. If a PET scan performed as a preventive measure indicates abnormally high levels of tau protein deposits, immunotherapy can be started and a follow-up PET scan performed, as in patients diagnosed with tauopathy. If a PET scan performed as a preventive measure shows levels of tau protein deposits within normal limits, further PET scans can be performed at intervals of six months to 10 years, preferably 1-5 years, as before or in response for signs and symptoms of tauopathy or mild cognitive impairment. By combining a preventative scan with the administration of tau-targeted immunotherapy, if and when higher than normal levels of tau deposits are detected, the level of tau deposits can be reduced to or closer to normal levels, or at least kept from increasing further, and the patient may be free of tauopathy for a longer period of time than would be the case without prophylactic scanning and tau-targeted immunotherapy (eg, at least 5, 10, 15 or 20 years or the rest of the patient's life).

Normal levels of tau protein deposits can be determined by the number of neurofibrillary tangles or tau inclusions in the brains of a representative sample of individuals in the general population who have not been diagnosed with a specific tauopathy (eg, Alzheimer's disease) and who are not considered to be at increased risk of developing such a disease ( for example, a representative sample of healthy individuals under 50 years of age). Alternatively, a normal level may be recognized in an individual patient if the PET signal according to the present methods in a region of the brain in which tau protein deposits are known to develop does not differ (within the accuracy of measurement) from the signal from a region of the brain , in which, as is known, such deposits usually do not form. Elevated levels in humans can be recognized by comparison with normal levels (eg, beyond the mean and standard deviation variance) or simply by increased signal beyond experimental error in a brain region associated with tau protein deposits compared to an area that not associated with sediments. For the purpose of comparing levels of tau protein deposits between an individual and a population, tau protein deposits are preferably determined in the same region(s) of the brain, which regions include at least one region in which tau deposits are known to form -proteins associated with a specific tauopathy (eg, Alzheimer's disease). A patient with elevated levels of tau protein deposits is a candidate for initiation of immunotherapy.

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Once immunotherapy is started, a decrease in the level of tau protein deposits can initially be taken as an indicator that the treatment is having the desired effect. The observed reduction may be, for example, in the range of 1-100%, 1-50% or 1-25% of the initial value. Such effects may be measured in one or more areas of the brain in which deposits are known to form, or may be measured as an average over such areas. The overall treatment effect can be approximated by adding the percentage reduction from baseline to the increase in tau protein deposits that would otherwise occur in the average untreated patient.

Maintaining tau protein deposits at approximately constant levels or even a slight increase in tau protein deposits may also be a sign of response to treatment, albeit a suboptimal response. Such responses can be compared with the dynamics of levels of tau protein deposits in patients with certain tauopathy (eg, Alzheimer's disease) who have not received treatment to determine whether immunotherapy has an effect in inhibiting further increases in tau protein deposits.

Monitoring changes in tau protein deposits allows immunotherapy or other treatment regimens to be adjusted in response to treatment. PET monitoring allows you to determine the nature and extent of response to treatment. It can then be determined whether treatment should be adjusted and, if desired, 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, MRI, or cognitive measures produce a noticeable response. A significant change means that comparing the value of a parameter after treatment compared with baseline provides some evidence that the treatment did or did not lead to a beneficial effect. In some cases, a change in parameter values in the patient himself indicates that the treatment did or did not lead to 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 not receiving immunotherapy. The difference in response in a given patient from the normal response in a control patient (eg, mean plus standard deviation variance) can also provide evidence that the immunotherapy regimen does or does not achieve a beneficial effect in the patient.

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

If monitoring indicates that the patient's tau deposit levels have already been reduced to normal or near-normal levels, the immunotherapy regimen may be changed from induction (i.e., reducing the level of tau deposits) to maintenance (i.e., maintaining tau deposits). -protein at approximately constant levels). This pattern can be influenced by reducing the dose and/or frequency of immunotherapy.

In other patients, monitoring may show that immunotherapy has some benefit, but the effect is suboptimal. The optimal effect can be defined as the percentage reduction in the level of tau deposits within the top half or quartile of change in tau deposits (measured or calculated over the entire brain or its representative region(s) in which tau deposits are known to form. protein) observed in a representative sample of patients with tauopathy undergoing immunotherapy at a given time after initiation of therapy. A patient who experiences less of a decrease or a patient whose tau protein deposits remain unchanged or even increase but to a lesser extent than expected in the absence of immunotherapy (eg, from a control group of patients who did not receive immunotherapy) may be classified as having a positive but suboptimal response. In such patients, an adjustment to the regimen may optionally be made, in which the dose and or frequency of use of the drug is increased.

In some patients, tau protein deposits may increase to a similar or greater extent than tau deposits in patients not receiving immunotherapy. If such an increase persists over a period of time, such as 18 months or 2 years, even after any increase in frequency or dosage of agents, immunotherapy may be discontinued in favor of other treatment options if desired.

The above description of diagnosis, monitoring and adjustment of treatment of tauopathies was mainly devoted to the use of PET scanning. However, any other method for visualizing and/or measuring tau protein deposits that allows the use of anti-tau antibodies of the invention (for example, murine, humanized, chimeric or venous antibodies 9F5, 10C12, 2D11, 12C4, 17C12 or 14H3) can be used instead PET scanning to implement such methods.

Methods for detecting an immune response to tau in a patient suffering from or predisposed to diseases associated with tau are also provided. Methods can be used to

- 78 047155 monitoring the progress of therapeutic and prophylactic treatment with the agents proposed in this document. The antibody profile following passive immunization typically shows an immediate peak in antibody concentration followed by an exponential decline. Without additional dosing, the decline approaches pre-treatment levels over a period of days to months depending on the half-life of the administered antibody. For example, the half-life of some human antibodies is about 20 days.

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

Methods for detecting tau in a subject are also provided, for example, by measuring tau in a sample obtained from the subject or by imaging tau in vivo in the subject. Such methods are useful for diagnosing or confirming a diagnosis of tau-associated diseases or predispositions thereto. These methods can also be used in asymptomatic subjects. The presence of tau indicates susceptibility to symptomatic disease in the future. The methods are also useful for monitoring disease progression and/or response to treatment in subjects who have previously been diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic 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/Guam parkinsonism dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, disease variant Alzheimer's with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP).

Biological samples obtained from a subject who has, is suspected of having, or is at risk of developing 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 disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophil granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease, or progressive supranuclear palsy (PSP), can be contacted with the antibodies disclosed herein to assess the presence of tau. For example, tau levels in such subjects can be compared with levels present in healthy subjects. Alternatively, tau levels in such subjects receiving treatment for the disease can be compared with tau levels in subjects not previously treated for Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, post-encephalitic parkinsonism, post-traumatic dementia, or boxer's dementia. Pick disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia, frontotemporal lobar degeneration, argyrophil granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with body parts Lewy body variant Alzheimer's disease (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease, or progressive supranuclear palsy (PSP). Some of these tests involve a biopsy of tissue taken from such subjects. ELISA assays may also be useful methods, for example, for assessing tau content in liquid samples.

VII. Sets

The invention also provides kits (eg, containers) comprising the antibody disclosed herein and accompanying materials, such as instructions for use (eg, package insert). The instructions for use may contain, for example, instructions for administering the antibody and, optionally, one or more additional agents. Containers may be unit doses, multiple dose packages (eg, multi-dose packages), or partial doses.

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Package insert refers to instructions typically included in commercial packaging of therapeutic products that contain information regarding the indications, use, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products

The kits may also include a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWI), phosphate buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

VIII. Other uses

Antibodies can be used to detect tau or its fragments 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 an indicator that the biological sample contains tau deposits. The binding of antibodies to a biological sample can be compared with the binding of antibodies to a control sample. The control sample and the biological sample may contain cells originating from the same tissue. Control samples and biological samples may be obtained from the same person or from different people and from the same case or from different cases. If desired, multiple biological samples and multiple control samples are evaluated multiple times to protect against random variations independent of differences between samples. A direct comparison can then be made between the biological sample(s) and the control sample(s) to determine whether antibody binding (i.e., presence of tau) to the biological sample(s) has increased, decreased, or remained the same compared to binding antibodies with control sample(s). Increased antibody binding to the biological sample(s) compared to the control sample(s) indicates the presence of tau in the biological sample(s). In some cases, increased antibody binding is statistically significant. Optionally, antibody binding to the biological sample is at least 1.5, 2, 3, 4, 5, 10, 20, or 100 times greater than antibody binding 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 used to treat a patient diagnosed with Alzheimer's disease, Down 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/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD) ), dementia with Lewy bodies, variant Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (11H11). Biological sample obtained from a patient diagnosed with Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic or boxer dementia, Pick disease, Niemann-Pick disease type C, supranuclear palsy, frontotemporal dementia , frontotemporal lobar degeneration, disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease, or progressive supranuclear palsy (PSP), are assessed to establish the baseline level of antibody binding to the sample (i.e., the baseline level of tau present in the sample) before initiating therapy with a therapeutic agent. In some cases, multiple biological samples obtained from a patient are assessed multiple times to establish both a baseline and a measure of random variation independent of treatment. Then the therapeutic agent is administered according to the scheme. The regimen may involve repeated administration of the agent over a period of time. Optionally, antibody binding (ie, the presence of tau) is assessed on multiple occasions in multiple biological samples obtained from a patient, both to determine a measure of random variation and to identify trends in response to immunotherapy. Various estimates of antibody binding to biological samples are then compared. If only two assessments are performed, a direct comparison between the two assessments can be made to determine whether antibody binding (ie, tau presence) increased, decreased, or remained the same between the two assessments. If more than two measurements are taken, the measurements can be analyzed as a time interval starting before treatment with a therapeutic agent and continuing throughout the course of therapy. In patients with decreased antibody binding to biological samples (i.e., presence of tau), it can be concluded that the therapeutic agent was effective in the treatment of Alzheimer's disease, Down 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, frontal

- 80 047155 temporal dementia, frontotemporal lobar degeneration, argyrophil granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant Alzheimer's disease with Lewy bodies (LBVAD), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease, or progressive supranuclear palsy (PSP) in the 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%. Antibody binding assessment may be performed in conjunction with assessment of other signs and symptoms of Alzheimer's disease, Down syndrome, mild cognitive impairment, primary age-related tauopathy, postencephalitic parkinsonism, post-traumatic or Boxer dementia, Pick's disease, Niemann-Pick disease type C, supranuclear palsy, frontal -temporal dementia, frontotemporal lobar degeneration, disease characterized by the appearance of argyrophilic granules, globular glial tauopathy, amyotrophic lateral sclerosis/Guam Parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant of Alzheimer's disease with Lewy bodies (LBVAD) ), chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP).

Antibodies can also be used as research reagents for laboratory studies to detect tau or its fragments. In such cases, antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotopes and can be provided as a kit with all the necessary reagents to perform the detection assay. Antibodies can also be used to purify tau or tau binding partners, for example using affinity chromatography.

All patent applications filed, other publications, accession numbers, etc. cited above or below are, in their entirety and in all respects, incorporated by reference to the same extent as if each individual item had been specifically and separately indicated for its inclusion via link. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the time of the effective filing date of this application is assumed to be the version associated with the accession number. Effective filing date means the earlier of the actual filing date or the filing date of the priority application referring to the reference number, if applicable. Likewise, if different versions of a publication, website, etc. published at different times, refers to the version published closest to the actual filing date of the application, unless otherwise noted. Any feature, step, element, embodiment or aspect of the invention may be used in combination with any others unless expressly stated otherwise. Although the present invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be apparent that certain changes and modifications may be made within the scope of the appended claims in practice.

Examples

Example 1: Identification and Screening of Tau Monoclonal Antibodies

Immunizations were carried out using recombinant human tau (4R0N) from 383 a/k, tagged at the N-terminus with a His tag, containing the P3O1S mutation [immunogen A] or recombinant human tau (4R0N) from 383 a/k, containing the P301S mutation, lacking N-terminal His tag [immunogen B]. The immunogens were emulsified in RIBI adjuvant.

Five-week-old female A/J mice were immunized intraperitoneally with 25 μg of immunogen A on day 0 and 10 μg of immunogen A each on days 7, 14, 21, 27, 34, 48, and 55. For antibody 10C12 on day 43 in mice blood was drawn and titrated against immunogen A. Animals with the highest titers were given a booster dose on day 54 with a terminal immunization of 50 μg of immunogen A, which was administered 1/2 intraperitoneally and 1/2 intravenously.

For antibodies 9F5, 17C12, 2D11, 14H3 and 12C4, animals were immunized with an additional 10 μg of immunogen A on day 62 and 10 μg of immunogen B on days 76 and 90. On days 43 and 98, mice were bled and titrated against immunogen A. Animals with The highest titers were given as a booster dose on day 101 using a terminal immunization with 50 μg of immunogen B, which was administered 1/2 intraperitoneally and 1/2 intravenously. For all antibodies, fusion hybridomas were screened by ELISA against both immunogens.

Internalization analysis using fluorescence-activated cell sorting (FACS) was performed to evaluate the ability of various antibodies to block the internalization of tau by neurons. Antibodies that block internalization are likely to block tau transmission. pHrodo-labeled soluble tau oligomer P301L (final concentration 1.5 μg/ml) was preincubated with anti-tau antibodies (dose titration: initial concentration 80 μg/ml followed by 4-fold serial dilutions) for 30 min at room temperature in cell culture medium. The TAU/antibody mixture was then added to B103 neuroblastoma cell lines at a final concentration of 500,000 cells/ml and incubated for 3-4 hours at 37°C in a tissue culture incubator (5% CO ₂ ). Then the cells were washed 3 times with culture medium followed by 10

- 81 047155 minute incubation in culture medium and washed twice with FACS buffer (1% FBS in PBS). Cells were resuspended in 100 μl FACS buffer and at mean Texas Red fluorescence intensity measured by FACS LSR II. Texas red pHrodo fluorescence is activated by low pH associated with endolysosomal compartments upon internalization. Since FACS detects cells and pHrodo only fluoresces when internalized, only tau internalized by cells will be detected. The lower the average fluorescence intensity, the lower the amount of internalized tau, suggesting higher blocking activity of the antibody being tested. As shown in table. 55 and in fig. 3, mouse antibody 3D6 (WO 2017/191560) and mouse antibody 9F5 blocked neuronal internalization significantly more than other antibodies tested. Murine 9F5 was selected based on its novel epitope and pHrodo assay results.

Table 55. Results of internalization assay with mouse antibodies 9F5, 3D6, 5G8, 16G7 and 16B5 All values indicate % of maximum signal compared to untreated cells

Control 3D6 9F5 5G8 16G7 16B5 Antibody concentration (µg/ml) 62.5 87.7±4.8 14.2±1.7 18.1±5 77.7±3.2 33.9±1.3 61.9±5.7 15,625 86.2±2.6 31±1.6 24.2±3.9 88.1±7.6 51.4±0.8 72.8±6.2 3.9 81.2±5.3 41.2±2.2 35.9±4.2 94.3±5.3 62.2±6.5 80.1±3.1 0.98 75.4±4.8 64.3±0.6 71±6.0 78.8±3.7 56±4.3 88.1±9.1 0.244 82.9±1.5 82.2±9.2 71.7±6.5 86±4.6 74.6±7.6 85.9±1.6 0.061 84.5±4.9 78.4±9.2 78.1±8.6 86.3±4.1 85.3±6.7 88.2±1.3 0 93.3±0.2 93.3±0.2 93.3±0.2 93.3±0.2 93.3±0.2 93.3±0.2

As shown in table. 56 and FIG. 15, mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11, mouse antibody 17C12 and mouse antibody 14H3 blocked neuronal internalization to the same extent as antibody 9F5. Internalization of tau into neurons is an important initial step in tau propagation and pathogenesis, and antibodies that block this process may share common features. Mouse antibody 10C12, mouse antibody 12C4, mouse antibody 2D11, mouse antibody 17C12 and mouse antibody 14H3 were selected based on the results of the pHrodo assay.

Table 56. Results of internalization assay with mouse antibodies 9F5, 10C12, 17C12, 2D11, 14H3 and 12C4

All values show % of maximum signal compared to untreated cells Control 9F5 10С12 17С12 2D11 14NZ 12С4 Antibody concentration (µg/ml) 62.5 87.7±4.8 18.1=1=5 34.5±3.7 30.6±1.8 28.4±3.6 48.4±3.5 37.3±3.5 15,625 86.2±2.6 24.2±3.9 38.9±1.5 32.4±4.8 28.6±2.9 53.2±3.2 36.6±1.9 3.9 81.2±5.3 35.9±4.2 55.2±1.7 36.8±2.5 43±3.3 63.1±9.9 42.6±3.2 0.98 75.4±4.8 71±6.0 90.5±3.6 63.9±4.2 62.7±4.8 67.8±9.7 69.7±7.0 0.244 82.9±1.5 71.7±6.5 9.09±4.9 78.6±0.8 79.5±8.2 66±5.6 78.6±5.1 0.061 84.5±4.9 78.1±8.6 86.7±4.0 79.8±4.7 71.5±6.4 79.7±6.8 78.2±7.1 0 93.3±0.2 93.3±0.2 93.3±0.1 9 93.7±0.7 93.3±0.2 93.3±0.2 93.7±0.7

Example 2: Epitope mapping of mouse antibodies 9F5, 2D11, 10C12, 17C12, 12C4 and 14H3 using ELISA

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Peptides spanning the entire length of tau were used for ELISA mapping. The peptides contained 15 amino acids with an overlap of 5 amino acids. To ensure binding to the streptavidin surface, the peptides also contained N-terminal biotin. Peptides were incubated on a streptavidin-coated plate and the plate was blocked using 1% BSA in 1x PBS. After washing, antibodies were incubated on plates for 1 h at room temperature and washed. The plates were then coated with goat anti-mouse horseradish peroxidase (HRP) and washed. The plates were developed using OPD and absorbance was read at 490 nm.

ELISA data show that mouse antibody 9F5, mouse antibody 10C12, mouse antibody 2D11, mouse antibody 17C12, mouse antibody 12C4 and mouse antibody 14H3 strongly bind peptides containing residues 302-316, as well as residues 383-397 of the longest tau isoform in CNS (441aa, Uniprot ID P10636-8; SEQ ID NO: 1). Both peptides 302-316 and 383-397 contain an IVYK repeat (SEQ ID NO: 276), which has been shown to be important for tau aggregation and self-association, and forms the core of the B-sheet of tau aggregates; in addition, it has been demonstrated to be an important seeding site for tau. This may allow mouse antibody 9F5, mouse antibody 10C12, mouse antibody 2D11, mouse antibody 17C12, mouse antibody 12C4 and mouse antibody 14H3 to bind a range of aggregated conformers compared to other antibodies binding regions in the MTBR. Additionally, the IVYK repeat (SEQ ID NO: 276) has been demonstrated to be an important site for tau seeding, suggesting that antibody binding to this region may interrupt the progression of tau pathology or be used for diagnostic purposes to identify species capable of I will sow.

Example 3: Epitope mapping of mouse antibodies 9F5, 2D11, 10C12 and 17C12 using peptide microarray analysis

Way

Analysis of 9F5, 2D11, 10C12 and 17C12 epitopes was performed using peptide microarray analysis. The full-length human tau sequence (441 amino acids) was linked and extended using neutral linkers GSGSGSG (SEQ ID NO: 59) at the C- and N-terminus to avoid truncated peptides. The bound and extended antigen sequence was translated into peptides of 15 amino acids with a peptide-peptide overlap of 14 amino acids. The resulting peptide microarrays contained 441 different peptides printed in duplicate (882 peptide spots) and were flanked by additional HA control peptides (YPYDVPDYAG, SEQ ID NO: 60, 82 spots).

After synthesis, the microarray was blocked to prevent nonspecific binding (Rockland catalog number MB-070). Then the mouse antibody 9F5 was applied to the microarray at a concentration of 1 μg/ml together with the mouse monoclonal antibody to HA (12CA5) DyLight800 (0.5 μg/ml) for 16 h at 4°C with shaking at 140 rpm. The microarray was washed and a secondary antibody (goat anti-mouse IgG (H+L) DyLight680 (0.2 μg/ml) was applied for 45 min at room temperature. After additional washing, the microarray was visualized using a Licor Odyssey imaging system.

Spot intensity quantification and peptide annotation were based on 16-bit gray scale tiff files at scan intensity 7/7, which exhibit higher dynamic range than 24-bit color tiff files; Microarray image analysis was performed using a PepSlide® analyzer. The software algorithm separates the fluorescence intensities of each spot into raw signal, foreground signal, and background signal and calculates the averaged median foreground intensity and inter-position variances of spot replicates (see Raw Data tabs). A heat map was created from the averaged average foreground intensities, and interactions in the peptide map were color-coded with intensity: red to indicate high intensity and white to indicate low intensity spots. A maximum deviation from spot to spot of 40% was allowed, otherwise the corresponding intensity value was reset to zero.

results

In the case of mouse antibody 9F5, mouse antibody 10C12 and mouse antibody 17C12, a very strong monoclonal antibody response was observed against two epitope-like spots formed by adjacent peptides with very similar consensus motifs QIVYKP (SEQ ID NO: 57) and EIVYKSP (SEQ ID NO: 58), corresponding to amino acid sequences 307-312 and 391397, respectively, of the longest tau isoform (441aa, Uniprot ID P10636-8; SEQ ID NO: 1). This indicates the epitope (Q/E)IVYK(S/P) (SEQ ID NO: 56) for mouse 9F5, mouse 10C12 and mouse 17C12.

In the case of the mouse antibody 2D11, a very strong monoclonal antibody response was observed against two epitope-like spots formed by adjacent peptides with very similar consensus motifs QIVYKP (SEQ ID NO: 57) and EIVYKS (SEQ ID NO: 277), corresponding to amino acid sequences 307-312 and 391 -396, corresponding to the longest tau isoform (441aa, Uniprot ID P10636-8; SEQ ID NO: 1). This indicates the epitope (Q/E)IVYK(S/P) (SEQ ID NO: 56) for we

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Example 4: Construction of Humanized 9F5 Antibodies

The original or donor antibody for humanization was mouse antibody 9F5. The amino acid sequence of the mature m9F5 heavy chain variable region is provided as SEQ ID NO: 7. The amino acid sequence of the mature m9F5 variable light chain is provided as SEQ ID NO: 11. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 according to the Kabat/Chothia composite definition are presented as SEQ ID NO: 8-10 respectively. The Kabat light chain amino acid sequences CDR1, CDR2 and CDR3 are shown as SEQ ID NO: 12-14, respectively. Kabat numbering is used throughout the text.

Variable kappa (Vk) 9F5 belongs to mouse Kabat subgroup 2, which corresponds to human Kabat subgroup 2, and variable heavy chain (Vh) belongs to mouse Kabat subgroup 2c, which corresponds to human Kabat subgroup 1 [Kabat E.A., et al ., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. Chothia CDR-L1 16 residues similar to canonical Chothia class 4, Chothia CDR-L2 7 residues similar to canonical Chothia class 1, Chothia CDR-L3 9 residues similar to canonical Chothia class 1 [Martin ACR. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.. [Martin, 2010]. CDR-H1 no Chothia 10 residues similar to canonical Chothia class 1, CDR-H2 Chothia 17 residues similar to canonical Chothia class 2 [Martin, 2010]]. The Chothia 3 residue CDR-H3 has no canonical classes. A search was conducted for protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33:D233-7] to find structures that would provide an approximate structural model of 9F5. The antibody fab crystal structure pdb 50BF [Vicentini, et al., 2017, unpublished] was used for both Vh and Vk structures because it had good resolution (1.92A°) and overall sequence similarity to Vh and Vk 9F5, keeping the same canonical structures for loops.

The 9F5 VH scaffolds have a high degree of sequence similarity to the corresponding Fab regions of the humanized 48G7 PDB: 2RCS constructed by Wedemayer, G.J. et al. (1997, Science 276: 1665-1669) and the AAN16432 heavy chain cloned by McElhiney, J. et al (2002, Appl. Environ. Microbiol. 68 (11), 5288-5295). Variable domains 9F5 and Fab AAN16432 and 48G7 also have the same length with respect to the CDR-H1, H2 loops. Similarly, the 9F5 VL scaffolds have a high degree of sequence similarity to the corresponding VL regions of human antibodies CAB51297 and VL 1911357B cloned by Capello et al (GenBank accession no. CAB51297, filed July 20, 1999, unpublished) and Cooper L.J., et al. . (1993, J. Immunol. 150 (6), 2231-2242) respectively. The light chain variable domain of antibodies 9F5, CAB51297 and 1911357B also has the same length with respect to the CDR-L1, L2 and L3 loops. Accordingly, the framework regions VH AAN16432 and VH 48G7 (2RCS-VH), as well as VL CAB51297 and VL 1911357B, were selected as hybrid acceptor sequences for CDR 9F5. A model of 9F5 CDRs grafted onto corresponding human scaffolds for VH and VL was constructed and used as a guide for further back mutations.

Heavy and light chain variant sequences resulting from the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains, as specified in the WHO INN committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: https://www.who.int/medicines/services/inn/BioRev2014.pdf) Residues have been modified to align with the corresponding human germline sequence where possible to enhance humanization and reduce potential immunogenicity. For the humanized variants VLv2, VLv3, VLv4, VLv5, VLv6, VLv7, VLv8 and VLv9, mutations were introduced to make the sequences more similar to the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). For the humanized variants VHv2, VHv3, VHv4, VHv5, VHv6, VHv7, VHv8, VHv9 and VHv10, mutations were introduced to make the sequences more similar to the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33)

Additional versions of hu9F5-VH and hu9F5-VL were designed to allow the evaluation of various scaffold residues for their contributions to antigen binding, thermostability, and immunogenicity, and to optimize deamination, oxidation, N-glycosylation, proteolysis, and aggregation. Positions considered for mutation include those that... Positions considered for mutation include those that define canonical CDR conformations (summarized in Martin, A.C.R. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.), are in the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99.), are localized at the VH/VL domain interface (summarized in Leger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. B: Shepherd P and Dean C (eds).

- 84 047155 clonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.), are sensitive to post-translational modifications such as glycosylation or pyroglutamination, are occupied by residues predicted to conflict with CDRs, according to the 9F5 CDR model grafted onto VH and VL scaffolds, or are occupied by residues which are rare among sequenced human antibodies, where either the parental mouse 9F5 residue or some other residue is much more common in the human antibody repertoire.

Alignments of mouse 9F5 and various humanized antibodies are shown for light chain variable regions (Table 7 and FIGS. 2A-2B, 5A-5B, 6A-6C) and heavy chain variable regions (Table 6 and FIGS. 1A-1B, 4A-6C) 4B).

10 variants of the humanized heavy chain variable region and 9 variants of the humanized light chain variable region were constructed, containing various substitution permutations: hu9F5VHv1, hu9F5VHv2, hu9F5VHv3, hu9F5VHv4, hu9F5VHv5, hu9F5VHv6, hu9F5VHv7, 5VHv8, hu9F5VHv9 or 9F5VHv10 (SEQ ID NO: 15-22 , SEQ ID NO: 127-128, respectively); and hu9F5VLv1, hu9F5VLv2, hu9F5VLv3, hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, hu9F5VLv8 or hu9F5VLv9 (SEQ ID NO: 23-29, SEQ ID NO: 130-131, respectively) (Table 6 and 7). Examples of humanized Vk and Vh constructs with back mutations and other mutations based on selected human scaffolds are shown in Table 1. 6 and 7 respectively. Areas highlighted in bold in the table. 6 and 7 denote CDR, according to the Kabat/Chothia composite definition. Sign - in the columns of the table. 6 and 7 indicates that there is no residue at the indicated position. SEQ ID NOs: 15-22 and SEQ ID NOs: 127-128 and SEQ ID NOs: 23-29 and SEQ ID NOs: 130-131 contain back mutations and other mutations, as shown in table. 8. Amino acids at positions in hu9F5VHv1, hu9F5VHv2, hu9F5VHv3, hu9F5VHv4, hu9F5VHv5, hu9F5VHv6, hu9F5VHv7, hu9F5VHv8, hu9F5VHv9 and hu9F5VHv10 are given in Table. 9. Amino acids at positions in hu9F5VLv1, hu9F5VLv2, hu9F5VLv3, hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, hu9F5VLv8 and hu9F5VLv9 are given in Table. 10. Hu9F5VLv8 with N60D replacement is also known as hu9F5VLv9.

Percentage of humanization degree for humanized VH chains hu9F5VHv1, hu9F5VHv2, hu9F5VHv3, hu9F5VHv4, hu9F5VHv5, hu9F5VHv6, hu9F5VHv7, hu9F5VLv8, hu9F5VHv9 and hu9F5VHv10 (SE Q ID NO: 15-22 and SEQ ID NO: 127-128 respectively) relative to the most similar human germline gene IGHV1-69-2*01 (SEQ ID NO: 33), and for humanized VL chains hu9F5VLv1, hu9F5VLv2, hu9F5VLv3, hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, hu9F5VLv8 and hu9F5VLv9 (SEQ ID NO: 23-29 and SEQ ID NO: 130-131, respectively) with respect to the most similar human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37), shown in table. eleven.

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Table 6

Balance according to Kabat No. Linear remainder no. FR or CDR VH murine 9F5 (SEQ ID NO:7) IMGT No. IGHVl-69-2*01 (SEQ ID AAN16432-VH huFrwk (SEQ ID NO:31) 2RCS-VH huFrwk (SEQ ID NO:32) Hu9F5VHvl (SEQ ID NO: 15) Hu9F5VHv2 (SEQ ID NO: 16) Hu9F5VHv3 (SEQ ID NO: 17) Hu9F5VHvv4 (SEQ ID NO: 18) Hu9F5VHvv5 (SEQ ID NO: 19) Hu9F5VHv6 (SEQ ID NO:20) Hu9F5VHv7 (SEQ ID NO:21) Hu9F5VHv8 (SEQ ID NO:22) Hu9F5VHv9 (SEQ ID NO: 127) Hu9F5VHvlO (SEQ ID NO: 128) 1 1 Frl E E E Q Q E E E E E E E E E 2 2 Frl 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 4 4 Frl L L L L L L L L L L L L L L 5 5 Frl Q V V Q Q Q Q Q Q Q V V V V 6 6 Frl Q Q Q Q Q Q Q Q Q Q Q Q Q Q 7 7 Frl s s s s s s s s s s s s s s 8 8 Frl G G G G G G G G G G G G G G 9 9 Frl A A A A A A A A A A A A A A 10 10 Frl E E E E E E E E E E E E E E AND AND Frl L V V L L L L L L L V V V V 12 12 Frl V TO TO V V V V V V V TO TO TO TO 13 13 Frl R To To TO TO TO TO TO TO TO To To To To 14 14 Frl 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 16 16 Frl A A A A A A A A A A A A A A 17 17 Frl S T S S S T T T T T T T T T 18 18 Frl 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 20 20 Frl L I V L L I I I I I I I I I

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21 21 Frl 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 23 23 Frl T To To T T T T To To To To To To To 24 24 Frl A V V 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 26 26 CDR -Hl G G G G G G G G G G G G G G 27 27 CDR -Hl F Y Y F F F F F F F F F F F 28 28 CDR -Hl N T T N N N N T T T N N N N 29 29 CDR -Hl I F L I I I I I I I I I I I thirty thirty CDR -Hl TO T T TO TO TO TO TO TO TO TO TO TO TO 31 31 CDR -Hl D D E D D D D D D D D D D D 32 32 CDR -Hl D Y L T D D D D D D D D D D 33 33 CDR -Hl Y Y S Y Y Y Y Y Y Y Y Y Y Y 34 34 CDR M M M M M M M M M M M M M M -Hl 35 35 CDR -Hl N H H H N N N N N N N N N N 35A CDR -Hl - - 35V CDR -Hl - - 36 36 Fr2 W w w w W W W W W W W W W W 37 37 Fr2 V V V V V V V V V V V V V V 38 38 Fr2 To Q R To To TO To TO To To R R Q To 39 39 Fr2 Q Q Q Q Q Q Q Q Q Q Q Q Q Q 40 40 Fr2 R A A R R R R R R A A A R R 41 41 Fr2 P P P P P P P P P P P P P P 42 42 Fr2 E G G E E E E E E E G G G E 43 43 Fr2 R TO TO Q Q Q Q Q TO TO TO TO TO TO 44 44 Fr2 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 46 46 Fr2 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 48 48 Fr2 I M M I I I I I I M I I I I 49 49 Fr2 G G G G G G G G G G G G G G 50 50 CDR W L G R W W W W W W W W W W

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-H2 51 51 CDR -H2 I V F I I I I I V V V V I I 52 52 CDR -H2 D D D D D D D D D D D D D D 52A 53 CDR -H2 P P P P P P P P P P P P P P 52V CDR -H2 - 52C CDR -H2 53 54 CDR -H2 E E E A E E E E E E E E E E 54 55 CDR -H2 N D D N N N N N D D D N N N 55 56 CDR -H2 G G G G G G G G G G G G G G 56 57 CDR -H2 D E E N D D D D E E E D D D 57 58 CDR -H2 T T T T T T T T T T T T T T 58 59 CDR E I I TO E E E E E E E E E E -H2 59 60 CDR -H2 Y Y Y Y Y Y Y Y Y Y Y Y Y Y 60 61 CDR -H2 A A A D A A A A A A A A A A 61 62 CDR -H2 S E Q P S S S S S S S S S S 62 63 CDR -H2 TO TO To TO TO TO TO TO TO TO TO TO TO TO 63 64 CDR -H2 F F F F F F F F F F F F F F 64 65 CDR -H2 Q Q Q Q Q Q Q Q Q Q Q Q Q Q 65 66 CDR -H2 G G G G G G G G G G G G G G 66 67 Fr3 TO R R TO TO TO R R R R R R R R 67 68 Fr3 A V V A A A A A A A A A A A 68 69 Fr3 T T T T T T T T T T T T T T 69 70 Fr3 M I M I I M M M M M M M M M 70 71 Fr3 T T T T T T T T T T T T T T 71 72 Fr3 A A E A A A A A A A A A A A 72 73 Fr3 D D D D D D D D D D D D D D

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73 74 Fr3 T T T T T T T T T T T T T T 74 75 Fr3 s s s s s s s s s s s s s s 75 76 Fr3 s T T s s T T T T T T T T T 76 77 Fr3 N D D N N N N N D D D D N N 77 78 Fr3 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 79 80 Fr3 Y Y Y Y Y Y Y Y Y Y Y Y Y Y 80 81 Fr3 L M M L L L L L M M M M M M 81 82 Fr3 Q E E Q Q Q E E E E E E E E 82 83 Fr3 F L L L L L L L L L L L L L 82A 84 Fr3 s S S s s s S S S S S S S S 82V 85 Fr3 s s s s s s S S s s s s s S 82С 86 Fr3 L L L L L L L L L L L L L L 83 87 Fr3 T R R T T T T R R R R R R R 84 88 Fr3 s S S s s s S S S S S S S S 85 89 Fr3 E E E E E E E E E E E E E E 86 90 Fr3 D D D D D D D D D D D D D D 87 91 Fr3 T T 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 89 93 Fr3 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 91 95 Fr3 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 93 97 Fr3 T A A A A T T T T T T T T T 94 98 Fr3 T T G s s T T T T T T T T T 95 99 CDR -H3 s - Y Y s s s s s s s s s s 96 CDR -H3 - - R Y - 97 CDR -H3 - - S G - 98 CDR -H3 - - P - - - - - - - - - 99 CDR -H3 - - M - - - - - - - - - 100 CDR -H3 - - P - 100A CDR -H3 T - 100V CDR -H3 - - A - 100С CDR -H3 - 100D CDR -

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-H3 100E CDR -H3 100F CDR -H3 - 100G CDR -H3 - 100H CDR -H3 - 1001 CDR -H3 100J CDR -H3 100K CDR -H3 - 101 100 CDR -H3 N Q N I N N N N N N N N N N 102 101 CDR -H3 G H TO Y G G G G G G G G G G 103 102 Fr4 W w w w W W W W W W W W W W 104 103 Fr4 G G G G G G G G G G G G G G 105 104 Fr4 Q Q Q Q Q Q Q Q Q Q Q Q Q Q 106 105 Fr4 G G G G G G G G G G G G G G 107 106 Fr4 T T T T T T T T T T T T T T 108 107 Fr4 L L L T T T T T L L L L L L 109 108 Fr4 V V V L L V V V V V V V V V 110 109 Fr4 T T T T T T T T T T T T T T 111 110 Fr4 V V V V V V V V V V V V V V 112 111 Fr4 s s s s s s s s s s s s s s 113 112 Fr4 T s s s s s s s s s s s s s

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Table 7

Balance according to Kabat No. Linear remainder no. FR or CDR VL mouse 9F5 (SEQ ID NO: 11) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) CAB51297-VL huFrwk (SEQ ID NO:35) 1911357B-VL huFrwk (SEQ ID NO:36) Hu9F5VLvl (SEQ ID NO:23) Hu9F5VLv2 (SEQ ID NO:24) Hu9F5VLv3 (SEQ ID NO:25) Hu9F5VLvv4 (SEQ ID NO:26) Hu9F5VLv5 (SEQ ID NO:27) Hu9F5VLv6 (SEQ ID NO:28) Hu9F5VLv7 (SEQ ID NO:29) Hu9F5VLv8 (SEQ ID NO: 130) Hu9F5VLv9 (SEQ ID NO: 131) 1 1 Frl D D D D D D D D D D D D D 2 2 Frl I I I I I I I I I I I I I 3 3 Frl V V V V V V V V V V V V V 4 4 Frl M M M M M M M M M M M M M 5 5 Frl T T T T T T T T T T T T T 6 6 Frl Q Q Q Q Q Q Q Q Q Q Q Q Q 7 7 Frl A s s A A s s s s s s s s 8 8 Frl A P P A A P P P P P P P P 9 9 Frl F L L F F F F F F F L F F 10 10 Frl S s s S S s s s s s s s s AND AND Frl N L L N N N N L L L L L L 12 12 Frl P P P P P P P P P P P P P 13 13 Frl V V V V V V V V V V V V V 14 14 Frl T T T T T T T T T T T T T 15 15 Frl L P P L L P P P P P P P P 16 16 Frl G G G G G G G G G G G G G 17 17 Frl T E E T T T E E E E E E E 18 18 Frl S P P S S s S S S S P S S 19 19 Frl A A A A A A A A A A A A A 20 20 Frl S S S S S S S S S S S S S 21 21 Frl I I I I I I I I I I I I I

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22 22 Frl S s S S s s s s s S S S s 23 23 Frl c c C c c c c c c c c c c 24 24 CD B- ΕΙ V V V V V V V V V V V V V 25 25 CD B- ΕΙ s s s s s s s s s s s s s 26 26 CD B- ΕΙ s s s s s s s s s s s s s 27 27 CD B- ΕΙ To Q Q To To To To To To To To To To 27 A 28 CD B- ΕΙ s s s N s s s s s s s s s 27V 29 CD B- ΕΙ L L L L L L L L L L L L L 27 WITH thirty CD B- L L L L L L L L L L L L L LI 27 D 31 CD B- ΕΙ H H H H H H H H H H H H H 27E 32 CD B- ΕΙ s S S S s s s s s s s s s 27F CD B- ΕΙ 28 33 CD B- ΕΙ N N N N N N N N N N N N N 29 34 CD B- ΕΙ G G G G G G G G G G G G G thirty 35 CD B- ΕΙ I Y Y I I I I I Y Y I I I 31 36 CD B- ΕΙ T N N T T T T T T T N T T

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32 37 CD R- L1 Y Y Y F Y Y Y Y Y Y Y Y Y 33 38 CD R- L1 L L L L L L L L L L L L L 34 39 CD R- L1 Y D D Y Y Y Y Y Y Y Y Y Y 35 40 Fr2 W W W W w w w w w w w w w 36 41 Fr2 Y Y Y Y Y Y Y Y Y Y Y Y Y 37 42 Fr2 L L L L L L L L L L L L L 38 43 Fr2 Q Q Q Q Q Q Q Q Q Q Q Q Q 39 44 Fr2 To To To R R R R R R R To To To 40 45 Fr2 P P P P P P P P P P P P P 41 46 Fr2 G G G G G G G G G G G G G 42 47 Fr2 Q Q Q Q Q Q Q Q Q Q Q Q Q 43 48 Fr2 s s s s s s s s s s s s s 44 49 Fr2 P P P P P P P P P P P P P 45 50 Fr2 Q Q Q Q Q Q Q Q Q Q Q Q Q 46 51 Fr2 L L L L L L L L L L L L L 47 52 Fr2 L L L L L L L L L L L L L 48 53 Fr2 I I I I I I I I I I I I I 49 54 Fr2 Y Y Y Y Y Y Y Y Y Y Y Y Y 50 55 CD R- L2 Q L L R Q Q Q Q Q Q Q Q Q 51 56 CD R- L2 M G G V M M M G G G G M M 52 57 CD R- L2 s S S s s s s s s s s s s 53 58 CD R- L2 N N N N N N N N N N N N N 54 59 CD R- L2 L R R L L L L R R R R L L 55 60 CD R- L2 A A A A A A A A A A A A A 56 61 CD S S S S S S S S S S S S S

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R- L2 57 62 Fr3 G G G G G G G G G G G G G 58 63 Fr3 V V V V V V V V V V V V V 59 64 Fr3 P P P P P P P P P P P P P 60 65 Fr3 D D D N N N N N N N D N D 61 66 Fr3 R R R R R R R R R R R R R 62 67 Fr3 F F F F F F F F F F F F F 63 68 Fr3 S S S S S S S S S S S S S 64 69 Fr3 S G G G S S G G G G G S S 65 70 Fr3 s S S S s s S S S S S s s 66 71 Fr3 G G G E G G G G G E G G G 67 72 Fr3 s S S S s s S S S S S s s 68 73 Fr3 G G G G G G G G G G G G G 69 74 Fr3 T T T T T T T T T T T T T 70 75 Fr3 D D D D D D D D D D D D D 71 76 Fr3 F F F F F F F F F F F F F 72 77 Fr3 T T T T T T T T T T T T T 73 78 Fr3 L L L L L L L L L L L L L 74 79 Fr3 R TO TO R R R R R R R TO TO TO 75 80 Fr3 I I I I I I I I I I I I I 76 81 Fr3 s s s s s s s s s s s s s 77 82 Fr3 R R R R R R R R R R R R R 78 83 Fr3 V V V V V V V V V V V V V 79 84 Fr3 E E E E E E E E E E E E E 80 85 Fr3 A A A A A A A A A A A A A 81 86 Fr3 E E E E E E E E E E E E E 82 87 Fr3 D D D D D D D D D D D D D 83 88 Fr3 V V V V V V V V V V V V V 84 89 Fr3 G G G G G G G G G G G G G 85 90 Fr3 V V V V V V V V V V V V V 86 91 Fr3 Y Y Y Y Y Y Y Y Y Y Y Y Y 87 92 Fr3 Y Y Y Y Y Y Y Y Y Y Y Y Y 88 93 Fr3 c c c c c c c c c c c c c 89 94 CD R- L3 A M M A A A A A A A A A A 90 95 CD R- L3 Q Q Q Q Q Q Q Q Q Q Q Q Q 91 96 CD R- N A A L N N N N N N N N N

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L3 92 97 CD R- L3 L L L L L L L L L L L L L 93 98 CD R- L3 E Q Q E E E E E E E E E E 94 99 CD R- L3 L T T L L L L L L L L L L 95 100 CD R- L3 P P P P P P P P P P P P P 95 A CD R- L3 95V CD R- L3 95 WITH CD R- L3 95 D CD R- L3 95E CD R- L3 95F CD R- L3 96 101 CD R- L3 L Y L Y L L L L L L L L L 97 102 CD R- L3 T T T T T T T T T T T T T 98 103 Fr4 F F F F F F F F F F F F F 99 104 Fr4 G G G G G G G G G G G G G 100 105 Fr4 A Q G G G Q Q Q Q Q Q Q Q 101 106 Fr4 G G G G G G G G G G G G G 102 107 Fr4 T T T T T T T T T T T T T 103 108 Fr4 TO TO TO TO TO TO TO TO TO TO TO TO TO 104 109 Fr4 L L V L L L L L L L L L L 105 110 Fr4 E E E E E E E E E E E E E 106 111 Fr4 L I I I I I I I I I I I I 106 A Fr4 - 107 112 Fr4 TO To TO TO TO TO TO TO TO TO TO TO TO

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Table 8. Backmutations VH, VL and other mutations for humanized 9F5

Option Vh or V _L Vh or V _l exon acceptor sequence Changes in acceptor framework residues (or CDRs) (based on composite CDR definitions by Kabat/Chothia) Hu9F5VHvl (SEQ ID NO: 15) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) Absent Hu9F5VHv2 (SEQ ID NO: 16) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H17, H20, H69, H75, N93, N94, N109 Hu9F5VHv3 (SEQ ID NO: 17) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H17, H20, H66, H69, H75, H81, H93, H94, H109 Hu9F5VHv4 (SEQ ID NO: 18) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H17, H20, H23, H28, H66, H69, H75, H81, H83, H93, H94, H109

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Hu9F5VHv5 (SEQ ID NO: 19) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H17, H20, H23, H28, H43, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H109, H109 Hu9F5VHv6 (SEQ ID NO:20) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H17, H20, H23, H28, H40, H43, H48, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108, H109 Hu9F5VHv7 (SEQ ID NO:21) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) H1,H5, H11,H12, H17, H20, H23, H38, H40, H42, H43, H51, H54, H56, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108, H109 Hu9F5VHv8 (SEQ ID NO:22) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) H1,H5, H11,H12, H17, H20, H23, H38, H40, H42, H43, H51, H66, H69, H75, H76, H80, H81, H83, H93, H94, H108, H109 Hu9F5VHv9 (SEQ ID NO: 127) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) H1,H5, H11,H12, H17, H20, H23, H38, H42, H43, H66, H69, H75, H80, H81, H83, H93, H94, H108, H109 Hu9F5VHvlO (SEQ ID NO: 128) GenBank accession number AAN16432-VH_huFrwk (SEQ ID NO:31) PDB ID 2RCS-VH_huFrwk (SEQ ID NO:32) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) H1,H5, H11,H12, H17, H20, H23, H43, H66, H69, H75, H80, H81, H83, H93, H94, H108, H109;

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Hu9F5VLvl (SEQ ID NO:23) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VLhuFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L64, L66 Hu9F5VLv2 (SEQ ID NO:24) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VLhuFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, L15, L64, L66, L100 Hu9F5VLv3 (SEQ ID NO:25) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VLhuFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, L15, L17, L66, L100 Hu9F5VLv4 (SEQ ID NO:26) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VL_huFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, Lil, L15, L17, L51, L54, L66, L100 Hu9F5VLv5 (SEQ ID NO:27) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VL_huFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, Lil, L15, L17, L30, L51,L54, L66, L100 Hu9F5VLv6 (SEQ ID NO:28) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VL_huFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, Lil, L15, L17, L30, L51,L54, L100

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Hu9F5VLv7 (SEQ ID NO:29) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VLhuFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, L9, Lil, L15, L17, L18, L31, L39, L51, L54, L60, L66, L74, L100 Hu9F5VLv8 (SEQ ID NO: 130) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VLhuFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, Lil, L15, L17, L39, L64, L66, L74, L100 Hu9F5VLv9 (SEQ ID NO:131) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number 1911357B-VLhuFrwk (SEQ ID NO:36) IMGT No. IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L7, L8, Lil, L15, L17, L39, L60, L64, L66, L74, LI00

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Table 9. Kabat numbering of framework residues (or CDRs) (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the heavy chains of humanized 9F5 antibodies

Remainder AAN16432-VH huFrwk (SEQ ID NO:31) 2RCS-VH huFrwk (SEQ ID NO:32) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) VH murine 9F5 (SEQ ID NO:7) Hu9F5VHvl (SEQ ID NO: 15) Hu9F5VHv2 (SEQ ID NO: 16) Hu9F5VHv3 (SEQ ID NO: 17) Hu9F5VHvv4 (SEQ ID NO: 18) Hu9F5VHvv5 (SEQ ID NO: 19) Hu9F5VHv6 (SEQ ID NO:20) Hu9F5VHv7 (SEQ ID NO:21) Hu9F5VHv8 (SEQ ID NO:22) Hu9F5VHv9 (SEQ ID NO: 127) Hu9F5VHvlO (SEQ ID NO: 128) H1 E Q E E Q E E E E E E E E E H5 V Q V Q Q Q Q Q Q Q V V V V NI V L V L L L L L L L V V V V H12 TO V TO V V V V V V V TO TO TO TO H17 s s T S S T T T T T T T T T H20 V L I L L I I I I I I I I I H23 To T To T T T T To To To To To To To H28 T N T N N N N T T T N N N N H38 R TO Q TO TO TO TO To To To R R Q TO H40 A R A R R R R R R A A A R R H42 G E G E E E E E E E G G G E H43 TO Q TO R Q Q Q Q TO TO TO TO TO TO H48 M I M I I I I I I M I I I I H51 F I V I I I I I V V V V I I H54 D N D N N N N N D D D N N N H56 E N E D D D D D E E E D D D H66 R TO R TO TO TO R R R R R R R R H69 M I I M I M M M M M M M M M H75 T s T s s T T T T T T T T T H76 D N D N N N N N D D D D N N H80 M L M L L L L L M M M M M M H81 E Q E Q Q Q E E E E E E E E H83 R T R T T T T R R R R R R R H93 A A A T A T T T T T T T T T H94 G s T T s T T T T T T T T T H108 L T L L T T T T L L L L L L H109 V L V V L V V V V V V V V V

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Table 10. Kabat numbering of framework residues (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the light chains of humanized 9F5 antibodies

Remainder CAB51297-VL huFrwk (SEQ ID NO:35) 1911357B-VL huFrwk (SEQ ID NO:36) and UjKJZUI (MAJ W NO:37) VL mouse 9F5 (SEQ ID NO: 11) Hu9F5VLvl (SEQ ID NO:23) Hu9F5VLv2 (SEQ ID NO:24) Hu9F5VLv3 (SEQ ID NO:25) Hu9F5VLvv4 (SEQ ID NO:26) Hu9F5VLv5 (SEQ ID NO:27) Hu9F5VLv6 (SEQ ID NO:28) Hu9F5VLv7 (SEQ ID NO:29) Hu9F5VLv8 (SEQ ID NO: 130) Hu9F5VLv9 (SEQ ID NO: 131) L7 S A S A A S S S S S S S S L8 P A P A A P P P P P P P P L9 L F L F F F F F F F L F F L11 L N L N N N N L L L L L L L15 P L P L L P P P P P P P P L17 E T E T T T E E E E E E E L18 P S P S S s S S S S P S S L30 Y I Y I I I I I Y Y I I I L31 N T N T T T T T T T N T T L39 TO R TO To R R R R R R TO To To L51 G V G M M M M G G G G M M L54 R L R L L L L R R R R L L L60 D N D D N N N N N N D N D L64 G G G s S S G G G G G S s L66 G E G G G G G G G E G G G L74 TO R TO R R R R R R R TO TO TO L100 G G Q A G Q Q Q Q Q Q Q Q

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Table 11. Percentage indicator of the degree of humanization of heavy and light chains of humanized 9F5 antibodies

Option Vh or Vl Degree Hu9F5VHvl (SEQ Ш NO: 15) 66.3% Hu9F5VHv2 (SEQ Ш NO: 16) 69.4% Hu9F5VHv3 (SEQ Ш NO: 17) 71.4% Hu9F5VHv4 (SEQ Ш NO: 18) 74.5% Hu9F5VHv5 (SEQ Ш NO: 19) 80.6% Hu9F5VHv6 (SEQ Ш NO:20) 82.7% Hu9F5VHv7 (SEQ Ш NO:21) 84.7% Hu9F5VHv8 (SEQ Ш NO:22) 82.7% Hu9F5VHv9 (SEQ ID NO: 127) 80.6% Hu9F5VHvlO (SEQ ID NO: 128) 78.6% Hu9F5VLvl (SEQ ID NO:23) 78.0% Hu9F5VLv2 (SEQ ID NO:24) 81.0% Hu9F5VLv3 (SEQ ID NO:25) 83.0% Hu9F5VLv4 (SEQ ID NO:26) 86.0% Hu9F5VLv5 (SEQ ID NO:27) 87.0% Hu9F5VLv6 (SEQ ID NO:28) 86.0% Hu9F5VLv7 (SEQ ID NO:29) 92.0% Hu9F5VLv8 (SEQ ID NO: 130) 85% Hu9F5VLv9 (SEQ ID NO: 131) 86%

Positions at which canonical, vernier, or interface residues differ between mouse and human acceptor sequences are candidates for replacement. Examples of interacting canonical/CDR residues include Kabat residues H54 and H94 in Table 1. 6. Examples of vernier residues include Kabat residues H28, H48, H69, H93 and H94 in Table. 6 and L64 and L66 in table. 7. Examples of interface/package residues (VH+VL) include Kabat H93 residue in Table. 6.

Justification for the choice of provisions indicated in table. 6, in the heavy chain variable region, candidates for replacement are as follows:

hu9F5VHv1 heavy chain variable regions;

consists of 9F5-VH CDR-H1, H2 and H3 loops grafted onto an AAN16432VH and RCS-VH scaffold;

hu9F5VHv2;

returns all framework substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or are localized at the VH/VL domain interface, or contribute to structural stability. hu9F5VHv2 includes back mutations or substitutions at various positions listed below, allowing the contribution of these positions to antigen binding affinity and immunogenicity to be assessed.

hu9F5VHv3, hu9F5VHv4, hu9F5VHv5, hu9F5VHv6, hu9F5VHv7, hu9F5VHv8, hu9F5VHv9 and hu9F5VHv10, consists of additional substitutions and either to increase Ab stability and/or optimize deamination, oxidation, N-glycosylation, proteolysis and aggregation;

Q1E: is a stability-enhancing mutation to reduce the potential for pyroglutamate formation and reduce N-terminal heterogeneity (Liu, above).

Q5V: is a germline-aligning mutation. Val is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

L11V: is a germline-aligning mutation. Val is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

V12K: is a germline straightening mutation. Lys is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

S17T: is a germline straightening mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position. Replacing Thr can by

- 102 047155 increase stability compared to Ser in this position.

L20I: is a germline-aligning mutation. Not found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

T23K: is a frequency and germline leveling mutation. Lys is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

N28T: This is a replacement of the CDR-H1 residue to Thr and a germline alignment mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

K38R: Arg is found in AAN16432-VH_huFrwk (SEQ ID NO: 31) at this position and may increase stability compared to Lys at this position.

K38Q: K38Q is a germline-aligning mutation. Gln is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position. This substitution makes the antibody more similar to the human sequence while maintaining the function of the antibody.

R40A: is a germline frequency and alignment mutation. Ala is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

E42G: is a germline-aligning mutation. Gly is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

Q43K: is a germline frequency and alignment mutation. Lys is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position. Lys in this position may increase stability.

I48M: is a frequency and germline alignment residue mutation in the Vernier zone. Met is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

I51V: This is a replacement of the remainder of CDR-H2 by Val. This position is predicted to be a non-antigenic contact position according to the homology model.

This is a germline straightening mutation. Val is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

N54D and D56E: These are substitutions of CDR-H2 residues and are predicted to be non-antigenic contact positions according to the homology model. It is assumed that the N54D and D56E substitutions stabilize the antibody structure.

K66R: Arg at this position is predicted to form H-bonds with Ser 82a and Thr 83 in addition to forming an H-bond and salt bridge with Asp 86.

I69M is a reverse mutation of a Vernier zone residue.

S75T: Ser at this position is predicted to form an H-bond with Asp 72 and Ar 76. Thr at this position is predicted to also make such contacts, but being a surface accessible Thr residue may increase the stability of the antibody. S75T is a germline-aligning mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position. Thr is found in the acceptor sequence AAN16432-VH_huFrwk at this position.

N76D: is a germline straightening mutation. Asp is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position. Asp at this position may reduce N-glycosylation potential.

L80M: is a germline-aligning mutation. Met is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

Q81E: Glu is predicted to form an H-bond plus a salt bridge with Lys19, therefore Glu at this position increases the stability of the antibody.

T83R: is a frequency- and germline-aligning mutation. Arg is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

A93T: Position H93 represents the residue of the heavy chain/light chain interface. A back mutation to the mouse Thr residue at this position preserves this interface. A93T is also a germline-aligning mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position. A93T in this position makes the antibody more similar to a human antibody.

S94T: is a reverse mutation of a canonical structural residue and a vernier residue as defined by Chothia. S94T is also a germline-aligning mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

T108L: is a germline-aligning mutation. Leu is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

L109V: is a frequency- and germline-aligning mutation. Val is most common in this position. Val is found in the human germline gene IGHV169-2*01 (SEQ ID NO: 33) at this position.

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Justification for the choice of provisions indicated in table. 7, in the light chain variable region, candidates for replacement are as follows:

kappa light chain variable regions hu9F5VLv1 consists of the CDR-L1, L2 and L3 loops of 9F5-VL grafted onto the VL backbone of CAB51297 and VL 1911357B along with the return of all backbone substitutions at positions that are key to defining canonical Chothia classes are part Vernier zones or located at the VH/VL domain interface.

hu9F5VLv2, hu9F5VLv3, hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, hu9F5VLv8 and hu9F5VLv9 also include substitutions that promote structural stability of antigen binding affinity and immunogenicity. The substitutions made in these variations are listed below.

A7S: is a germline-aligning mutation. Ser is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. The Ser substitution may increase stability because the Ser side chain forms an H-bond with the Phe9 backbone, making the loop more stable.

A8P: is a frequency and germline leveling mutation. Pro is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

F9L: is a germline-aligning mutation. Leu is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

N11L: is a frequency- and germline-aligning mutation. Leu is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

L15P: is a frequency- and germline-aligning mutation. Pro is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. The Pro in this position may be better suited to support the squirrel bend in this position.

T17E: represents a frequency mutation. Glu is often found at this position, and Glu at this position is believed to form an H-bond with T14 and a salt bridge with Lys 107, both light chain residues, and increase the stability of the antibody.

S18P: is a germline-aligning mutation. Pro is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

I30Y: This is a replacement for the CDR-L1 residue thought to be in the antigen interface. Tyr at this position forms stacking with Tyr32 and His27D, thereby increasing stability. Because Ile and Tyr are hydrophobic, Tyr replacement may be well tolerated.

T31N: This is a replacement for the remainder of CDR-L1. Asn in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

R39K: is a germline-aligning mutation. Lys is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. Lys may provide structural support equivalent to Arg.

M51G: This is a replacement for the CDR-L2 residue and is thought to be oriented away from the antigen interface. Gly is also a germline-aligning mutation. Gly is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

This position is assumed to be accessible on the surface. Gly in this position, being neutral, reduces the oxidation potential of Met.

L54R: This is a replacement for the CDR-L2 residue and is thought to be oriented away from the antigen interface. Arg is also a germline-aligning mutation. Arg in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. Arg is predicted to form H-bonds with strands Asn60 and Phe62, increasing the stability of the interstrand loop.

N60D: is a germline straightening mutation. Asp is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

G64S is a reverse mutation of a Vernier zone residue. The G64S back mutation retains the CDR conformation.

E66G is a reverse mutation of a Vernier zone residue. The back mutation E66G retains the CDR conformation. E66G is a germline-aligning mutation. Gly is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

R74K: is a germline-aligning mutation. Lys is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

G100Q: is a frequency- and germline-aligning mutation. Gln is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. It is assumed that this residue is surface accessible, and therefore Gln at this position may be better for antibody solubility since it is hydrophilically neutral

- 104 047155 no. Gln in this position may enhance stability.

Based on these human scaffolds, the following variants were developed: heavy chain variable regions > hu9F5VHvl (SEQ ID NO: 15)

QVQLQQSGAELVKPGASVKLSCTASGFNIKDDYMNWVKQRPEQGLEWIGWIDPENGD

TEYASKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCASSNGWGQGTTLTVSS > hu9F5VHv2 (SEQ ID NO: 16)

EVQLQQSGAELVKPGATVKISCTASGFNIKDDYMNWVKQRPEQGLEWIGWIDPENGDT EYASKFQGKATMTADTSTNTAYLQLSSLTSEDTAVYYCTTSNGWGQGTTVTVSS > hu9F5VHv3 (SEQ ID NO: 17)

EVQLQQSGAELVKPGATVKISCTASGFNIKDDYMNWVKQRPEQGLEWIGWIDPENGDT EYASKFQGRATMTADTSTNTAYLELSSLTSEDTAVYYCTTSNGWGQGTTVTVSS > hu9F5VHv4 (SEQ ID NO: 18)

EVQLQQSGAELVKPGATVKISCKASGFTIKDDYMNWVKQRPEQGLEWIGWIDPENGDT EYASKFQGRATMTADTSTNTAYLELSSLRSEDTAVYYCTTSNGWGQGTTVTVSS > hu9F5VHv5 (SEQ ID NO: 19)

EVQLQQSGAELVKPGATVKISCKASGFTIKDDYMNWVKQRPEKGLEWIGWVDPEDGET EYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS > hu9F5VHv6 (SEQ ID NO:20)

EVQLQQSGAELVKPGATVKISCKASGFTIKDDYMNWVKQAPEKGLEWMGWVDPEDGE TEYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS > hu9F5VHv7 (SEQ ID NO:21)

EVQLVQSGAEVKKPGATVKISCKASGFNIKDDYMNWVRQAPGKGLEWIGWVDPEDGE TEYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS > hu9F5VHv8 (SEQ Ш NO:22)

EVQLVQSGAEVKKPGATVKISCKASGFNIKDDYMNWVRQAPGKGLEWIGWVDPENGD TEYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS > hu9F5VHv9 (SEQ ID NO: 127)

EVQLVQSGAEVKKPGATVKISCKASGFNIKDDYMNWVQQRPGKGLEWIGWIDPENGD TEYASKFQGRATMTADTSTNTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS > hu9F5VHvlO (SEQ ID NO: 128), (also known as hu9F5VHv9_ Q38K G42E) EVKKPGATVKISCKASGFNIKDDYMNWVKQRPEKGLEWIGWIDPENGDT EYASKFQGRATMTADTSTNTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS kappa light chain variable regions

- 105 047155 > hu9F5VLvl (SEQ ID NO:23)

DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQRPGQSPQLLIYQMSNLA

SGVPNRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGGGTKLEIK > hu9F5VLv2 (SEQ ID NO:24)

DIVMTQSPFSNPVTPGTSASISCRSSKSLLHSNGITYLYWYLQRPGQSPQLLIYQMSNLAS

GVPNRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv3 (SEQ ID NO:25)

DIVMTQSPFSNPVTPGESASISCRSSKSLLHSNGITYLYWYLQRPGQSPQLLIYQMSNLAS

GVPNRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv4 (SEQ ID NO:26)

DIVMTQSPFSLPVTPGESASISCRSSKSLLHSNGITYLYWYLQRPGQSPQLLIYQGSNRAS

GVPNRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv5 (SEQ ID NO:27)

DIVMTQSPFSLPVTPGESASISCRSSKSLLHSNGYTYLYWYLQRPGQSPQLLIYQGSNRAS

GVPNRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv6 (SEQ ID NO:28)

DIVMTQSPFSLPVTPGESASISCRSSKSLLHSNGYTYLYWYLQRPGQSPQLLIYQGSNRAS

GVPNRFSGSESGTDFTLRISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv7 (SEQ ID NO:29)

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGINYLYWYLQKPGQSPQLLIYQGSNRAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv8 (SEQ ID NO: 130)

DIVMTQSPFSLPVTPGESASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS

GVPNRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK > hu9F5VLv9 (SEQ ID NO: 131), also known as hu9F5VLv8_N60D

DIVMTQSPFSLPVTPGESASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS

GVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGQGTKLEIK

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

Example 5. Immunogenicity of hu9F5VHv4/hu9F5VLv2

The amino acid sequences of the heavy chain variable region (SEQ ID NO: 18) and light chain variable region (SEQ ID NO: 24) of hu9F5VHv4/hu9F5VLv2 were analyzed using the iedb.org deimmunization tool (Dhanda et al, Immunology. 2018 Jan;153(l) :118-132). In table 12 shows peptides that can be selected to deimmunize hu9F5VLv2, i.e. areas where additional substitutions can be made to reduce potential immunogenicity are indicated.

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Table 12. Results of immunogenicity assay identifying potential peptides in hu9F 5VLv2 for deimmunization

Start position (linear) End position (linear) Median percentile rank Peptide 51 65 9,895 LLIYQMSNLASGVPN 46 60 15.18 GQSPQLLIYQMSNLA 26 40 16,055 SKSLLHSNGITYLYW 76 90 18.23 FTLRISRVEAEDVGV 36 50 19,455 T YL YW YLQRPGQ SPQ 86 100 19,535 EDVGVYYCAQNLELP

Based on the analysis results presented in table. 12, light chain variable region variants hu9F5VLv2 were constructed targeting the amino acid residues highlighted in bold in Table. 13. Each variant includes one of the following amino acid substitutions, as shown in table. 14.

Table 13. Amino acid residues intended for replacement

Target residues of the light chain variable region hu9F5VLv2 Original SEQ Ш NO DIVMTQSPFSNPVTPGTSASISCRSSKSLL HSNGITYLYWYLQRP GQSPQLLIYQMSNLA SGVPNRFS S SGSGTDFTLRISRVEAED VGV YYCAQNLELPLTFGQGTKLEIK 24

Table 14. Amino acid substitutions made in the hu9F5VLv2 light chain variable region to reduce immunogenicity

Linear number Number according to Kabat SEQ ID NO of the resulting light chain variable region M56E M51E SEQIDNO:61 M56D M51D SEQ ID NO:62 L30D L27cD SEQ ID NO:63 L30G L27cG SEQ ID NO:64 L30S L27cS SEQ ID NO:65 L30E L27cE SEQ ID NO:66 13 5E 13 0E SEQ ID NO:67 13 5K 13 OK SEQ ID NO:68 L30T L27cT SEQ ID NO:69 L30N L27cN SEQ ID NO:70 L29D L27bD SEQIDNO:71 13 5G 13 0G SEQ ID NO:72

- 107 047155

L38N L33N SEQ ID NO:73 L30A L27cA SEQ ID NO:74 L38T L33T SEQ ID NO:75 L38S L33S SEQ ID NO:76 L38R L33R SEQ ID NO:77 13 5Q 13 0Q SEQ ID NO:78 L29T L27bT SEQ ID NO:79 T36G T31G SEQ ID NO:80 L29Q L27bQ SEQIDNO:81 L38G L33G SEQ ID NO:82 L30P L27cP SEQ ID NO:83 V83R V78R SEQ ID NO:84 I80D I75D SEQIDNO:85 V83D V78D SEQ ID NO:86 V83E V78E SEQ ID NO:87 V83P V78P SEQIDNO:88 V83K V78K SEQ ID NO:89 R82D R77D SEQ ID NO:90 V83G V78G SEQIDNO:91 S81P S76P SEQ ID NO:92 I80P I75P SEQ ID NO:93 I80Q I75Q SEQ ID NO:94 I80G I75G SEQ ID NO:95 L78P L73P SEQ ID NO:96 L78G L73G SEQ ID NO:97 V83Q V78Q SEQ ID NO:98 S81G S76G SEQ ID NO:99 L97D L92D SEQ ID NO: 100 Y91T Y86T SEQ ID NO: 101 L97E L92E SEQ ID NO: 102 L97G L92G SEQ ID NO: 103 L97Q L92Q SEQ ID NO: 104 L99G L93G SEQ ID NO: 105 V90G V85G SEQ ID NO: 106 L97T L92T SEQ ID NO: 107 A94G A89G SEQ ID NO: 108

Similarly, hu9F5VHv4 was analyzed for potential immunogenicity, and Table. 15 shows a peptide identified for potential deimmunization.

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Table 15. Results of immunogenicity assay identifying potential peptides in hu9F 5VHv4 for deimmunization

Start position (linear) End position (linear) Median percentile rank Peptide 76 90 11,925 TNTAYLELS SLRSED

Based on these results, hu9F5VHv4 heavy chain variable region and hu9F5VHv5 heavy chain variable region variants were constructed targeting the amino acid residues highlighted in bold in Table 1. 16. Each variant includes one of the following amino acid substitutions, as shown in table. 17.

Table 16. Amino acid residues intended for replacement.

Target heavy chain variable region residues Original SEQ ID NO hu9F5VHv4 18 EVQLQQSGAELVKPGATVKISCKASGFTIK DDYMNWVKQRPEQGLEWIGWIDPENGDTEY ASKFQGRATMTADTSTNTAYLELSSLRSED TAVYYCTTSNGWGQGTTVTVSS hu9F5VHv5 EVQLQQSGAELVKPGATVKISCKASGFTIK DDYMNWVKQRPEKGLEWIGWVDPEDGETEY ASKFQGRATMTADTSTDTAYMELS SLRSED TAVYYCTTSNGWGQGTLVTVSS 19

Table 17. Amino acid substitutions made in the heavy chain variable region of hu9F5VHv4 or v5, as indicated, to reduce immunogenicity

Linear number Number by Kabat Variant 9F5, in which replacements were made SEQ ID NO of the resulting variable region heavy chain L81P L80P hu9F5VHv4 SEQ ID NO: 109 L81D L80D hu9F5VHv4 SEQ ID NO: 110 L86G L82cG hu9F5VHv4 SEQIDNO:111 L86D L82cD hu9F5VHv4 SEQ ID NO: 112 L83P L82P hu9F5VHv4 SEQ ID NO: 113 L81G L80G hu9F5VHv4 SEQ ID NO: 114 L83K L82K hu9F5VHv4 SEQ ID NO: 115 L83R L82R hu9F5VHv4 SEQ ID NO: 116 L83E L82E hu9F5VHv4 SEQ ID NO: 117 L83N L82N hu9F5VHv4 SEQ ID NO: 118 Y80D Y79D hu9F5VHv4 SEQ ID NO: 119 Y80N Y79N hu9F5VHv4 SEQ ID NO: 120 Y80G Y79G hu9F5VHv4 SEQ ID NO: 121

- 109 047155

M81E M80E hu9F5VHv5 SEQ ID NO: 122 M81G M80G hu9F5VHv5 SEQ ID NO: 123 L86S L82cS hu9F5VHv4 SEQ ID NO: 124 Y80Q Y79Q hu9F5VHv4 SEQ ID NO: 125 S84G S82aG hu9F5VHv4 SEQ ID NO:126

Example 6. Immunogenicity of hu9F5VLv8 and hu9F5VHv10

The amino acid sequences of the heavy chain variable region (SEQ ID NO: 128) and light chain variable region (SEQ ID NO: 130) of hu9F5VHv10/hu9F5VLv8 were analyzed using the iedb.org deimmunization tool (Dhanda et al, Immunology. 2018 Jan;153(1) :118-132). In table 18 shows peptides that can be selected to deimmunize hu9F5VLv8, i.e. areas where additional substitutions can be made to reduce potential immunogenicity are indicated.

Table 18. Results of immunogenicity assay identifying potential peptides in hu9F 5VLv8 for deimmunization

Start position (linear) End position (linear) Median percentile rank Peptide 51 65 9,895 LLIYQMSNLASGVPN 46 60 15.18 GQSPQLLIYQMSNLA 26 40 16,055 SKSLLHSNGITYLYW 1 15 17,995 DIVMTQSPFSLPVTP 86 100 19,535 EDVGVYYCAQNLELP 36 50 19.84 TYLYWYLQKPGQSPQ

Based on the analysis results presented in table. 19, light chain variable region variants hu9F5VLv8 were constructed targeting the amino acid residues highlighted in bold in Table. 19. Each variant includes one or more amino acid substitutions at the residues highlighted in bold in the table. 19, as shown in table. 20-21. Hu9F5VLv8 with N60D replacement is also known as hu9F5VLv9.

Table 19. Amino acid residues intended for replacement

Target residues of the light chain variable region hu9F5VLv8 SEQ ID NO: hu9F5VLv8 DIVMTQSPFSLPVTPGESASISCRSSKSLL HSNGITYLYWYLQKP GQSPQLLIYQMSNLA SGVPNRFSSSGSGTDFTLKISRVEAEDVGV YYCAQNLELPLTFGQGTKLEIK 130

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Table 20. Amino acid substitutions made in the hu9F5VLv8 light chain variable region to reduce immunogenicity

Option name Substitutions in additional variants in hu9F5VLv8 intended to reduce immunogenicity (Kabat mutation number(s) indicated) SEQ ID NO of the resulting light chain variable region hu9F5VLv8_DIMl V3Q, L27cS, L37Q, M51G, L54G, L92I SEQ ID NO: 132 hu9F5VLv8_DIM2 V3Q, L27cS, L37Q, M51G, L54R, L92I SEQ ID NO: 133 hu9F5VLv8_DIM3 V3Q, L27cS, L37Q, M51G, L54T, L92I SEQ ID NO: 134 hu9F5VLv8_DIM4 V3Q, L27cS, L37Q, M51G, L54R, L92G SEQ ID NO: 135 hu9F5VLv8_DIM5 V3Q, L27cG, L37Q, M51G, L54R, L92I SEQ ID NO: 136 hu9F5VLv8_DIM6 V3Q, L27cD, L37Q, M51G, L54R, L92I SEQ ID NO: 137 hu9F5VLv8_DIM7 V3Q, L27cD, L37Q, M51K, L54R, L92I SEQ ID NO: 138 hu9F5VLv8_DIM8 V3Q, L27cG, L37Q, M51K, L54R, L92I SEQ ID NO: 139 hu9F5VLv8_DIM9 V3Q, L27cG, L37Q, M51K, L54G, L92I SEQ ID NO: 140 hu9F5VLv8_DIM10 V3Q, L27cS, L37Q, M51K, L54G, L92I SEQ ID NO: 141 hu9F5VLv8_DIMl 1 V3Q, L27cG, L37G, M51G, L54R, L92I SEQ ID NO: 142 hu9F5VLv8_DIM12 V3Q, L27cG, L37G, M51G, L54R, L92G SEQ ID NO: 143 hu9F5VLv8_DIM13 V3Q, L27cG, L37G, M51G, L54R SEQ ID NO: 144 hu9F5VLv8_DIM14 V3Q, L27cG, L37G, M51G, L54T, L92I SEQ ID NO: 145 hu9F5VLv8_DIM15 V3Q, L27cG, L37G, M51G, L54T, L92G SEQ ID NO: 146 hu9F5VLv8_DIM16 V3Q, L27cG, L37G, M51G, L54T SEQ ID NO: 147 hu9F5VLv8_DIM17 V3Q, L27cS, L37G, M51G, L54T, L92I SEQ ID NO: 148 hu9F5VLv8_DIM18 V3Q, L27cD, L37G, M51G, L54R, L92I SEQ ID NO: 149 hu9F5VLv8_DIM19 V3Q, L27cS, L37I, M51I, L54R, L92I SEQ ID NO: 150 hu9F5VLv8_DIM20 V3Q, L27cS, L37Q, M51I, L54G, L92I SEQ ID NO: 151 hu9F5VLv8_DIM21 V3Q, L27cS, L37Q, M51I, L54G SEQ ID NO: 152 hu9F5VLv8_DIM22 V3Q, L27cS, L37Q, M51E, L54R, L92I SEQ ID NO: 153 hu9F5VLv8_DIM23 V3Q, L27cG, L37Q, M51E, L54G, L92I SEQ ID NO: 154 hu9F5VLv8_DIM24 V3Q, L27cG, L37I, M51E, L54R, L92I SEQ ID NO: 155 hu9F5VLv8_DIM25 V3Q, L27cG, L37I, M51E, L54R, L92G SEQ ID NO: 156 hu9F5VLv8_DIM26 V3Q, L27cl, L37I, M51E, L54R SEQ ID NO: 157 hu9F5VLv8_DIM27 V3Q, L37Q, M51G, L54R, L92I SEQ ID NO: 158 hu9F5VLv8_DIM28 V3Q, L27cS, M51G, L54R, L92I SEQ ID NO: 159 hu9F5VLv8_DIM29 V3Q, L27cS, L37Q, L54R, L92I SEQ ID NO: 160 hu9F5VLv8_DIM30 V3Q, L27cS, L37Q, M51G, L92I SEQ ID NO: 161

Table 21. Amino acid substitutions made in the hu9F5VLv9 light chain variable region to reduce immunogenicity

Option name Substitutions in additional options in SEQ ID NO hu9F5VLv9, designed for received reduction in immunogenicity (number indicated variable region mutation(s) according to Kabat) light chain hu9F5VLv9_DIMl V3Q, L27cS, L37Q, M51G, L54G, L92I SEQ ID NO: 162 hu9F5VLv9_DIM2 V3Q, L27cS, L37Q, M51G, L54R, L92I SEQ ID NO: 163 hu9F5VLv9_DIM4 V3Q, L27cS, L37Q, M51G, L54R, L92G SEQ ID NO: 164 hu9F5VLv9_DIM5 V3Q, L27cG, L37Q, M51G, L54R, L92I SEQ ID NO: 165 hu9F5VLv9_DIM8 V3Q, L27cG, L37Q, M51K, L54R, L92I SEQ ID NO: 166 hu9F5VLv9_DIM10 V3Q, L27cS, L37Q, M51K, L54G, L92I SEQ ID NO: 167 hu9F5VLv9_DIMl 1 V3Q, L27cG, L37G, M51G, L54R, L92I SEQ ID NO: 168 hu9F5VLv9_DIM13 V3Q, L27cG, L37G, M51G, L54R SEQ ID NO: 169 hu9F5VLv9_DIM19 V3Q, L27cS, L37I, M51I, L54R, L92I SEQ ID NO: 170 hu9F5VLv9_DIM20 V3Q, L27cS, L37Q, M51I, L54G, L92I SEQ ID NO: 171

Justification for the choice of provisions indicated in table. 20 and table. 21, in the variable region of the lung

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The V3Q mutation was developed based on recommendations from the deimmunization analysis. Gln at position 3 is the second most abundant at this position in the human sequence, so the V3Q substitution is intended to reduce immunogenicity and maintain the sequence more similar to the human sequence. Immunogenicity prediction for hu9F5VLv8_V3Q substitutions shows elimination of N-terminal peptide 1-15.

The following mutations were designed based on recommendations from the deimmunization assay and the position of this residue in the homology model. Mutations have also been designed to preserve the functionality of the antibody by maintaining the size and charge/polarity of the substituted residue. Priority was given to a residue if it matched a residue in the human germline.

L27cI, L27cS, L27cD, L27cG: to reduce immunogenicity L37Q, L37G, L37I: to reduce immunogenicity M51G, M51K, M51I, M51E: to reduce immunogenicity M51G: This is a replacement for the CDR-L2 residue and is expected to be directed away from antigen interface. Gly is also a germline-aligning mutation. Gly is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. This position is assumed to be accessible on the surface. Gly in this position, being neutral, reduces the oxidation potential of Met.

L54G, L54R, L54T: to reduce immunogenicity

L54R: This is a replacement for the CDR-L2 residue and is thought to be oriented away from the antigen interface. Arg is also a germline-aligning mutation. Arg in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position. Arg is predicted to form H-bonds with strands Asn60 and Phe62, increasing the stability of the interstrand loop.

N60D: to reduce immunogenicity. N60D: is also a germline-aligning mutation. Asp is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

L92I, L92G: to reduce immunogenicity

Table 22. Results of immunogenicity assay identifying potential peptides in hu9F5VHv10 for deimmunization

Start position (linear) End position (linear) Median percentile rank Peptide 76 90 12.72 TNTA YMELS SLRSED

B table 22 shows peptides that can be selected to deimmunize hu9F5VHv10, i.e. areas where additional substitutions can be made to reduce potential immunogenicity are indicated.

Based on these results, a heavy chain variable region variant, hu9F5VHv10, was designed to target amino acid residue H82c, shown in bold in Table 1. 23. Option hu9F5VHv10_L82cG includes the replacement of L82cG, as shown in table. 24.

Table 23. Amino acid residues intended for replacement

Target heavy chain variable region residues Original SEQ ID NO >hu9F5VHvlO EVQLVQSGAEVKKPGATVKISCKASGFNIKDDYMNWVKQRPE KGLEWIGWIDPENGDTEYASKFQGRATMTADT STNT AYMELS S LRSEDTAVYYCTTSNGWGQGTLVTVSS 128

Table 24. Amino acid substitutions made in the heavy chain variable region of hu9F5VHv10 to reduce immunogenicity

Name option Substitutions in additional variants in hu9F5VHv!0 intended to reduce immunogenicity (Kabat mutation number(s) indicated) SEQ ID NO the resulting variable region of severe chains hu9F5VHvlO_L82c G L82cG SEQIDNO:129

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Example 7. Analysis of Humanized 9F5 Variants

Humanized 9F5 variants with predicted deimmunizing substitutions were analyzed for several characteristics, including target binding affinity, activity in cell-based assays, thermostability, expression characteristics, and number of substitutions. In all cases, the results were compared with the original hu9F5VHv9/hu9F5VLv8 sequence to determine whether there was a loss of activity or stability.

Target binding assays were performed using a Biacore T200 to compare the binding affinities of humanized 9F5 variants to recombinant human 4R0N tau. Anti-human Fc antibody was immobilized onto the CM3 sensor chip via amine coupling, and humanized 9F5 variants were captured to equivalent levels. Various concentrations of recombinant human tau 4R0N (ranging from 0.02 to 12.5 nM) were passed through the captured ligand at a rate of 50 μl/min in running buffer (HBS + 0.05% P-20, 1 mg/ml BSA) in for 180 s association/420 s dissociation as one cycle. Blank values for the irrelevant sensor containing no antibody and 0 nM analyte concentration were subtracted from the data. Analysis was performed using a 1:1 overall fit using Biacore Evaluation software.

Affinity testing identified a number of deimmunized variants that retained the affinity of the parent antibodies, as determined by comparing the KD of each antibody. In this case, the KD of antibodies were determined to be comparable within a 3-fold level with hu9F5VHv9/hu9F5VLv8 (Table 25), include hu9F5VHv9/hu9F5VLv8_DIM2, hu9F5VHv9/hu9F5VLv8_DIM6, hu9F5VHv9/hu9F5VLv8_DIM8, hu9F5 V Hv9/hu9F 5 VLv8_DIM 12, hu9F5 VHv9/ hu9F 5 VLv8_DIM 14, hu9F5VHv9/hu9F5VLv8_DIMl 8, hu9F5VHv9/hu9F5VLv8_DIM28, hu9F5VHv9/hu9F5VLv8_DIM5, hu9F5VHv9/hu9F5VLv8_DIM7, hu9F5VHv9/hu 9F5VLv8_DIMl 1, hu9F5VHv9/hu9F5VLv8_DIM13, hu9F5VHv9/hu9F5VLv8_DIM17, hu9F5VHv9/hu9F5VLv8_DIM27, hu9F5VHv9/hu9F5VLv8_DIM29, hu9F5VHv9/hu9F5 VLv8 DIM30 and hu9F5VHvlO/ hu9F5VLv9 DIM11.

Table 25. Affinity of humanized 9F5 variants

Humanized version 9F5 and SEQ ID NO: Substitutions in VH refer to BIO hu9F5VH v9 (mutation number(s) by Kabat) Substitutions in VL relative to hu9F5VLv8 (mutation number(s) according to Kabat) K _a (1/M C) K _d (1/s) K _D (Μ) hu9F5VHv9/hu9F 5VLv8 SEQ ID NO: 127/ SEQ ID NO: 130 1716666.6 67 0.0004293 33 2.69333 Ε-10 hu9F5VHv9/hu9F 5VLv8_DIMl SEQ ID NO: 127/ SEQ ID NO: 132 V3Q, L27cS, L37Q, M51G, L54G, L92I 6620000 0.00559 8.44Ε-10 hu9F5VHv9/hu9F 5VLv8_DIM2 SEQ ID NO: 127/ SEQ ID NO: 133 V3Q, L27cS, L37Q, M51G, L54R, L92I 1960000 0.000586 2.99Ε-10

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hu9F5VHv9/hu9F 5VLv8_DIM3 SEQ ID NO: 127/ SEQ ID NO: 134 V3Q, L27cS, L37Q, M51G, L54T, L92I 1130000 0.00068 6E-10 hu9F5VHv9/hu9F 5VLv8_DIM4 SEQ ID NO: 127/ SEQ ID NO: 135 V3Q, L27cS, L37Q, M51G, L54R, L92G 542000 0.00143 2.63E-09 hu9F5VHv9/hu9F 5VLv8_DIM5 SEQ ID NO: 127/ SEQ ID NO: 136 V3Q, L27cG, L37Q, M51G, L54R, L92I 1750000 0.000295 1.69E-10 hu9F5VHv9/hu9F 5VLv8_DIM6 SEQ ID NO: 127/ SEQ ID NO: 137 V3Q, L27cD, L37Q, M51G, L54R, L92I 1530000 0.000272 1.78E-10 hu9F5VHv9/hu9F 5VLv8_DIM7 SEQ ID NO: 127/ SEQ ID NO: 138 V3Q, L27cD, L37Q, M51K, L54R, L92I 2360000 0.000424 1.8E-10 hu9F5VHv9/hu9F 5VLv8_DIM8 SEQ ID NO:127/ SEQ ID NO: 139 V3Q, L27cG, L37Q, M51K, L54R, L92I 1320000 0.000576 4.35E-10 hu9F5VHv9/hu9F 5VLv8_DIM9 SEQ ID NO: 127/ SEQ ID NO: 140 V3Q, L27cG, L37Q, M51K, L54G, L92I 2290000 0.0116 5.07E-09 hu9F5VHv9/hu9F 5VLv8_DIM10 SEQ ID NO: 127/ SEQ ID NO: 141 V3Q, L27cS, L37Q, M51K, L54G, L92I 18900000 0.0741 3.92E-09 hu9F5VHv9/hu9F 5VLv8_DIMll SEQ ID NO: 127/ SEQ ID NO: 142 V3Q, L27cG, L37G, M51G, L54R, L92I 1420000 0.00011 7.72E-11 hu9F5VHv9/hu9F 5VLv8_DIM12 SEQ ID NO: 127/ SEQ ID NO: 143 V3Q, L27cG, L37G, M51G, L54R, L92G 1940000 0.000429 2.21E-10 hu9F5VHv9/hu9F 5VLv8_DIM13 SEQ ID NO: 127/ SEQ ID NO: 144 V3Q, L27cG, L37G, M51G, L54R 1650000 0.000439 2.66E-10 hu9F5VHv9/hu9F 5VLv8_DIM14 SEQ ID NO:127/ SEQ ID NO: 145 V3Q, L27cG, L37G, M51G, L54T, L92I 1330000 0.000188 1.42E-10

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hu9F5VHv9/hu9F 5VLv8_DIM15 SEQ ID NO: 127/ SEQ ID NO: 146 V3Q, L27cG, L37G, M51G, L54T, L92G 839000 0.000732 8.73E-10 hu9F5VHv9/hu9F 5VLv8_DIM16 SEQ ID NO: 127/ SEQ ID NO: 147 V3Q, L27cG, L37G, M51G, L54T 2270000 0.000808 3.55E-10 hu9F5VHv9/hu9F 5VLv8_DIM17 SEQ ID NO: 127/ SEQ ID NO: 148 V3Q, L27cS, L37G, M51G, L54T, L92I 2540000 0.000326 1.28E-10 hu9F5VHv9/hu9F 5VLv8_DIM18 SEQ ID NO: 127/ SEQ ID NO: 149 V3Q, L27cD, L37G, M51G, L54R, L92I 2100000 0.0000939 4.47E-11 hu9F5VHv9/hu9F 5VLv8_DIM19 SEQ ID NO: 127/ SEQ ID NO: 150 V3Q, L27cS, L37I, M51I, L54R, L92I 3610000 0.00215 5.97E-10 hu9F5VHv9/hu9F 5VLv8_DIM20 SEQ ID NO: 127/ SEQ ID NO: 151 V3Q, L27cS, L37Q, M51I, L54G, L92I 5640000 0.0694 1.23E-08 hu9F5VHv9/hu9F 5VLv8_DIM21 SEQ ID NO: 127/ SEQ ID NO:152 V3Q, L27cS, L37Q, M51I, L54G 1310000 1.12 0.000000 858 hu9F5VHv9/hu9F 5VLv8_DIM22 SEQ ID NO: 127/ SEQ ID NO: 153 V3Q, L27cS, L37Q, M51E, L54R, L92I 2250000 0.000878 3.9E-10 hu9F5VHv9/hu9F 5VLv8_DIM23 SEQ ID NO: 127/ SEQ ID NO:154 V3Q, L27cG, L37Q, M51E, L54G, L92I 66900000 0.0832 1.24E-09 hu9F5VHv9/hu9F 5VLv8_DIM24 SEQ ID NO: 127/ SEQ ID NO: 155 V3Q, L27cG, L37I, M51E, L54R, L92I 2190000 0.000993 4.54E-10 hu9F5VHv9/hu9F 5VLv8_DIM25 SEQ ID NO: 127/ SEQ ID NO: 156 V3Q, L27cG, L37I, M51E, L54R, L92G 13000000 0.0409 3.15E-09 hu9F5VHv9/hu9F 5VLv8_DIM26 SEQ ID NO:127/ SEQ ID NO: 157 V3Q, L27cl, L37I, M51E, L54R 3840000 0.004 1.04E-09

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hu9F5VHv9/hu9F 5VLv8_DIM27 SEQ ID NO: 127 SEQ ID NO: 158 V3Q, L37Q, M51G, L54R, L92I 2410000 0.000385 1.6E-10 hu9F5VHv9/hu9F 5VLv8_DIM28 SEQ ID NO: 127/ SEQ ID NO: 159 V3Q, L27cS, M51G, L54R, L92I 3130000 0.000217 6.92E-11 hu9F5VHv9/hu9F 5VLv8_DIM29 SEQ ID NO: 127/ SEQ ID NO: 160 V3Q, L27cS, L37Q, L54R, L92I 1980000 0.000534 2.7E-10 hu9F5VHv9/hu9F 5VLv8_DIM30 SEQ ID NO: 127/ SEQ ID NO: 161 V3Q, L27cS, L37Q, M51G, L92I 1020000 0.000447 4.38E-10 hu9F5VHvlO/hu9 F5VLv9_DIMl SEQ ID NO: 128/ SEQ ID NO: 162 Q38K, G42E V3Q, L27cS, L37Q, M51G, L54G, N60D, L92I 2690000 0.00395 1.47E-09 hu9F5VHvlO/hu9 F5VLv9_DIM2 SEQ ID NO: 128/ SEQ ID NO: 163 Q38K, G42E V3Q, L27cS, L37Q, M51G, L54R, N60D, L92I 2720000 0.00184 6.79E-10

hu9F5VHvl0/hu9 F5VLv9_DIM4 SEQ ID NO: 128/ SEQ ID NO: 164 Q38K, G42E V3Q, L27cS, L37Q, M51G, L54R, N60D, L92G 2530000 0.00439 1.74E-09 hu9F5VHvl0/hu9 F5VLv9_DIM5 SEQ ID NO: 128/ SEQ ID NO: 165 Q38K, G42E V3Q, L27cG, L37Q, M51G, L54R, N60D, L92I 1700000 0.000919 5.42E-10 hu9F5VHvl0/hu9 F5VLv9_DIM8 SEQ ID NO: 128/ SEQ ID NO: 166 Q38K, G42E V3Q, L27cG, L37Q, M51K, L54R, N60D, L92I 2710000 0.00232 8.59E-10 hu9F5VHvl0/hu9 F5VLv9_DIM10 SEQ ID NO:/SEQ ID NO: 167 Q38K, G42E V3Q, L27cS, L37Q, M51K, L54G, N60D, L92I 6890000 0.026 3.77E-09 hu9F5VHvl0/hu9 F5VLv9_DIMl 1 SEQ ID NO: 128/ SEQ ID NO: 168 Q38K, G42E V3Q, L27cG, L37G, M51G, L54R, N60D, L92I 1760000 0.000287 1.63E-10 hu9F5VHvl0/hu9 F5VLv9_DIM13 SEQ ID NO: 128/ SEQ ID NO: 169 Q38K, G42E V3Q, L27cG, L37G, M51G, L54R, N60D, 2280000 0.00116 5.08E-10

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hu9F5VHvl0/hu9 F5VLv9_DIM19 SEQ ID NO: 128/ SEQ ID NO: 170 Q38K, G42E V3Q, L27cS, L37I, M51I, L54R, N60D, L92I 3490000 0.00688 1.97E-09 hu9F5VHvl0/hu9 F5VLv9_DIM20 SEQ ID NO: 128/ SEQ ID NO: 171 Q38K, G42E V3Q, L27cS, L37Q, M51I, L54G, N60D, L92I 3470000 0.0334 9.62E-09

In addition, as a secondary characteristic, heat stability and titer were analyzed for all deimmunized variants. Heat stability and titer levels were compared for antibodies that were comparable to Hu9F5VHv9/Hu9F5VLv8 based on affinity measurements, and the antibodies in Table. The 26 are listed in order based on deviation from Tm of Hu9F5VHv9/Hu9F5VLv8.

Thermal stability values were determined using differential scanning calorimetry (DSC). All DSC scans were performed using a VP-Capillary DSC system (Malvern). All samples were prepared at a concentration of 0.5 mg/ml in 1xPBS and compared with the 1xPBS standard. Approximately 0.5 ml of protein solution and buffer was injected into the sample and reference cell. Calorimetric scanning was carried out at a scanning speed of 60°C/h, at temperatures from 25 to 110°C at constant pressure. Analysis was performed using Origin software. The reported values represent the temperature at which the maximum heat capacity of the Fab peak is recorded.

The titer was determined as follows. After expression in 293 suspension cells, the antibodies were purified using protein A binding chromatography using standard methods. After purification, the antibodies were replaced with 1xPBS, and protein concentrations were determined by absorbance at 280 nm. Titers were calculated by dividing the final yield of purified protein by the initial volume of expression culture and expressed in milligrams per liter.

Table 26. Thermal stability and titer of humanized 9F5 variants

Humanized variant 9F5 Substitutions in VH refer to hu9F5VH v9 (number of mutation(s) according to Kabat) Substitutions in VL relative to hu9F5VLv8 (number of mutation(s) by Kabat) T _m (°C)—deviation of the Fab peak from hu9F5VHv9/ hu9F5VLv8 titer (mg/l) hu9F5VHv9/hu9F5VL v8 SEQ ID NO:127/SEQ IDNO:130 0 491.6 hu9F5VHv9/hu9F5VL v8_DIM18 SEQ ID NO:127/SEQ Ш NO: 149 V3Q, L27cD, L37G, M51G, L54R, L92I -1.55 579.60 hu9F5VHv9/hu9F5VL v8_DIMll SEQ ID NO:127/SEQ Ш NO: 142 V3Q, L27cG, L37G, M51G, L54R, L92I -3.06 446.40 hu9F5VHv9/hu9F5VL v8_DIM28 SEQ ID NO:127/SEQ IDNO:159 V3Q, L27cS, M51G, L54R, L92I -0.27 348.70

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hu9F5VHv9/hu9F5VL v8_DIM17 SEQ ID NO:127/SEQ ID NO: 148 V3Q, L27cS, L37G, M51G, L54T, L92I -3.83 423.80 hu9F5VHv9/hu9F5VL v8_DIM6 SEQ ID NO:127/SEQ IDNO:137 V3Q, L27cD, L37Q, M51G, L54R, L92I -0.77 569.80 hu9F5VHv9/hu9F5VL v8_DIM14 SEQ ID NO:127/SEQ ID NO: 145 V3Q, L27cG, L37G, M51G, L54T, L92I -5.65 282.40 hu9F5VHv9/hu9F5VL v8_DIM5 SEQ ID NO:127/SEQ IDNO:136 V3Q, L27cG, L37Q, M51G, L54R, L92I -1.83 287.30 hu9F5VHv9/hu9F5VL v8_DIM7 SEQ ID NO:127/SEQ IDNO:138 V3Q, L27cD, L37Q, M51K, L54R, L92I -2.05 636.60 hu9F5VHv9/hu9F5VL v8_DIM27 SEQ ID NO: 127/SEQ IDNO:158 V3Q, L37Q, M51G, L54R, L92I -0.16 351.20 hu9F5VHv9/hu9F5VL v8_DIM12 SEQ ID NO:127/SEQ ID NO: 143 V3Q, L27cG, L37G, M51G, L54R, L92G -6.38 369.40 hu9F5VHv9/hu9F5VL V8DIM13 SEQ ID NO: 127/SEQ ID NO: 144 V3Q, L27cG, L37G, M51G, L54R -1.7 595.80 hu9F5VHv9/hu9F5VL v8_DIM2 SEQ ID NO:127/SEQ IDNO:133 V3Q, L27cS, L37Q, M51G, L54R, L92I -0.3 474.30 hu9F5VHv9/hu9F5VL v8_DIM29 SEQ ID NO:127/SEQ ID NO: 160 V3Q, L27cS, L37Q, L54R, L92I -1.83 327.50 hu9F5VHv9/hu9F5VL v8_DIM30 SEQ ID NO:127/SEQ IDNO:161 V3Q, L27cS, L37Q, M51G, L92I -0.92 372.50 hu9F5VHv9/hu9F5VL v8_DIM8 SEQ ID NO:127/SEQ IDNO:139 V3Q, L27cG, L37Q, M51K, L54R, L92I -4.89 451.50

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hu9F5VHvlO/hu9F5V Lv9_DIMl 1 SEQ ID NO: 128/SEQ Ш NO: 168 Q38K, G42E V3Q, L27cG, L37G, M51G, L54R, N60D, L92I -11.94 244.60

Example 8: Neutralizing Activity of Humanized 9F5 Variants

The neutralizing activity of humanized 9F5 variants is analyzed in a cellular model of tau internalization. To assess the ability of antibodies to block the internalization of tau into neurons, an internalization assay using fluorescence-activated cell sorting (FACS) is performed. Antibodies that block internalization are likely to block tau transmission. pHrodo-labeled soluble human tau oligomer P301L 4R0N (final concentration 1.5 μg/ml) is preincubated with anti-tau antibodies (dose titration: initial concentration 80 μg/ml followed by 4-fold serial dilutions) for 30 min at room temperature in cell culture medium. The tau/antibody mixture is then added to B103 neuroblastoma cell lines at a final concentration of 500,000 cells/ml and incubated for 3-4 hours at 37°C in a tissue culture incubator (5% CO2). The cells are then washed 3 times with culture medium, followed by a 10-minute incubation in culture medium and washed twice with FACS buffer (1% FBS in PBS). Cells were resuspended in 100 μl FACS buffer and at mean Texas Red fluorescence intensity measured by FACS LSR II. Texas red pHrodo fluorescence is activated by low pH associated with endolysosomal compartments upon internalization. Since FACS detects cells and pHrodo only fluoresces when internalized, only tau internalized by cells will be detected. The lower the average fluorescence intensity, the lower the amount of internalized tau, suggesting higher blocking activity of the antibody being tested.

Example 9. Construction of humanized antibodies 10C12

The original or donor antibody for humanization was mouse antibody 10C12. The amino acid sequence of the mature m10C12 heavy chain variable region is provided as SEQ ID NO: 7. The amino acid sequence of the mature m10C12 variable light chain is provided as SEQ ID NO: 11. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 according to the Kabat/Chothia composite definition are presented as SEQ ID NO : 8-10, respectively. The Kabat light chain amino acid sequences CDR1, CDR2 and CDR3 are shown as SEQ ID NO: 12-14, respectively. Kabat numbering is used throughout the text.

Variable kappa (Vk)10C12 belongs to mouse Vk subgroup 2, which corresponds to human Vk subgroup 2, and heavy chain variable (Vh) belongs to mouse Vh subgroup 2c, which corresponds to human Vh subgroup 1 [Kabat E. A., et al ., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. Chothia CDR-L1 16 residues similar to canonical Chothia class 4, Chothia CDR-L2 7 residues similar to canonical Chothia class 1, Chothia CDR-L3 9 residues similar to canonical Chothia class 1 [Martin ACR. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.. [Martin, 2010]. Chothia CDR-H1 is 10 residues similar to canonical Chothia class 1, Chothia CDR-H2 is 17 residues similar to canonical Chothia class 2 [Martin, 2010]. The Chothia 3 residue CDR-H3 has no canonical classes. A search was conducted for protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33:D233-7] to find structures that would provide an approximate structural model of 10C12. The antibody fab crystal structure pdb code 5OBF [Vicentini, et al., 2017, unpublished] was used for both the Vh and Vk structures because it had good resolution (1.92A°) and overall sequence similarity to Vh and Vk 10C12, keeping the same canonical structures for loops.

The VH 10C12 frameworks have a high degree of similarity to the corresponding regions of the immunoglobulin heavy chain variable region [Homo sapiens] CAC20421, cloned by Arnold-Schild et al [Cancer Res. 60 (15), 4175-4178 (2000)]. The variable domains VH 10C12 and CAC20421 also have the same length with respect to the CDR-H1, H2 loops. Likewise, the VL 10C12 scaffolds have a high degree of sequence similarity to the corresponding regions of the human antibody CAB51297 cloned by Capello et al. [Identification of three subgroups of B-cell chronic lymphocytic leukemia based upon mutations of BCL-6 and IGV genes. Unpublished (direct submission to GenBank)]. The light chain variable domain of antibodies 10C12 and CAB51297 also has the same length in relation to the CDRL1, L2 and L3 loops. Accordingly, the framework regions of both VH CAC20421 and VL CAB51297 were selected as acceptor sequences for CDR 10C12. A model of 10C12 CDRs grafted onto the corresponding human scaffolds for VH and VL was constructed and used as a guide for further back mutations.

Sequences of heavy and light chain variants obtained from the humani process

- 119 047155 antibody tions were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains, as specified in the WHO INN committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: https://www.who.int/medicines/services/inn/BioRev2014.pdf) Residues have been modified to align with the corresponding human germline sequence where possible to enhance humanization and reduce potential immunogenicity. For the humanized variants VLv1 and VLv2, mutations were introduced to make the sequences more similar to the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). For the humanized variants of VHv1 and VHv2, mutations were introduced to make the sequences more similar to the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33)

Additional versions of hu10C12-VH and hu10C12-VL were designed to enable the evaluation of various scaffold residues for their contribution to antigen binding, thermostability and immunogenicity, and to optimize deamination, oxidation, Nglycosylation, proteolysis and aggregation. Positions considered for mutation include those that define canonical CDR conformations (summarized in Martin, A.C.R. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany : Springer International Publishing AG.), are located in the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99.), localized on VH/VL domain interface (summarized in Leger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. B: Shepherd P and Dean C (eds). Monoclonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.), are sensitive to post-translational modifications such as glycosylation or pyroglutamination, are occupied by residues predicted to conflict with CDRs, according to the CDR 10C12 model, grafted onto VH and VL scaffolds, or are occupied by residues that are rare among sequenced human antibodies, where either the parental mouse 10C12 residue or some other residue is much more common in the human antibody repertoire.

Alignments of mouse 10C12 and various humanized antibodies are shown for light chain variable regions (Table 28 and FIG. 8) and heavy chain variable regions (Table 27 and FIG. 7).

2 humanized heavy chain variable region variants and 2 humanized light chain variable region variants were constructed containing different substitution permutations: hu10C12VHv1 or hu10C12VHv2, (SEQ ID NO: 214-215, respectively); and hu10C12VLv1 or hu10C12VLv2, (SEQ ID NO: 216-217, respectively) (Tables 27 and 28). Examples of humanized Vk and Vh constructs with back mutations and other mutations based on selected human scaffolds are shown in Table 1. 27 and 28, respectively. Areas highlighted in bold in the table. 27 and 28 denote CDR, according to the Kabat/Chothia composite definition. Sign - in the columns of the table. 27 and 28 indicate that there is no residue at the indicated position. SEQ ID NO: 214-215 and SEQ ID NO: 216217 contain back mutations and other mutations, as shown in table. 29. The amino acids at positions in hu10C12VHv1 and hu10C12VHv2 are listed in Table. 30. The amino acids at positions in hu10C12VLv1 and hu10C12VLv2 are listed in Table. 31.

Percentage of degree of humanization for humanized VH chains hu10C12VHv1 and hu10C12VHv2 (SEQ ID NO: 214-215, respectively) relative to the most similar human germline gene IGHV1-69-2*01 (SEQ ID NO: 33), and for humanized VL chains hu10C12VLv1 and hu10C12VLv2 (SEQ ID NO: 216-217, respectively) in relation to the most similar human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37), shown in table. 32.

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Table 27

Balance according to Kabat No. Linear remainder no. FR or CDR VH murine 10C12 (SEQ ID NO:7) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) CAC20421-VH huFrwk (SEQ ID NO:218) HulOC12VHvl (SEQ ID NO:214) HulOC12VHv2 (SEQ ID NO:215) 1 1 Frl E E Q Q E 2 2 Frl V V V V V 3 3 Frl Q Q Q Q Q 4 4 Frl L L L L L 5 5 Frl Q V V V V 6 6 Frl Q Q Q Q Q 7 7 Frl s s s s s 8 8 Frl G G G G G 9 9 Frl A A A A A 10 10 Frl E E E E E AND AND Frl L V V V V 12 12 Frl V TO TO TO TO 13 13 Frl R To To To To 14 14 Frl P P P P P 15 15 Frl G G G G G 16 16 Frl A A A A A 17 17 Frl S T T T T 18 18 Frl V V V V V 19 19 Frl To To To To To 20 20 Frl L I I I I 21 21 Frl s s s s s 22 22 Frl c c c c c 23 23 Frl T To To To To 24 24 Frl A V V A A 25 25 Frl s s s s s 26 26 CDR-H1 G G G G G 27 27 CDR-H1 F Y Y F F 28 28 CDR-H1 N T T N N 29 29 CDR-H1 I F F I I thirty thirty CDR-H1 TO T T TO TO 31 31 CDR-H1 D D D D D 32 32 CDR-H1 D Y Y D D

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33 33 CDR-H1 Y Y Y Y Y 34 34 CDR-H1 M M M M M 35 35 CDR-H1 N H H N N 35A CDR-H1 - - - - - 35V CDR-H1 - - - - - 36 36 Fr2 W w w w w 37 37 Fr2 V V V V V 38 38 Fr2 To Q Q Q Q 39 39 Fr2 Q Q Q Q Q 40 40 Fr2 R A A A A 41 41 Fr2 P P P P P 42 42 Fr2 E G G G G 43 43 Fr2 R TO TO TO TO 44 44 Fr2 G G G G G 45 45 Fr2 L L L L L 46 46 Fr2 E E E E E 47 47 Fr2 W W W W W 48 48 Fr2 I M M I I 49 49 Fr2 G G G G G 50 50 CDR-H2 W L L W W 51 51 CDR-H2 I V V I I 52 52 CDR-H2 D D D D D 52A 53 CDR-H2 P P P P P 52V CDR-H2 - - - - - 52C CDR-H2 - - - - - 53 54 CDR-H2 E E E E E 54 55 CDR-H2 N D D N N 55 56 CDR-H2 G G G G G 56 57 CDR-H2 D E E D D 57 58 CDR-H2 T T T T T 58 59 CDR-H2 E I I E E 59 60 CDR-H2 Y Y Y Y Y 60 61 CDR-H2 A A A A A 61 62 CDR-H2 S E E S S 62 63 CDR-H2 TO TO TO TO TO 63 64 CDR-H2 F F F F F 64 65 CDR-H2 Q Q Q Q Q 65 66 CDR-H2 G G G G G 66 67 Fr3 TO R R R R 67 68 Fr3 A V V A A 68 69 Fr3 T T T T T 69 70 Fr3 M I I M M 70 71 Fr3 T T T T T 71 72 Fr3 A A A A A

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72 73 Fr3 D D D D D 73 74 Fr3 T T T T T 74 75 Fr3 S S S s S 75 76 Fr3 s T T T T 76 77 Fr3 N D D D D 77 78 Fr3 T T T T T 78 79 Fr3 A A A A A 79 80 Fr3 Y Y Y Y Y 80 81 Fr3 L M M M M 81 82 Fr3 Q E E E E 82 83 Fr3 F L L L L 82A 84 Fr3 s S S S S 82V 85 Fr3 s s s S s 82C 86 Fr3 L L L L L 83 87 Fr3 T R R R R 84 88 Fr3 s S S S S 85 89 Fr3 E E E E E 86 90 Fr3 D D D D D 87 91 Fr3 T T T T T 88 92 Fr3 A A A A A 89 93 Fr3 V V V V V 90 94 Fr3 Y Y Y Y Y 91 95 Fr3 Y Y Y Y Y 92 96 Fr3 c c c c c 93 97 Fr3 T A A T T 94 98 Fr3 T T R T T 95 99 CDR-H3 s - I s s 96 CDR-H3 - - P - - 97 CDR-H3 - - L - - 98 CDR-H3 - - F - - 99 CDR-H3 - - G - - 100 CDR-H3 - - R - - 100 A CDR-H3 - - - - - 100V CDR-H3 - - - - - 100 With CDR-H3 - - - - - 100 D CDR-H3 - - - - - 100E CDR-H3 - - - - - 100F CDR-H3 - - - - - 100 G CDR-H3 - - - - - 100 CDR-H3 - - - - -

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Η 1001 CDR-H3 - - - - - 100J CDR-H3 - - - - - 100 k CDR-H3 - - - - - 101 100 CDR-H3 N Q D N N 102 101 CDR-H3 G H H G G 103 102 Fr4 W w W W W 104 103 Fr4 G G G G G 105 104 Fr4 Q Q Q Q Q 106 105 Fr4 G G G G G 107 106 Fr4 T T T T T 108 107 Fr4 L L L L L 109 108 Fr4 V V V V V 110 109 Fr4 T T T T T 111 110 Fr4 V V V V V 112 111 Fr4 s s s s s FROM 112 Fr4 T s s s s

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Table 28

Balance according to Kabat No. Linear remainder no. FR or CDR VL murine 10C12 (SEQ ID NO:11) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) CAB51297-VL huFrwk (SEQ ID NO:35 HulOC12VLvl (SEQ ID NO:216) HulOC12VLv2 (SEQ ID NO:217) 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 M M M M M 5 5 Frl T T T T T 6 6 Frl Q Q Q Q Q 7 7 Frl A s s s s 8 8 Frl A P P P P 9 9 Frl F L L L L 10 10 Frl S s s s s AND AND Frl N L L L L 12 12 Frl P P P P P 13 13 Frl V V V V V 14 14 Frl T T T T T 15 15 Frl L P P P P 16 16 Frl G G G G G 17 17 Frl T E E E E 18 18 Frl S P P P P 19 19 Frl A A A A A 20 20 Frl S S S S S 21 21 Frl I I I I I 22 22 Frl s s s s s

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23 23 Frl C C C C C 24 24 CDR-L1 R R R R R 25 25 CDR-L1 S S S S S 26 26 CDR-L1 S S S S S 27 27 CDR-L1 TO Q Q TO TO 27A 28 CDR-L1 s s s s s 27V 29 CDR-L1 L L L L L 27C thirty CDR-L1 L L L L L 27D 31 CDR-L1 H H H H H 27E 32 CDR-L1 S S S S S 27F CDR-L1 - - - - 28 33 CDR-L1 N N N N N 29 34 CDR-L1 G G G G G thirty 35 CDR-L1 I Y Y I I 31 36 CDR-L1 T N N T T 32 37 CDR-L1 Y Y Y Y Y 33 38 CDR-L1 L L L L L 34 39 CDR-L1 Y D D Y Y 35 40 Fr2 w W W w w 36 41 Fr2 Y Y Y Y Y 37 42 Fr2 L L L L L 38 43 Fr2 Q Q Q Q Q 39 44 Fr2 To To To To To 40 45 Fr2 P P P P P 41 46 Fr2 G G G G G 42 47 Fr2 Q Q Q Q Q 43 48 Fr2 s s s s s 44 49 Fr2 P P P P P 45 50 Fr2 Q Q Q Q Q 46 51 Fr2 L L L L L 47 52 Fr2 L L L L L 48 53 Fr2 I I I I I 49 54 Fr2 Y Y Y Y Y 50 55 CDR-L2 Q L L Q Q 51 56 CDR-L2 M G G M M 52 57 CDR-L2 s S S s s 53 58 CDR-L2 N N N N N 54 59 CDR-L2 L R R L L 55 60 CDR-L2 A A A A A 56 61 CDR-L2 S S S S S 57 62 Fr3 G G G G G 58 63 Fr3 V V V V V 59 64 Fr3 P P P P P 60 65 Fr3 D D D D D

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61 66 Fr3 R R R R R 62 67 Fr3 F F F F F 63 68 Fr3 S S s S S 64 69 Fr3 S G G S S 65 70 Fr3 s S S s s 66 71 Fr3 G G G G G 67 72 Fr3 s S S s s 68 73 Fr3 G G G G G 69 74 Fr3 T T T T T 70 75 Fr3 D D D D D 71 76 Fr3 F F F F F 72 77 Fr3 T T T T T 73 78 Fr3 L L L L L 74 79 Fr3 R TO TO TO TO 75 80 Fr3 I I I I I 76 81 Fr3 s s s s s 77 82 Fr3 R R R R R 78 83 Fr3 V V V V V 79 84 Fr3 E E E E E 80 85 Fr3 A A A A A 81 86 Fr3 E E E E E 82 87 Fr3 D D D D D 83 88 Fr3 V V V V V 84 89 Fr3 G G G G G 85 90 Fr3 V V V V V 86 91 Fr3 Y Y Y Y Y 87 92 Fr3 Y Y Y Y Y 88 93 Fr3 c c c c c 89 94 CDR-L3 A M M A A 90 95 CDR-L3 Q Q Q Q Q 91 96 CDR-L3 N A A N N 92 97 CDR-L3 L L L L L 93 98 CDR-L3 E Q Q E E 94 99 CDR-L3 L T T L L 95 100 CDR-L3 P P P P P 95A CDR-L3 - - - - - 95V CDR-L3 - - - - - 95С CDR-L3 - - - - - 95D CDR-L3 - - - - - 95E CDR-L3 - - - - - 95F CDR-L3 - - - - - 96 101 CDR-L3 L Y L L L 97 102 CDR-L3 T T T T T 98 103 Fr4 F F F F F

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99 104 Fr4 G G G G G 100 105 Fr4 A Q G G G 101 106 Fr4 G G G G G 102 107 Fr4 T T T T T 103 108 Fr4 TO TO TO TO TO 104 109 Fr4 L L V V L 105 110 Fr4 E E E E E 106 111 Fr4 L I I I I 106 A Fr4 - - - - - 107 112 Fr4 TO TO TO TO TO

Table 29. Back mutations VH, VL and other mutations for humanized 10C12

Option Vh or V _L V _H or V _l exon acceptor sequence Changes in acceptor framework residues (or CDRs) (based on composite CDR definitions by Kabat/Chothia) HulOC12VHvl (SEQ ID NO:214) HulOC12VHv2 (SEQ ID NO:215) HulOC12VLvl (SEQ ID NO:216) HulOC12VLv2 (SEQ ID NO:217) GenBank accession number CAC20421-VH_huFrwk (SEQ ID NO:218) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) GenBank accession number CAC20421-VH_huFrwk (SEQ ID NO:218) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) GenBank accession number CAB51297-VL_huFrwk (SEQ ID NO:35) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) H24, H48, H67, H69, H93, H94 Hl, H24, H48, H67, H69, H93, H94 L64 L64, LI04

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Table 30. Kabat numbering of framework residues (or CDRs) (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the heavy chains of humanized 10C12 antibodies

Remainder CAC20421-VH_huFrwk (SEQ ID NO:218) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) VH murine 10C12 (SEQ ID NO:7) HulOC12VHvl (SEQ ID NO:214) HulOC12VHv2 (SEQ ID NO:215) H1 Q E E Q E H24 V V A A A H48 M M I I I H67 V V A A A H69 I I M M M H93 A A T T T H94 R T T T T

Table 31. Kabat numbering of framework residues (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the light chains of humanized 10C12 antibodies

Remainder (S£ON iTT ALYA) ΊΑΚ.Ι.ΐηΐΓ ΐ Λ-/А71<Я\Л ID NO:37) (ΙΙΌΝ (TT Olya) 7TL0T OYUNNPPCHK LL HulOC12VLvl (SEQ ID NO:216) HulOC12VLv2 (SEQ ID NO:217) L64 G G S S S L104 V L L V L

Table 32. Percentage indicator of the degree of humanization of heavy and light chains of humanized antibodies 10C12

Option V _H or V _L Degree HulOC12VHvl (SEQ ID NO:214) 81.6% HulOC12VHv2 (SEQ ID NO:215) 82.7% HulOC12VLvl (SEQ IDNO:216) 88.0% HulOC12VLv2(SEQ ID NO:217) 88.0%

Positions at which canonical, vernier, or interface residues differ between mouse and human acceptor sequences are candidates for replacement. Examples of interacting canonical/CDR residues include Kabat residues H24 and H94 in Table 1. 27. Examples of vernier residues include Kabat residues H48, H67, H69, H93 and H94 in Table. 27 and L64 in table. 28. Examples of interface/package residues (VH+VL) include Kabat H93 residue in Table. 27.

Justification for the choice of provisions indicated in table. 27, in the heavy chain variable region, candidates for replacement are as follows:

- 129 047155 heavy chain variable regions hu10C12VHv1 consists of the CDR-H1, H2 and H3 loops of 10C12-VH grafted onto a CAC20421-VH backbone. In addition, hu10C12VHv1 also returns all scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability.

V24A: is a reverse mutation of a Vernier zone residue. M48I: is a reverse mutation of a Vernier zone residue.

V67A: is a back mutation of a Vernier zone residue and undergoes back mutation to maintain the CDR conformation.

I69M: is a back mutation of a Vernier zone residue, and the back mutation is made to preserve the integrity of the CDR packaging.

A93T: Position H93 is a remnant of the VH/VL interface and is backmutated to Thr to maintain the integrity of the antibody interface.

R94T: is a reverse mutation of a canonical Chothia structural residue. R94T is also a germline-aligning mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

hu10C12VHv2

10C12VHv2 retains all returned scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability. In addition, hu10C12VHv2 includes back mutations or substitutions to the most abundant residue at a given position and to optimize deamination, oxidation, N-glycosylation, proteolysis, and aggregation. Mutations in VHv2 that are not included in hu10C12VHv1:

Q1E: is a stability-enhancing mutation to reduce the potential for pyroglutamate formation and reduce N-terminal heterogeneity (Liu, above).

Justification for the choice of provisions indicated in table. 28, in the light chain variable region, candidates for replacement are as follows.

kappa light chain variable regions hu10C12VLv1 consists of CDR-L1, L2 and L3 loops of 10C12-VL grafted onto a CAB51297-VL scaffold. In addition, hu10C12VLv1 also returns all scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability.

G64S: is a reverse mutation of a Vernier zone residue. The G64S back mutation retains the CDR packaging.

hu10C12VLv2 retains all returned scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability. In addition, hu10C12VLv2 includes back mutations or substitutions to the most abundant residue at a given position and to optimize deamination, oxidation, N-glycosylation, proteolysis, and aggregation. Mutations in VLv2 that are not included in hu10C12VLv1:

V104L: is a germline-aligning mutation. Leu is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

Based on these human scaffolds, the following variants were developed: heavy chain variable regions >hulOC12VHvl (SEQ ID NO:214)

QVQLVQSGAEVKKPGATVKISCKASGFNIKDDYMNWVQQAPGKGLEWIGWIDPENGD

TEYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS >hulOC12VHv2 (SEQ ID NO :215)

EVQLVQSGAEVKKPGATVKISCKASGFNIKDDYMNWVQQAPGKGLEWIGWIDPENGD

TEYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS kappa light chain variable regions

- 130 047155 >hulOC12VLvl (SEQ Ш NO:216)

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS

GVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK >hulOC12VLv2 (SEQ Ш NO:217)

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS

GVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKLEIK

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

Example 10. Construction of humanized antibodies 12C4

The original or donor antibody for humanization was mouse antibody 12C4. The amino acid sequence of the mature m12C4 heavy chain variable region is provided as SEQ ID NO: 219. The amino acid sequence of the mature m12C4 light chain variable region is provided as SEQ ID NO: 11. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 according to the composite definition of Kabat/Chothia K are presented as SEQ ID NO: 8, 220 and 10, respectively. The Kabat light chain amino acid sequences CDR1, CDR2 and CDR3 are shown as SEQ ID NO: 12-14, respectively. Kabat numbering is used throughout the text.

Variable kappa (Vk) 12C4 belongs to mouse Vk subgroup 2, which corresponds to human Vk subgroup 2, and heavy chain variable (Vh) belongs to mouse Vh subgroup 2c, which corresponds to human Vh subgroup 1 [Kabat E. A., et al ., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. Chothia CDR-L1 16 residues similar to canonical Chothia class 4, Chothia CDR-L2 7 residues similar to canonical Chothia class 1, Chothia CDR-L3 9 residues similar to canonical Chothia class 1 [Martin ACR. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.. [Martin, 2010].

Chothia CDR-H1 is 10 residues similar to canonical Chothia class 1, Chothia CDR-H2 is 17 residues similar to canonical Chothia class 2 [Martin, 2010]. The Chothia 3 residue CDR-H3 has no canonical classes. A search was conducted for protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33:D233-7] to find structures that would provide an approximate structural model of 12C4. The antibody fab crystal structure pdb code 5OBF [Vicentini, et al., 2017, unpublished] was used for both the Vh and Vk structures because it had good resolution (1.92A°) and overall sequence similarity to Vh and Vk 12C4, keeping the same canonical structures for loops.

The VH 12C4 frameworks have a high degree of similarity to the corresponding regions of the immunoglobulin heavy chain variable region [Homo sapiens] CAC20421, cloned by Arnold-Schild et al. [Cancer Res. 60 (15), 4175-4178 (2000)]. Similarly, the 12C4 VL scaffolds have a high degree of sequence similarity to the corresponding VL regions of the human antibody CAB51297 cloned by Capello et al. (GenBank ref number CAB51297, filed July 20, 1999, unpublished). The light chain variable domain of antibodies 12C4 and CAB51297 also has the same length with respect to the CDR-L1, L2 and L3 loops. Accordingly, the framework regions and VH of CAC20421 and VL of CAB51297 were selected as acceptor sequences for the VL 12C4 CDR. A model of 12C4 CDRs grafted onto the corresponding human scaffolds for VH and VL was constructed and used as a guide for further back mutations.

Heavy and light chain variant sequences resulting from the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains, as specified in the WHO INN committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: https://www.who.int/medicines/services/inn/BioRev2014.pdf) Residues have been modified to align with the corresponding human germline sequence where possible to enhance humanization and reduce potential immunogenicity. For the humanized variants VLv1 and VLv2, mutations were introduced to make the sequences more similar to the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). For the humanized variants VHv1 and VHv2, mutations were introduced to make the sequences more similar to the human germline gene IGHV-69-2*01 (SEQ ID NO: 33)

Additional versions of hu12C4-VH and hu12C4-VL were designed to allow the evaluation of various scaffold residues for their contribution to antigen binding, thermostability and immunogenicity, and to optimize deamination, oxidation, Nglycosylation, proteolysis and aggregation. Provisions considered for mutation include those

- 131 047155 which define canonical CDR conformations (summarized in Martin, A.C.R. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG. ), are located in the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99), localized at the VH/VL domain interface (summarized in Leger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. B: Shepherd P and Dean C (eds). Oxford University Press.), are sensitive to post-translational modifications such as glycosylation or pyroglutamination, are occupied by residues that are predicted to conflict with CDRs, according to the 12C4 CDR model grafted onto the VH and VL scaffolds, or are occupied by residues that are rare among sequenced human antibodies, where either the parental mouse 12C4 residue or some other residue is much more common in the human antibody repertoire.

Alignments of murine 12C4 and various humanized antibodies are shown for light chain variable regions (Table 34 and FIG. 10) and heavy chain variable regions (Table 33 and FIG. 9).

2 humanized heavy chain variable region variants and 2 humanized light chain variable region variants were constructed containing different substitution permutations: hu12C4VHv1 or hu12C4VHv2, (SEQ ID NO: 221-222, respectively); and hu12C4VLv1 or hu12C4VLv2, (SEQ ID NO: 223-224, respectively) (Tables 33 and 34). Examples of humanized Vk and Vh constructs with back mutations and other mutations based on selected human scaffolds are shown in Table 1. 33 and 34 respectively. Areas highlighted in bold in the table. 33 and 34 denote CDR, according to the Kabat/Chothia composite definition. Sign - in the columns of the table. 33 and 34 indicate that there is no residue at the indicated position. SEQ ID NO: 221-222 and SEQ ID NO: 223224 contain back mutations and other mutations, as shown in table. 35. The amino acid positions in hu12C4VHv1 and hu12C4VHv2 are listed in Table. 36. The amino acid positions in hu12C4VLv1 and hu12C4VLv2 are listed in Table. 37.

Percentage of degree of humanization for humanized VH chains hu12C4VHv1 and hu12C4VHv2 (SEQ ID NO: 221-222, respectively) relative to the most similar human germline gene IGHV1-69-2*01 (SEQ ID NO: 33), and for humanized VL chains hu12C4VLv1 and hu12C4VLv2 (SEQ ID NO: 223-224, respectively) in relation to the most similar human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37), shown in table. 38.

Table 33

Balance according to Kabat No. Linear remainder no. FR or CDR VH murine 12C4 (SEQ ID NO:219) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) CAC20421 VH (SEQ ID NO:218) Hul2C4VHvl (SEQ ID NO:221) Hul2C4VHv2 (SEQ ID NO:222) 1 1 Frl E E Q Q E 2 2 Frl V V V V V 3 3 Frl Q Q Q Q Q

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4 4 Frl L L L L L 5 5 Frl Q V V V V 6 6 Frl Q Q Q Q Q 7 7 Frl s s s s s 8 8 Frl G G G G G 9 9 Frl A A A A A 10 10 Frl E E E E E AND AND Frl L V V V V 12 12 Frl V TO TO TO TO 13 13 Frl R To To To To 14 14 Frl P P P P P 15 15 Frl G G G G G 16 16 Frl A A A A A 17 17 Frl S T T T T 18 18 Frl V V V V V 19 19 Frl To To To To To 20 20 Frl L I I I I 21 21 Frl s s s s s 22 22 Frl c c c c c 23 23 Frl T To To To To 24 24 Frl A V V V V 25 25 Frl s s s s s 26 26 CDR-H1 G G G G G 27 27 CDR-H1 F Y Y F F 28 28 CDR-H1 N T T N N 29 29 CDR-H1 I F F I I thirty thirty CDR-H1 TO T T TO TO 31 31 CDR-H1 D D D D D 32 32 CDR-H1 D Y Y D D 33 33 CDR-H1 Y Y Y Y Y 34 34 CDR-H1 M M M M M 35 35 CDR-H1 N H H N N 35A CDR-H1 - - - - - 35V CDR-H1 - - - - - 36 36 Fr2 W w w W W 37 37 Fr2 V V V V V 38 38 Fr2 R Q Q Q Q 39 39 Fr2 Q Q Q Q Q 40 40 Fr2 R A A A A 41 41 Fr2 P P P P P 42 42 Fr2 E G G G G 43 43 Fr2 R TO TO TO TO 44 44 Fr2 G G G G G 45 45 Fr2 L L L L L 46 46 Fr2 E E E E E 47 47 Fr2 W W W W W

- 133 047155

48 48 Fr2 I M M M I 49 49 Fr2 G G G G G 50 50 CDR-H2 W L L W W 51 51 CDR-H2 I V V I I 52 52 CDR-H2 D D D D D 52A 53 CDR-H2 P P P P P 52V CDR-H2 - - - - - 52C CDR-H2 - - - - - 53 54 CDR-H2 E E E E E 54 55 CDR-H2 N D D N N 55 56 CDR-H2 G G G G G 56 57 CDR-H2 D E E D D 57 58 CDR-H2 T T T T T 58 59 CDR-H2 A I I A A 59 60 CDR-H2 Y Y Y Y Y 60 61 CDR-H2 A A A A A 61 62 CDR-H2 S E E S S 62 63 CDR-H2 TO TO TO TO TO 63 64 CDR-H2 F F F F F 64 65 CDR-H2 Q Q Q Q Q 65 66 CDR-H2 G G G G G 66 67 Fr3 TO R R R R 67 68 Fr3 A V V V V 68 69 Fr3 T T T T T 69 70 Fr3 M I I I I 70 71 Fr3 T T T T T 71 72 Fr3 A A A A A 72 73 Fr3 D D D D D 73 74 Fr3 T T T T T 74 75 Fr3 s s s s s 75 76 Fr3 s T T T T 76 77 Fr3 N D D D D 77 78 Fr3 T T T T T 78 79 Fr3 A A A A A 79 80 Fr3 Y Y Y Y Y 80 81 Fr3 L M M M M 81 82 Fr3 Q E E E E 82 83 Fr3 F L L L L 82A 84 Fr3 S S S S S 82V 85 Fr3 s s s s s 82C 86 Fr3 L L L L L 83 87 Fr3 T R R R R 84 88 Fr3 s S S S S 85 89 Fr3 E E E E E 86 90 Fr3 D D D D D 87 91 Fr3 S T T T T

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

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Table 34

Balance according to Kabat No. Linear remainder no. FR or CDR VL murine 12C4 (SEQ ID NO: 11) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) CAB51297 VL (SEQ ID NO:35) Hul2C4VLvl (SEQ ID NO:223) Hul2C4VLv2 (SEQ ID NO:224) 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 M M M M M 5 5 Frl T T T T T 6 6 Frl Q Q Q Q Q 7 7 Frl A s s s s 8 8 Frl A P P P P 9 9 Frl F L L L L 10 10 Frl S s s s s AND AND Frl N L L L L 12 12 Frl P P P P P 13 13 Frl V V V V V 14 14 Frl T T T T T 15 15 Frl L P P P P 16 16 Frl G G G G G 17 17 Frl T E E E E 18 18 Frl S P P P P 19 19 Frl A A A A A 20 20 Frl S S S S S 21 21 Frl I I I I I

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22 22 Frl S S S S S 23 23 Frl C c c c c 24 24 CDR-L1 R R R R R 25 25 CDR-L1 S S S S S 26 26 CDR-L1 s s s s s 27 27 CDR-L1 To Q Q To To 27A 28 CDR-L1 s s s s s 27V 29 CDR-L1 L L L L L 27C thirty CDR-L1 L L L L L 27D 31 CDR-L1 H H H H H 27E 32 CDR-L1 s S S s s 27F CDR-L1 - - - - - 28 33 CDR-L1 N N N N N 29 34 CDR-L1 G G G G G thirty 35 CDR-L1 I Y Y I I 31 36 CDR-L1 T N N T T 32 37 CDR-L1 Y Y Y Y Y 33 38 CDR-L1 L L L L L 34 39 CDR-L1 Y D D Y Y 35 40 Fr2 w W W w w 36 41 Fr2 Y Y Y Y Y 37 42 Fr2 L L L L L 38 43 Fr2 Q Q Q Q Q 39 44 Fr2 To To To To To 40 45 Fr2 P P P P P 41 46 Fr2 G G G G G 42 47 Fr2 Q Q Q Q Q 43 48 Fr2 s s s s s 44 49 Fr2 P P P P P 45 50 Fr2 Q Q Q Q Q 46 51 Fr2 L L L L L 47 52 Fr2 L L L L L 48 53 Fr2 I I I I I 49 54 Fr2 Y Y Y Y Y 50 55 CDR-L2 Q L L Q Q 51 56 CDR-L2 M G G M M 52 57 CDR-L2 s S S s s 53 58 CDR-L2 N N N N N 54 59 CDR-L2 L R R L L 55 60 CDR-L2 A A A A A 56 61 CDR-L2 S S S S S 57 62 Fr3 G G G G G

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58 63 Fr3 V V V V V 59 64 Fr3 P P P P P 60 65 Fr3 D D D D D 61 66 Fr3 R R R R R 62 67 Fr3 F F F F F 63 68 Fr3 S S S S S 64 69 Fr3 s G G G s 65 70 Fr3 s S S S s 66 71 Fr3 G G G G G 67 72 Fr3 s S S S s 68 73 Fr3 G G G G G 69 74 Fr3 T T T T T 70 75 Fr3 D D D D D 71 76 Fr3 F F F F F 72 77 Fr3 T T T T T 73 78 Fr3 L L L L L 74 79 Fr3 R TO TO TO TO 75 80 Fr3 I I I I I 76 81 Fr3 s s s s s 77 82 Fr3 R R R R R 78 83 Fr3 V V V V V

79 84 Fr3 E E E E E 80 85 Fr3 A A A A A 81 86 Fr3 E E E E E 82 87 Fr3 D D D D D 83 88 Fr3 V V V V V 84 89 Fr3 G G G G G 85 90 Fr3 V V V V V 86 91 Fr3 Y Y Y Y Y 87 92 Fr3 Y Y Y Y Y 88 93 Fr3 c c c c c 89 94 CDR-L3 A M M A A 90 95 CDR-L3 Q Q Q Q Q 91 96 CDR-L3 N A A N N 92 97 CDR-L3 L L L L L 93 98 CDR-L3 E Q Q E E 94 99 CDR-L3 L T T L L 95 100 CDR-L3 P P P P P 95A CDR-L3 - - - - - 95B CDR-L3 - - - - - 95C CDR-L3 - - - - - 95D CDR-L3 - - - - -

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95E CDR-L3 - - - - - 95F CDR-L3 - - - - - 96 101 CDR-L3 L Y L L L 97 102 CDR-L3 T T T T T 98 103 Fr4 F F F F F 99 104 Fr4 G G G G G 100 105 Fr4 A Q G G G 101 106 Fr4 G G G G G 102 107 Fr4 T T T T T 103 108 Fr4 TO TO TO TO TO 104 109 Fr4 L L V V L 105 BY Fr4 E E E E E 106 111 Fr4 L I I I I 106 A Fr4 - - - - - 107 112 Fr4 TO TO TO TO TO

Table 35. Back mutations VH, VL and other mutations for humanized 12C4

Option V _H or Vl Vh or Vl exon acceptor sequence Changes in acceptor framework residues (or CDRs) (based on composite CDR definitions by Kabat/Chothia) Hul2C4VHvl (SEQ ID NO:221) GenBank accession number CAC20421 VH (SEQ ID NO:218) Absent Hul2C4VHv2 (SEQ Ш NO:222) GenBank Accession Number CAC20421 VH (SEQ ID NO:218) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H48, H93, H94 Hul2C4VLvl (SEQ Ш NO:223) GenBank accession number CAB51297 VL (SEQ ID NO:3 5) Absent Hul2C4VLv2 (SEQ Ш NO:224) GenBank accession number CAB51297 VL (SEQ ID NO:3 5) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L64, L104

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Table 36. Kabat numbering of framework residues (or CDRs) (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the heavy chains of humanized 12C4 antibodies

Remainder CAC20421 VH (SEQ ID NO:218) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) VH murine 12C4 (SEQ ID NO:219) Hul2C4VHvl (SEQ ID NO:221) Hul2C4VHv2 (SEQ ID NO:222) H1 Q E E Q E H48 M M I M I H93 A A T A T H94 R T T R T

Table 37. Kabat numbering of frame residues (based on CDR according to composite definition

Table 38. Percentage indicator of the degree of humanization of heavy and light chains of humanized 12C4 antibodies

Option Vh or V _l Degree Hul2C4VHvl (SEQ ID NO:221) 86.6% Hul2C4VHv2 (SEQ ID NO:222) 85.7% Hul2C4VLvl (SEQ ID NO:223) 89.0% Hul2C4VLv2 (SEQ ID NO:224) 88.0%

Positions at which canonical, vernier, or interface residues differ between mouse and human acceptor sequences are candidates for replacement. Examples of interacting canonical residues/CDR residues include the residue of Kabat H94 in table. 33. Examples of vernier residues include Kabat residues H48, H93 and H94 in Table. 33 and L64 in table. 34. Examples of interface/package residues (VH+VL) include Kabat H93 residue in Table. 33.

Justification for the choice of provisions indicated in table. 33, in the heavy chain variable region, candidates for replacement are as follows:

heavy chain variable regions hu12C4VHv1 consists of CDR-H1, H2 and H3 loops of 12C4-VH grafted onto a CAC20421 scaffold.

hu12C4VHv2 returns all scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, or contribute to structural stability. 12C4-VH_v2 includes back mutations or

- 140 047155 replacement with the most frequently occurring residue in the indicated position.

Q1E: is a stability-enhancing mutation to reduce the potential for pyroglutamate formation and reduce N-terminal heterogeneity (Liu, above)

M48I: is a reverse mutation of a Vernier zone residue.

A93T: is a back mutation of the VH/VL interface residue and undergoes back mutation to maintain the interface.

R94T: is a reverse mutation of a Vernier zone residue. R94T is also a germline-aligning mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

Justification for the choice of provisions indicated in table. 34, in the light chain variable region, candidates for replacement are as follows.

kappa light chain variable regions hu12C4VLv1 consists of CDR-L1, L2 and L3 loops of 12C4-VL grafted onto the VL backbone of CAB51297 along with the return of all backbone substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or located on the VH/VL domain interface. For hu12C4VLv1, no position other than G64 meets this criterion, so no back mutation is performed. Based on homology model analysis, Gly at position 64 may be transferable, so changes at position 64 are not made in 12C4VLv1, an attempt to replace it with Ser is made in hu12C4VLv2.

Hu12C4VLv2 hu12C4VLv2 includes substitutions that promote structural stability or increase the degree of humanization of the antibody.

G64S: is a reverse mutation of a Vernier zone residue. The G64S back mutation retains the CDR packaging.

V104L: is a germline-aligning mutation. Leu is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

Based on these human scaffolds, the following variants were developed: heavy chain variable regions >hul2C4VHvl (SEQ ID NO:221)

QVQLVQSGAEVKKPGATVKISCKVSGFNIKDDYMNWVQQAPGKGLEWMGWIDPENG

DTAYASKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARSNGWGQGTLVTVSS >hul2C4VHv2 (SEQ ID NO :222)

EVQLVQSGAEVKKPGATVKISCKVSGFNIKDDYMNWVQQAPGKGLEWIGWIDPENGD

TAYASKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTLVTVSS kappa light chain variable regions >hul2C4VLvl (SEQ ID NO:223)

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK >hul2C4VLv2 (SEQ ID NO:224)

DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLAS

GVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKLEIK

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

Example 11. Construction of humanized antibodies 17C12

The original or donor antibody for humanization was mouse antibody 17C12. The amino acid sequence of the mature m17C12 heavy chain variable region is provided as SEQ ID NO: 225. The amino acid sequence of the mature m17d2 light chain variable region is provided as SEQ ID NO: 228. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 according to the Kabat/Chothia K composite definition are presented as SEQ ID NO: 226, 227 and 10, respectively. The Kabat light chain amino acid sequences CDR1, CDR2 and CDR3 are provided as SEQ ID NO: 229-231, respectively. Kabat numbering is used throughout the text.

Variable kappa (Vk) 17C12 belongs to mouse Vk subgroup 2, which corresponds to human Vk subgroup 3, and heavy chain variable (Vh) belongs to mouse Vh subgroup 2c, which corresponds to human Vh subgroup 1 [Kabat E. A., et al ., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. CDR-L1 by Chothia 16 rest

- 141 047155 kov similar to canonical class 4 of Chothia, CDR-L2 of Chothia 7 residues similar to canonical class 1 of Chothia, CDR-L3 of Chothia 9 residues similar to canonical class 1 of Chothia [Martin ACR. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.. [Martin, 2010]. Chothia CDR-H1 is 10 residues similar to canonical Chothia class 1, Chothia CDR-H2 is 17 residues similar to canonical Chothia class 2 [Martin, 2010]. The Chothia 3 residue CDR-H3 has no canonical classes. A search was conducted for protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33:D233-7] to find structures that would provide an approximate structural model of 17C12. Crystal structure of fab antibody with pdb code 3PP3 [Niederfellner, et al., 2011,. Blood 118: 358-367] was used for both the Vh and Vk structures because it had good resolution (2.51A°) and overall sequence similarity to Vh and Vk of 17C12, retaining the same canonical structures for the loops.

The 17C12 VH frameworks have a high degree of similarity to the corresponding regions of the human antibody CAC20421 cloned by Arnold-Schild, et al [Cancer Res. 60 (15), 4175-4178 (2000)]. The variable domains of the heavy chains 17C12 and CAC20421 also have the same length with respect to the CDR-H1, H2 loops. Similarly, the 17C12 VL scaffolds have a high degree of sequence similarity to the corresponding VL regions of the human antibody QDO16713 cloned by Cho et al [Cell Rep 28 (4), 909-922.e6 (2019)]. The light chain variable domain of antibodies 17C12 and QDO16713 also has the same length with respect to the CDR-L1, L2 and L3 loops. Accordingly, the framework regions and VH of CAC20421 and VL of QDO16713 were selected as acceptor sequences for CDR 17C12. A model of 17C12 CDRs grafted onto corresponding human scaffolds for VH and VL was constructed and used as a guide for further back mutations.

Heavy and light chain variant sequences resulting from the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains, as specified in the WHO INN committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: https://www.who.int/medicines/services/inn/BioRev2014.pdf) Residues have been modified to align with the corresponding human germline sequence where possible to enhance humanization and reduce potential immunogenicity. For the humanized variants VLv2 and VLv2, mutations were introduced to make the sequences more similar to the human germline gene IGKV2-29*02 and IGKJ4*01 (SEQ ID NO: 239). For the humanized variants VHv1 and VHv, mutations were introduced to make the sequences more similar to the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33).

Additional versions of hu17C12-VH and hu17C12-VL were designed to allow the evaluation of various scaffold residues for their contribution to antigen binding, thermostability and immunogenicity, and to optimize deamination, oxidation, Nglycosylation, proteolysis and aggregation. Positions considered for mutation include those that define canonical CDR conformations (summarized in Martin, A.C.R. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany : Springer International Publishing AG.), are located in the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99), localized at the interface VH/VL domain (summarized in Leger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. B: Shepherd P and Dean C (eds). Monoclonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.), are sensitive to post-translational modifications such as glycosylation or pyroglutamination, are occupied by residues predicted to conflict with CDRs, according to the 17C12 CDR model, grafted onto VH and VL scaffolds, or are occupied. residues that are rare among sequenced human antibodies, where either the parental mouse 17C12 residue or some other residue is much more common in the human antibody repertoire.

Alignments of murine 17C12 and various humanized antibodies are shown for light chain variable regions (Table 40 and FIG. 12) and heavy chain variable regions (Table 39 and FIG. 11).

2 humanized heavy chain variable region variants and 2 humanized light chain variable region variants were constructed containing different substitution permutations: hu17C12VHv1 or hu17C12VHv2 (SEQ ID NO: 232-233, respectively); and hu17C12VLv1 or hu17C12VLv2 (SEQ ID NO: 234-235, respectively) (Tables 39 and 40). Examples of humanized const

- 142 047155 versions of Vk and Vh with back mutations and other mutations based on selected human scaffolds are shown in Table. 39 and 40 respectively. Areas highlighted in bold in the table. 39 and 40 denote CDR, according to the Kabat/Chothia composite definition. Sign - in the columns of the table. 39 and 40 indicate that there is no residue at the indicated position. SEQ ID NO: 232-233 and SEQ ID NO: 234235 contain back mutations and other mutations, as shown in table. 41. The amino acid positions in hu17C12VHv1 and hu17C12VHv2 are listed in Table. 42. The amino acid positions in hu17C12VLv1 and hu17C12VLv2 are listed in Table. 43.

Percentage of degree of humanization for humanized VH chains hu17C12VHv1 and hu17C12VHv2 (SEQ ID NO: 232-233, respectively) relative to the most similar human germline gene IGHV1-69-2*01 (SEQ ID NO: 33), and for humanized VL chains hu17C12VLv1 and hu17C12VLv2 (SEQ ID NO: 234-235, respectively) in relation to the most similar human germline gene IGKV2-29*02 and IGKJ4*01 (SEQ ID NO: 239), shown in table. 44.

Table 39

Balance according to Kabat No. Linear remainder no. FR or CDR VH murine 17C12 (SEQ ID NO:225) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) CAC20421-VH huFrwk (SEQ ID:218) Hul7C12VHvl (SEQ ID NO:232) Hul7C12VHv2 (SEQ ID NO:233) 1 1 Frl TO E Q Q E 2 2 Frl I V V I I 3 3 Frl Q Q Q Q Q 4 4 Frl L L L L L 5 5 Frl Q V V V V 6 6 Frl Q Q Q Q Q 7 7 Frl s s s s s 8 8 Frl G G G G G 9 9 Frl A A A A A 10 10 Frl E E E E E AND AND Frl L V V V V 12 12 Frl V TO TO TO TO 13 13 Frl R To To To To 14 14 Frl P P P P P 15 15 Frl G G G G G 16 16 Frl A A A A A 17 17 Frl S T T T T 18 18 Frl V V V V V 19 19 Frl To To To To To 20 20 Frl L I I I I

- 143 047155

21 21 Frl S S s s s 22 22 Frl c c c c c 23 23 Frl T To To To To 24 24 Frl A V V A A 25 25 Frl s s s s s 26 26 CDR-H1 A G G A A 27 27 CDR-H1 F Y Y F F 28 28 CDR-H1 N T T N N 29 29 CDR-H1 I F I I thirty thirty CDR-H1 TO T T TO TO 31 31 CDR-H1 D D D D D 32 32 CDR-H1 D Y Y D D 33 33 CDR-H1 Y Y Y Y Y 34 34 CDR-H1 M M M M M 35 35 CDR-H1 N H H N N 35A CDR-H1 - - - - - 35V CDR-H1 - - - - - 36 36 Fr2 W w w W W 37 37 Fr2 V V V V V 38 38 Fr2 To Q Q Q Q 39 39 Fr2 Q Q Q Q Q 40 40 Fr2 R A A A A 41 41 Fr2 P P P P P 42 42 Fr2 E G G G G 43 43 Fr2 R TO TO TO TO 44 44 Fr2 G G G G G 45 45 Fr2 L L L L L 46 46 Fr2 E E E E E 47 47 Fr2 W W W W W 48 48 Fr2 I M M I I 49 49 Fr2 G G G G G 50 50 CDR-H2 W L L W W 51 51 CDR-H2 I V V I I 52 52 CDR-H2 D D D D D 52A 53 CDR-H2 P P P P P 52V CDR-H2 - - - - - 52C CDR-H2 - - - - - 53 54 CDR-H2 E E E E E 54 55 CDR-H2 N D D N N 55 56 CDR-H2 G G G G G 56 57 CDR-H2 D E E D D 57 58 CDR-H2 T T T T T 58 59 CDR-H2 TO I I TO TO 59 60 CDR-H2 Y Y Y Y Y

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60 61 CDR-H2 A A A A A 61 62 CDR-H2 S E E S s 62 63 CDR-H2 TO TO TO TO To 63 64 CDR-H2 F F F F F 64 65 CDR-H2 Q Q Q Q Q 65 66 CDR-H2 G G G G G 66 67 Fr3 TO R R R R 67 68 Fr3 A V V A A 68 69 Fr3 T T T T T 69 70 Fr3 M I I M M 70 71 Fr3 T T T T T 71 72 Fr3 A A A A A 72 73 Fr3 D D D D D 73 74 Fr3 T T T T T 74 75 Fr3 S S S S S 75 76 Fr3 s T T T T 76 77 Fr3 N D D D D 77 78 Fr3 T T T T T 78 79 Fr3 A A A A A 79 80 Fr3 Y Y Y Y Y 80 81 Fr3 L M M M M 81 82 Fr3 Q E E E E 82 83 Fr3 L L L L L 82A 84 Fr3 s S S S S 82V 85 Fr3 s s s s s 82C 86 Fr3 L L L L L 83 87 Fr3 T R R R R 84 88 Fr3 s S S S S 85 89 Fr3 E E E E E 86 90 Fr3 D D D D D 87 91 Fr3 T T T T T 88 92 Fr3 A A A A A 89 93 Fr3 V V V V V 90 94 Fr3 Y Y Y Y Y 91 95 Fr3 Y Y Y Y Y 92 96 Fr3 c c c c c 93 97 Fr3 T A A T T 94 98 Fr3 T T R T T 95 99 CDR-H3 s I s s 96 CDR-H3 - - P - - 97 CDR-H3 - - L - - 98 CDR-H3 - - F - - 99 CDR-H3 - - G - - 100 CDR-H3 - - R - -

- 145 047155

100A CDR-H3 - - - - - 100V CDR-H3 - - - - - Yuos CDR-H3 - - - - - 100D CDR-H3 - - - - - 100E CDR-H3 - - - - - 100F CDR-H3 - - - - - 100G CDR-H3 - - - - - 100H CDR-H3 - - - - - 1001 CDR-H3 - - - - - 100J CDR-H3 - - - - - 100K CDR-H3 - - - - - 101 100 CDR-H3 N Q D N N 102 101 CDR-H3 G H H G G 103 102 Fr4 W w w W W 104 103 Fr4 G G G G G 105 104 Fr4 Q Q Q Q Q 106 105 Fr4 G G G G G 107 106 Fr4 T T T T T 108 107 Fr4 L L T T L 109 108 Fr4 V V V V V 110 109 Fr4 T T T T T 111 110 Fr4 V V V V V 112 111 Fr4 s s s s s 113 112 Fr4 A s R R s

Table 40

Remaining no Kabat No. Linear remainder no. FR or CDR VL murine 17C12 (SEQ ID NO: 228) IGKV2-29*02 and IGKJ4*01 (SEQ ID NO:239) QDO16713-VL_huFrwk (SEQ ID NO:238) Hul7C12VLvl (SEQ ID NO:234) Hul7C12VLv2 (SEQ ID NO:235) 1 1 Frl D D D D D 2 2 Frl V I I V V 3 3 Frl V V V V V 4 4 Frl M M M M M 5 5 Frl T T T T T

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6 6 Frl Q Q Q Q Q 7 7 Frl T T T T T 8 8 Frl P P P P P 9 9 Frl L L L L L 10 10 Frl T s s s s AND AND Frl L L L L L 12 12 Frl s S S S S 13 13 Frl V V V V V 14 14 Frl T T T T T 15 15 Frl I P P P P 16 16 Frl G G G G G 17 17 Frl Q Q Q Q Q 18 18 Frl P P P P P 19 19 Frl A A A A A 20 20 Frl s s s s s 21 21 Frl I I I I I 22 22 Frl s s s s s 23 23 Frl c c c c c 24 24 CDR-L1 T To To T T 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 27V 29 CDR-L1 L L L L L 27C thirty CDR-L1 L L L L L 27D 31 CDR-L1 H H H H H 27E 32 CDR-L1 S S S S S 27F CDR-L1 - - - - - 28 33 CDR-L1 N D D N N 29 34 CDR-L1 R G G R R thirty 35 CDR-L1 TO TO TO TO TO 31 36 CDR-L1 T T T T T 32 37 CDR-L1 Y Y Y Y Y 33 38 CDR-L1 L L L L L 34 39 CDR-L1 H Y Y H H 35 40 Fr2 W W W W W 36 41 Fr2 L Y Y L L 37 42 Fr2 L L L L L 38 43 Fr2 Q Q Q Q Q 39 44 Fr2 R To To TO TO 40 45 Fr2 P P P P P 41 46 Fr2 G G G G G 42 47 Fr2 Q Q Q Q Q 43 48 Fr2 s s P P s

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44 49 Fr2 P P P P P 45 50 Fr2 TO Q Q Q Q 46 51 Fr2 L L L L L 47 52 Fr2 L L L L L 48 53 Fr2 I I I I I 49 54 Fr2 Y Y Y Y Y 50 55 CDR-L2 L E E L L 51 56 CDR-L2 V V V V V 52 57 CDR-L2 s s s s s 53 58 CDR-L2 To s N To To 54 59 CDR-L2 L R R L L 55 60 CDR-L2 E F F E E 56 61 CDR-L2 S S S S S 57 62 Fr3 G G G G G 58 63 Fr3 V V V V V 59 64 Fr3 P P P P P 60 65 Fr3 D D D D D 61 66 Fr3 R R R R R 62 67 Fr3 F F F F F 63 68 Fr3 S S S S S 64 69 Fr3 G G G G G 65 70 Fr3 S S S S S 66 71 Fr3 G G G G G 67 72 Fr3 S S S S S 68 73 Fr3 G G G G G 69 74 Fr3 T T T T T 70 75 Fr3 D D D D D 71 76 Fr3 F F F F F 72 77 Fr3 T T T T T 73 78 Fr3 L L L L L 74 79 Fr3 TO TO TO TO TO 75 80 Fr3 I I I I I 76 81 Fr3 s s s s s 77 82 Fr3 R R R R R 78 83 Fr3 V V V V V 79 84 Fr3 E E E E E 80 85 Fr3 A A A A A 81 86 Fr3 E E E E E 82 87 Fr3 D D D D D 83 88 Fr3 L V V V V 84 89 Fr3 G G G G G 85 90 Fr3 V V V V V 86 91 Fr3 Y Y Y Y Y 87 92 Fr3 Y Y Y Y Y

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88 93 Fr3 C c c c c 89 94 CDR-L3 L M M L L 90 95 CDR-L3 Q Q Q Q Q 91 96 CDR-L3 T G s T T 92 97 CDR-L3 T I I T T 93 98 CDR-L3 H H Q H H 94 99 CDR-L3 F L L F F 95 100 CDR-L3 P P P P P 95A CDR-L3 - - - - - 95V CDR-L3 - - - - - 95С CDR-L3 - - - - - 95D CDR-L3 - - - - - 95E CDR-L3 - - - - - 95F CDR-L3 - - - - - 96 101 CDR-L3 R L P R R 97 102 CDR-L3 T T T T T 98 103 Fr4 F F F F F 99 104 Fr4 G G G G G 100 105 Fr4 G G G G G 101 106 Fr4 G G G G G 102 107 Fr4 T T T T T 103 108 Fr4 TO TO TO TO TO 104 109 Fr4 L V V V V 105 110 Fr4 E E E E E 106 111 Fr4 I I I I I 106 A Fr4 - - - - - 107 112 Fr4 TO TO TO TO TO

Table 41. Back mutations VH, VL and other mutations for humanized 17C12

Option V _H or V _L Vh or Vl exon acceptor sequence Changes in acceptor framework residues (or CDRs) (based on composite CDR definitions by Kabat/Chothia) Hul7C12VHvl (SEQ Ш NO:232) GenBank accession number CAC20421-VH_huFrwk (SEQ ID NO:218) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) H2, H24, H48, H67, H69, H93, H94

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Hul7C12VHv2 (SEQ Ш NO:232) GenBank accession number CAC20421-VH_huFrwk (SEQ ID NO:218) IMGT No. IGHV1-69-2*01 (SEQ ID NO:33) Hl, H2, H24, H48, H67, H69, H93, H94, H108, H113 Hul7C12VLvl (SEQ Ш NO:233) GenBank accession number QDO16713-VL_huFrwk (SEQ ID NO:238) L2, L36 Hul7C12VLv2 (SEQ Ш NO:234) GenBank accession number QDO16713-VL_huFrwk (SEQ ID NO:238) IGKV2-29*02 and IGKJ4*01 (SEQ ID NO:239) L2, L36, L43

Table 42. Kabat numbering of framework residues (or CDRs) (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the heavy chains of humanized 17C12 antibodies

Remainder CAC20421-VH huFrwk (SEQ ID NO:218) IMGT No. IGHVl-69-2*01 (SEQ ID NO:33) VH murine 17C12 (SEQ ID NO:225) Hul7C12VHvl (SEQ ID NO:232) Hul7C12VHv2 (SEQ ID NO:233) Hl Q E TO Q E H2 V V I I I H24 V V A A A H48 M M I I I H67 V V A A A H69 I I M M M H93 A A T T T H94 R T T T T H108 T L L T L BOTTOM R S A R S

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Table 43. Kabat numbering of framework residues (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the light chains of humanized 17C12 antibodies

Remainder QDO16713-VLhuFrwk (SEQ ID NO:238) IGKV2-29*02 and IGKJ4*01 (SEQ ID NO:239) L2 I I L36 Y Y L43 P s

VL murine 17C12 (SEQ ID NO:228) Hul7C12VLvl (SEQ ID NO:234) Hul7C12VLv2 (SEQ ID NO:235) V V V L L L S P S

Table 44. Percentage indicator of the degree of humanization of heavy and light chains of humanized antibodies 17C12

Option V _H or V _L Degree Hul7C12VHvl (SEQ ID NO:232) 79.6% Hul7C12VHv2 (SEQ ID NO:233) 80.6% Hul7C12VLvl (SEQ ID NO:234) 85.0% Hul7C12VLv2 (SEQ ID NO:235) 86.0%

Justification for the choice of provisions indicated in table. 39, in the variable region of the heavy chain, the candidates for replacement are the following:

heavy chain variable regions hu17C12VHv1 consists of CDR-H1, H2 and H3 loops of 17C12-VH grafted onto a CAC20421 scaffold. In addition, hu17C12VHv1 also returns all scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability.

V2I: is a reverse mutation of the remainder of the Vernier zone to preserve the Vernier zone.

V24A: is a back mutation of a residue of the canonical Chothia structure, and the back mutation can improve stability.

M48I: is a reverse mutation of a Vernier zone residue.

V67A: is a back mutation of a Vernier zone residue and undergoes back mutation to maintain the CDR conformation.

I69M: is a back mutation of a Vernier zone residue, and the back mutation is made to preserve the integrity of the CDR packaging.

A93T: is a back mutation of the VH/VL interface residue and undergoes back mutation to maintain the integrity of the antibody interface.

R94T: is a reverse mutation of a canonical Chothia structural residue. R94T is also a germline-aligning mutation. Thr is found in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

hu17C12VHv2 hu17C12VHv2 also retains all returned scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability. In addition, hu17C12VHv2 includes back mutations or substitutions to the most abundant residue at a given position and to optimize deamination, oxidation, N-glycosylation, proteolysis, and aggregation. Mutations in hu17C12VHv2 that are not included in hu17C12VHv1:

Q1E: is a stability-enhancing mutation to reduce the potential for pyroglutamate formation and reduce N-terminal heterogeneity (Liu, above).

T108L: is a germline-aligning mutation. Leu is in the gene

- 151 047155 human germline IGHV1-69-2*01 (SEQ ID NO: 33) at this position.

R113S: is a frequency- and germline-aligning mutation. Arg at this position is rare, and Ser in the human germline gene IGHV1-69-2*01 (SEQ ID NO: 33) occurs at this position and is the most common residue at this position.

Justification for the choice of provisions indicated in table. 40, in the light chain variable region, candidates for replacement are as follows.

kappa light chain variable regions hu17C12VLv1 consists of the CDR-L1, L2 and L3 loops of 17C12-VL grafted onto the VL backbone of QDO16713 along with the return of all backbone substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone or located on the VH/VL domain interface.

I2V: is a back mutation of a Vernier zone residue and the back mutation is carried out to maintain the CDR packing conformation.

Y36L: is a back mutation of the remainder of the VH/VL interface and Vernier's area, and the back mutation is carried out to maintain the integrity of the interface.

hu17C12VLv2 hu17C12VLv2 includes substitutions that promote structural stability or increase the degree of humanization of the antibody.

P43S: is a germline frequency and alignment mutation. Pro at this position is rare and is replaced by Ser, which is present at this position in the human germline gene IGKV2-29*02 & IGKJ4*01 (SEQ ID NO: 239).

Based on these human scaffolds, the following variants have been developed: heavy chain variable regions >hul7C12VHvl

QIQLVQSGAEVKKPGATVKISCKASAFNIKDDYMNWVQQAPGKGLEWIGWIDPENGDT

KYASKFQGRATMTADTSTDTAYMELSSLRSEDTAVYYCTTSNGWGQGTTVTVSR >hul7C12VHv2

EVQLQQSGAELVKPGATVKISCTASGFNIKDDYMNWVKQRPEQGLEWIGWIDPENGDT

EYASKFQGKATMTADTSTNTAYLQLSSLTSEDTAVYYCTTSNGWGQGTTVTVSS kappa light chain variable regions >hul7C12VLvl

DWMTQTPLSLSVTPGQPASISCTSSQSLLHSNRKTYLHWLLQKPGQPPQLLIYLVSKLE

SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQTTHFPRTFGGGTKVEIK >hul7C12VLv2

DWMTQTPLSLSVTPGQPASISCTSSQSLLHSNRKTYLHWLLQKPGQSPQLLIYLVSKLE SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQTTHFPRTFGGGTKVEIK

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

Example 12: Construction of Humanized 14H3 Antibodies

The original or donor antibody for humanization was mouse antibody 14H3. The amino acid sequence of the mature m14113 heavy chain variable region is provided as SEQ ID NO: 240. The amino acid sequence of the mature m14113 light chain variable region is provided as SEQ ID NO: 244. The amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 according to the Kabat/Chothia composite definition are presented as SEQ ID NO: 241-243 respectively. The Kabat light chain amino acid sequences CDR1, CDR2 and CDR3 are provided as SEQ ID NO: 245-247, respectively. Kabat numbering is used throughout the text.

Variable kappa (Vk) 14H3 belongs to mouse Vk subgroup 2, which corresponds to human Vk subgroup 2, and variable heavy chain (Vh) belongs to mouse Vh subgroup 1b, which corresponds to human Vh subgroup 2 [Kabat E.A., et al ., (1991), Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242]. Chothia CDR-L1 is 16 residues similar to canonical Chothia class 4, Chothia CDR-L2 is 7 residues similar to canonical Chothia class 1, and Chothia CDR-L3 is 9 residues similar to canonical Chothia class 1 [Martin ACR. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany: Springer International Publishing AG.. [Martin, 2010].

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Chothia CDR-H1 is 12 residues similar to canonical Chothia class 3, Chothia CDR-H2 is 16 residues similar to canonical Chothia class 1 [Martin, 2010], and Chothia CDR-H3 is 4 residues and has no canonical classes. A search was conducted for protein sequences in the PDB database [Deshpande N, et al., (2005) Nucleic Acids Res. 33: D233-7] to find structures that would provide an approximate structural model of 14H3. Crystal structure of antibody fab with pdb code 2VQ1 [De Geus, D.C., et al. Proteins 76: 439; (2009)] was used for both the Vh and Vk structures because it had good resolution (2.5A°) and overall sequence similarity to Vh and Vk of 14H3, retaining the same canonical structures for the loops.

The 14H3 VH scaffolds have a high degree of similarity to the corresponding regions of the immunoglobulin heavy chain variable region [Homo sapiens] QDJ57937 cloned by Cassotta et al. [(2009) Cassotta, A., Mikol, V., Bertrand, T., et al. A single T cell epitope drives the neutralizing anti-idiotypic antibody response to natalizumab in patients with multiple sclerosis Unpublished (direct submission to GenBank (2019)]. The VH 14H3 and QDJ57937 variable domains also have the same length with respect to the CDRH1, H2 loops. Exactly The 14H3 VL frameworks have a high degree of sequence similarity to the corresponding regions of the human antibody ABC66914, cloned by Shriner et al. [Vaccine 24 (49-50), 7159-7166 (2006)]. for the CDR-L1, L2 and L3 loops. Accordingly, the framework regions of both the VH of QDJ57937 and the VL of ABC66914 were selected as acceptor sequences for the 14H3 CDRs grafted onto the corresponding human scaffolds for the VH and VL were constructed and used in. as a guide for further back mutations.

Heavy and light chain variant sequences resulting from the antibody humanization process were further aligned to human germline sequences using the IMGT Domain GapAlign tool to assess the degree of humanization of the heavy and light chains, as specified in the WHO INN committee guidelines. (WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review) (Internet) 2014. Available at: https://www.who.int/medicines/services/inn/BioRev2014.pdf) Residues have been modified to align with the corresponding human germline sequence where possible to enhance humanization and reduce potential immunogenicity. For the humanized variants VLv1 and VLv2, mutations were introduced to make the sequences more similar to the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37). For the humanized variants VHv1 and VHv2, mutations were introduced to make the sequences more similar to the human germline gene IGHV1-70*04 and IGHJ4*01 (SEQ ID NO: 254). Additional versions hu14H3-VH and hu14H3-VL were engineered to enable assessing various scaffold residues for their contribution to antigen binding, thermostability and immunogenicity, and to optimize deamination, oxidation, N-glycosylation, proteolysis and aggregation. Positions considered for mutation include those that define canonical CDR conformations (summarized in Martin, A.C.R. (2010) Protein sequence and structure analysis of antibody variable domains. B: Kontermann R and Dubel S (eds). Antibody Engineering. Heidelberg, Germany : Springer International Publishing AG.), are located in the Vernier zone (Foote J and Winter G. (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99.), localized on VH/VL domain interface (summarized in Leger OJP and Saldanha J. (2000) Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. B: Shepherd P and Dean C (eds). Monoclonal Antibodies: a Practical Approach. Oxford, UK: Oxford University Press.), are sensitive to post-translational modifications such as glycosylation or pyroglutamination, are occupied by residues that are predicted to conflict with CDRs, according to the 14H3 CDR model, grafted onto VH and VL scaffolds, or occupied by residues that are rare among sequenced human antibodies, where either the parental mouse 14H3 residue or some other residue is much more common in the human antibody repertoire.

Alignments of murine 14H3 and various humanized antibodies are shown for light chain variable regions (Table 46 and FIG. 14) and heavy chain variable regions (Table 45 and FIG. 13).

2 humanized heavy chain variable region variants and 2 humanized light chain variable region variants were constructed containing different substitution permutations: hu14H3VHv1 or hu14H3VHv2, (SEQ ID NO: 248-249, respectively); and hu14H3VLv1 or hu14H3VLv2, (SEQ ID NO: 250-251, respectively) (Tables 45 and 46). Examples of humanized Vk and Vh constructs with back mutations and other mutations based on selected human scaffolds are shown in Table 1. 45 and 46 respectively. Areas highlighted in bold in the table. 45 and 46, denote CDR, according to the composite definition according to Kabat/Chothia. Sign - in the columns of the table. 45

- 153 047155 and 46 indicates the absence of residue in the indicated position. SEQ ID NO: 248-249 and SEQ ID NO: 250251 contain back mutations and other mutations, as shown in table. 47. The amino acid positions in hu14H3VHv1 and hu14H3VHv2 are listed in Table. 48 The amino acid positions in hu14H3VLv1 and hu14H3VLv2 are listed in Table. 49

Percentage degree of humanization for humanized VH chains hu14H3VHv1 and hu14H3VHv2 (SEQ ID NO: 248-249, respectively) relative to the most similar human germline gene IGHV1-70*04 and IGHJ4*01 (SEQ ID NO: 254), and for humanized VL chains hu14H3VLv1 and hu14H3VLv2 (SEQ ID NO: 250-251, respectively) in relation to the most similar human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37), shown in table. 50.

Table 45

Balance according to Kabat No. Linear remainder no. FR or CDR VH murine 14H3 (SEQ ID NO:240) IGHV2-70*04 and IGHJ4*01 (SEQ ID NO:254) QDJ57937VH hFrwk (SEQ ID NO:x253 Hu4H3VHvl (SEQ ID NO:248) Hul4H3VHv2 (SEQ ID NO:249) 1 1 Frl Q Q Q Q Q 2 2 Frl V V V V V 3 3 Frl T T T T T 4 4 Frl L L L L L 5 5 Frl TO TO TO TO TO 6 6 Frl E E E E E 7 7 Frl S S S S S 8 8 Frl G G G G G 9 9 Frl P P P P P 10 10 Frl G A A A A AND AND Frl I L L L L 12 12 Frl L V V V V

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13 13 Frl Q To TO TO To 14 14 Frl P P P P P 15 15 Frl s T T T T 16 16 Frl Q Q Q Q Q 17 17 Frl T T T T T 18 18 Frl L L L L L 19 19 Frl S T T T T 20 20 Frl L L L L L 21 21 Frl T T T T T 22 22 Frl C C C C C 23 23 Frl s T T T T 24 24 Frl F F F F F 25 25 Frl S S S S S 26 26 CDR-H1 G G G G G 27 27 CDR-H1 F F F F F 28 28 CDR-H1 S S S S S 29 29 CDR-H1 L L L L L thirty thirty CDR-H1 S S S S S 31 31 CDR-H1 T T T T T 32 32 CDR-H1 Y s s Y Y 33 33 CDR-H1 G G G G G 34 34 CDR-H1 M M M M M 35 35 CDR-H1 G R R G G 35A 36 CDR-H1 V V V V V 35V 37 CDR-H1 G s s s s 36 38 Fr2 W w w w w 37 39 Fr2 I I I I I 38 40 Fr2 R R R R R 39 41 Fr2 Q Q Q Q Q 40 42 Fr2 P P P P P 41 43 Fr2 s P P P P 42 44 Fr2 G G G G G 43 45 Fr2 TO TO TO TO TO 44 46 Fr2 G A A A A 45 47 Fr2 L L L L L 46 48 Fr2 E E E E E 47 49 Fr2 W W W W W 48 50 Fr2 L L L L L 49 51 Fr2 A A A A A 50 52 CDR-H2 N R R N N 51 53 CDR-H2 I I I I I 52 54 CDR-H2 w D D w w

- 155 047155

52A CDR-H2 - - - - - 52V CDR-H2 - - - - - 52C CDR-H2 - - - - - 53 55 CDR-H2 W W W W W 54 56 CDR-H2 D D D D D 55 57 CDR-H2 D D D D D 56 58 CDR-H2 I D D I I 57 59 CDR-H2 TO TO TO TO TO 58 60 CDR-H2 Y F F Y Y 59 61 CDR-H2 Y Y Y Y Y 60 62 CDR-H2 N S S N N 61 63 CDR-H2 A T T A A 62 64 CDR-H2 A s s A A 63 65 CDR-H2 L L L L L 64 66 CDR-H2 TO TO TO TO TO 65 67 CDR-H2 S T T S S 66 68 Fr3 R R R R R 67 69 Fr3 L L L L L 68 70 Fr3 T T T T T 69 71 Fr3 I I I I I 70 72 Fr3 s s s s s 71 73 Fr3 To To To To To 72 74 Fr3 D D D D D 73 75 Fr3 T T T T T 74 76 Fr3 s s s s s 75 77 Fr3 To To To To To 76 78 Fr3 N N N N N 77 79 Fr3 Q Q Q Q Q 78 80 Fr3 V V V V V 79 81 Fr3 F V V V V 80 82 Fr3 L L L L L 81 83 Fr3 TO T T T T 82 84 Fr3 I M M M M 82A 85 Fr3 A T T T T 82V 86 Fr3 S N N N N 82С 87 Fr3 V M M M M 83 88 Fr3 D D D D D 84 89 Fr3 T P P P P 85 90 Fr3 A V V V V 86 91 Fr3 D D D D D 87 92 Fr3 T T T T T 88 93 Fr3 A A A A A

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

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Table 46

Balance according to Kabat No. Linear remainder no. FR or CDR VL murine 14H3 (SEQ ID NO:244) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) ABC66914VL hFwrk (SEQ ID NO:256) Hul4H3VLvl (SEQ ID NO:250) Hul4H3VLv2 (SEQ ID NO:251) 1 1 Frl D D D D D 2 2 Frl V I I V V 3 3 Frl V V V V V 4 4 Frl M M M M M 5 5 Frl T T T T T 6 6 Frl Q Q Q Q Q 7 7 Frl T s T T s 8 8 Frl P P P P P 9 9 Frl L L L L L 10 10 Frl s s s s s AND AND Frl L L L L L 12 12 Frl P P P P P 13 13 Frl V V V V V 14 14 Frl s T T T T 15 15 Frl L P P P P 16 16 Frl G G G G G 17 17 Frl D E E E E 18 18 Frl Q P P P P 19 19 Frl A A A A A 20 20 Frl S S S S S 21 21 Frl I I I I I

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22 22 Frl S S S S S 23 23 Frl C c c c c 24 24 CDR-L1 R R R R R 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 27V 29 CDR-L1 L L L L L 27C thirty CDR-L1 V L L V V 27D 31 CDR-L1 H H H H H 27E 32 CDR-L1 s S S s s 27F CDR-L1 - - - - - 28 33 CDR-L1 N N N N N 29 34 CDR-L1 G G G G G thirty 35 CDR-L1 N Y Y N N 31 36 CDR-L1 T N N T T 32 37 CDR-L1 F Y Y F F 33 38 CDR-L1 L L L L L 34 39 CDR-L1 H D D H H 35 40 Fr2 W W W W W 36 41 Fr2 Y Y Y Y Y 37 42 Fr2 L L L L Q 38 43 Fr2 Q Q Q Q Q 39 44 Fr2 To To To To To 40 45 Fr2 P P P P P 41 46 Fr2 G G G G G 42 47 Fr2 Q Q Q Q Q 43 48 Fr2 s s s s s 44 49 Fr2 P P P P P 45 50 Fr2 To Q Q Q Q 46 51 Fr2 L L L L L 47 52 Fr2 L L L L L 48 53 Fr2 I I I I I 49 54 Fr2 Y Y Y Y Y 50 55 CDR-L2 To L L To To 51 56 CDR-L2 V G G V V 52 57 CDR-L2 s S S s s 53 58 CDR-L2 N N N N N 54 59 CDR-L2 R R R R R 55 60 CDR-L2 F A A F F 56 61 CDR-L2 S S S S S 57 62 Fr3 G G G G G

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58 63 Fr3 V V V V V 59 64 Fr3 P P P P P 60 65 Fr3 D D D D D 61 66 Fr3 R R R R R 62 67 Fr3 F F F F F 63 68 Fr3 S S s S S 64 69 Fr3 G G G G G 65 70 Fr3 S S S S S 66 71 Fr3 G G G G G 67 72 Fr3 S S S S S 68 73 Fr3 G G G G G 69 74 Fr3 T T T T T 70 75 Fr3 D D D D D 71 76 Fr3 F F F F F 72 77 Fr3 T T T T T 73 78 Fr3 L L L L L 74 79 Fr3 TO TO TO TO TO 75 80 Fr3 I I I I I 76 81 Fr3 S s S S s 77 82 Fr3 R R R R R 78 83 Fr3 V V V V V 79 84 Fr3 E E E E E 80 85 Fr3 A A A A A 81 86 Fr3 E E E E E 82 87 Fr3 D D D D D 83 88 Fr3 L V V V V 84 89 Fr3 G G G G G 85 90 Fr3 V V V V V 86 91 Fr3 Y Y Y Y Y 87 92 Fr3 F Y Y F F 88 93 Fr3 C c c C C 89 94 CDR-L3 s M M s s 90 95 CDR-L3 Q Q Q Q Q 91 96 CDR-L3 s A A s s 92 97 CDR-L3 T L L T T 93 98 CDR-L3 L Q Q L L 94 99 CDR-L3 V T T V V 95 100 CDR-L3 P P P P P 95A CDR-L3 - - L - - 95V CDR-L3 - - T - - 95C CDR-L3 - - - - - 95D CDR-L3 - - - - -

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95E CDR-L3 - - - - - 95F CDR-L3 - - - - - 96 101 CDR-L3 W Y L w w 97 102 CDR-L3 T T T T T 98 103 Fr4 F F F F F 99 104 Fr4 G G G G G 100 105 Fr4 G Q G G Q 101 106 Fr4 G G G G G 102 107 Fr4 T T T T T 103 108 Fr4 TO TO TO TO TO 104 109 Fr4 L L V V L 105 110 Fr4 E E E E E 106 111 Fr4 I I I I I 106 A Fr4 - - - - - 107 112 Fr4 TO TO TO TO TO

Table 47. Back mutations VH, VL and other mutations for humanized 14H3

Option V _H or Vl Vh or Vl exon acceptor sequence Changes in acceptor framework residues (or CDRs) (based on composite CDR definitions by Kabat/Chothia) Hul4H3VHvl (SEQ Ш NO:248 GenBank accession number QDJ57937VH hFrwk (SEQ ID NO:253) IGHV2-70*04 and IGHJ4*01 (SEQ ID NO:254) Н35В Hul4H3VHv2 (SEQ Ш NO:249) GenBank accession number QDJ57937VH hFrwk (SEQ ID NO:253) IGHV2-70*04 and IGHJ4*01 (SEQ ID NO:254) H35B, H108, NIZ Hul4H3VLvl (SEQ Ш NO:250) GenBank Accession Number ABC66914VL_hFwrk (SEQ ID NO:256) L2, L87 Hul4H3VLv2 (SEQ ID NO:251) GenBank Accession Number ABC66914VL_hFwrk (SEQ ID NO:256) IGKV2-28*01 and IGKJ2*01 (SEQ ID NO:37) L2, L7, L37, L87, L100, L104

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Table 48. Kabat numbering of framework residues (or CDRs) (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the heavy chains of humanized 14H3 antibodies

Remainder QDJ57937VH hFrwk (SEQ ID NO:253) IGHV2-70*04 and IGHJ4*01 (SEQ ID NO:254) VH murine 14H3 (SEQ ID NO:240) Hul4H3VHvl (SEQ ID NO:248) Hul4H3VHv2 (SEQ ID NO:249) Н35В S S G S S H108 M L T M L Refinery L S S L S

Table 49. Kabat numbering of framework residues (based on CDRs according to the Kabat/Chothia composite definition) for back mutations and other mutations in the light chains of humanized 14H3 antibodies

Remainder AlitbbV 14 V Lill W1K (M-O ID NO:256) 1GKV2-28 ^X U1 and 1GKJ2 ^X U1 (SEQ ID NO:37) VL· mouse 14±ιό (Μίψ Ш ΝΟ:χ244) Hul4H3VLvl (SEQ ID NO:250) Hul4H3VLv2 (SEQ ID NO:251) L2 I I V V V L7 T s T T s L37 L L L L Q L87 Y Y F F F L100 G Q G G Q L104 V L L V L

Table 50. Percentage indicator of the degree of humanization of heavy and light chains of humanized 14H3 antibodies

Option Vh or Vl Degree Hul4H3VHvl (SEQ ID NO:248) 89.9% Hul4H3VHv2 (SEQ ID NO:249) 89.9% Hul4H3VLvl (SEQ ID NO:250) 85.0% Hul4H3VLv2 (SEQ ID NO:251) 85.0%

Positions at which canonical, vernier, or interface residues differ between mouse and human acceptor sequences are candidates for replacement. Examples of interacting canonical/CDR residues include the Kabat L2 residue in Table 1. 46. Examples of vernier residues include the Kabat L2 residue in Table. 46. Examples of interface/package residues (VH+VL) include Kabat L87 residue in Table. 46.

Justification for the choice of provisions indicated in table. 45, in the heavy chain variable region, candidates for replacement are as follows:

heavy chain variable regions hu14H3VHv1 consists of the CDR-H1, H2 and H3 loops of 14H3-VH grafted onto a QDJ57937-VH scaffold. Additionally contains a replacement for G35bS in CDR-H1

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G35bS: is a mutation of the CDR residue. The residue at position H35b is not expected to be in direct contact with the antigen based on the model. The human germline gene IGHV1-70*04 and IGHJ4*01 (SEQ ID NO: 254) have Ser at this position. L35bS is a germline-aligning mutation.

hu14H3VHv2 returns all scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, or contribute to structural stability. hu14H3VHv2 includes back mutations or substitutions at various positions listed below, allowing the contribution of these positions to antigen binding affinity and immunogenicity to be assessed;

returns all framework substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or are localized at the VH/VL domain interface, or contribute to structural stability. hu14H3VHv2 includes back mutations or substitutions at various positions listed below, allowing the contribution of these positions to antigen binding affinity and immunogenicity to be assessed. In addition, it includes back mutations or substitutions with the most common residue at the specified position.

M108L: is a germline-aligning mutation. Leu is found in the human germline gene IGHV1-70*04 and IGHJ4*01 (SEQ ID NO: 254) at this position.

L113S: is a germline-aligning mutation. Ser is found in the human germline gene IGHV1-70*04 and IGHJ4*01 (SEQ ID NO: 254) at this position.

Justification for the choice of provisions indicated in table. 46, in the light chain variable region, candidates for replacement are as follows:

kappa light chain variable regions hu14H3VLv1 consists of the CDR-L1, L2 and L3 loops of 14H3-VL grafted onto the VL backbone of ABC66914. In addition, hu14H3VLv1 also returns all scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or localize to the VH/VL domain interface, and contribute to structural stability.

I2V: is a reverse mutation of a residue of the canonical Chothia structure to preserve the canonical structure.

Y87F: is a reverse mutation of the VH/VL interface residue to preserve the heavy chain: light chain interface.

hu14H3VLv2

Stores all returned scaffold substitutions at positions that are key to defining canonical Chothia classes, are part of the Vernier zone, or reside in the VH/VL domain interface. In addition, hu14H3VLv2 includes back mutations or substitutions to the most common residue at a given position, which increases the marketability of the antibody. Mutations in hu14H3VLv2 that are not included in hu14H3VLv1:

T7S: is a germline straightening mutation. Ser is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

L37Q: Leu at this position is immunogenic, so it is replaced with Gln to reduce immunogenicity. L37Q is also a high frequency mutation. Gln is the most common residue at this position.

G100Q: is a germline-aligning mutation. Gln is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

V104L: is a germline-aligning mutation. Leu is found in the human germline gene IGKV2-28*01 and IGKJ2*01 (SEQ ID NO: 37) at this position.

The following variants have been developed from these human scaffolds: heavy chain variable regions >hul4H3VHvl

Q VTLKESGPALVKPTQTLTLTCTF SGF SLST YGMGVSWIRQPPGKALEWLANIWWDDIK

YYNAALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARNVDYWGQGTMVTVSL >hul4H3VHv2

Q VTLKESGPALVKPTQTLTLTCTF SGF SLST YGMGVSWIRQPPGKALEWLANIWWDDIK

YYNAALKSRLTISKDTSKNQVVLMTTNMDPVDTATYYCARNVDYWGQGTLVTVSS kappa light chain variable regions

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DWMTQTPLSLPVTPGEPASISCRSSQSLVHSNGNTFLHWYLQKPGQSPQLLIYKVSNRF

SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTLVPWTFGGGTKVEIK >hul4H3VLv2

DWMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTFLHWYQQKPGQSPQLLIYKVSNRF

SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTLVPWTFGQGTKLEIK

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

Example 13 Mouse antibody 9F5, mouse antibody 10C12, mouse antibody 12C4 and mouse antibody 2D11 prevent tau toxicity in primary neurons.

Embryonic day 16–17 cortical neurons were obtained from C57B16/J mouse fetuses as described in Pillot et al., 1999; Kriem et al., 2005; and Garcia et al., 2010. Briefly, dissociated cortical cells were seeded into 48-well plates (50,000 cells/well) precoated with 1.5 μg/mL polyornithine (Sigma). Cells were cultured in Dulbecco's modified Eagle's medium/P12, serum-free and supplemented with hormones, proteins, and salts. Cultures were stored at 35°C in a humidified atmosphere using 6% CO ₂ .

All treatments were performed in triplicate on days 6–7 in vitro (DIV). Neurons were incubated with either vehicle or human tau oligomers (final concentration 1 μM on a monomer basis) in the presence of 5 increasing concentrations of the indicated antibody for 24 h and in a final volume of 140 μl per well.

After incubation, neuronal viability was measured using 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) using published protocols (e.g., Mosmann et al., 1983 ). Cells were incubated at 35°C for 1 h using MTT. For this purpose, MTT was solubilized in PBS at a concentration of 5 mg/ml. 14 μL of MTT solution was added to each well. After incubation, the medium was removed and the cells were lysed using 150 μl of DMSO for 10 min, protected from light. After complete solubilization of the formazan product, the absorbance was determined at 570 nm. Viability in vehicle control was determined to be 100%. Data were expressed as % viability in vehicle control. The results are presented in table. 57 and in fig. 16.

Table 57. MTT assay using 9F5, 10C12, 2D11 and 12C4

All values indicate cell viability (% of control cells not treated with tau oligomers) ± SD 9F5 10С12 2D11 12С4 Quantity antibodies (molar equivalents) 5 93.1±1.5 86.8±1.7 92.2±1.1 81.1±4.5 3 92.6±3.4 69.2±3 64±3.6 78.7±2.1 1 64.9±4.5 56.4±2.6 50.9±2.4 62.3±2.9 0.33 58±2.6 51.3±2.4 59.5±2.3 53.2±4.5 0.2 47.4±1.6 47.1±2.1 52.8±1.1 48.6±2.1 0 48.9±2.8 48.9±1.9 50.9±2.6 50.9±3.4

All four antibodies demonstrated the ability to protect neurons from tau-induced toxicity. It was noted that 9F5 showed higher activity with the ability to completely inhibit toxicity at a lower concentration than other antibodies.

The release of lactate dehydrogenase (LDH) is an indicator of cell death. Decreased LDH levels indicate reduced cell death as a result of reduced internalization of tau. To measure lactate dehydrogenase (LDH) release, culture medium (110 μL) from each well was transferred to a 1.5 mL Eppendorf tube and replaced with fresh MTT assay medium. The collected medium was centrifuged at 800 g for 5 min, and the supernatant (100 μl of cell-free culture medium) was transferred to a 48-well plate, which was stored at 4°C, protected from light, for further analysis. Quantitative determination of LDH in the culture medium was carried out in accordance with the manufacturer's recommendations (cytotoxicity test kit [LDH], Roche part no. 11 644 793 001). The results are presented in table. 58 and in fig. 17.

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Table 58. LDH analysis using 9F5, 10C12, 2D11 and 12C4

All values indicate LDH release (% of control cells not treated with tau oligomers) ± SD 9F5 10С12 2D11 12С4 Amount of antibody (molar equiv.) 5 91.4±5.1 95.1±3.9 115.4±4.5 108.2±5.2 3 95.9±2.9 101.1±5.8 109±3.8 109.9±8.2 1 116.0±2.6 109±1.1 131.1±3.3 123.7±7.5 0.33 151.5±5.9 128.6±7.6 147.7±5.3 151.9±7.1 0.2 155.4±3.8 140±2.7 177±3.0 153.4±10.3 0 158.1±4.5 145.1±3.9 159.4±7.8 160.1±4.1

All four antibodies demonstrated the ability to protect neurons from tau-induced toxicity. It was noted that 9F5 showed higher activity with the ability to completely inhibit toxicity at a lower concentration than other antibodies and even reduced LDH release to below baseline levels at the highest added concentration.

Links

Pillot, T., Drouet, V., Queille, S., et al.: The nonfibrillar amyloid beta-peptide induces apoptotic neuronal cell death: involvement of its C-terminal fusogenic domain. J Neurochem. 73(4): 1626-34 (1999).

Kriem, V., Sponne, I, Fifre, A., et al.: Cytosolic phospholipase A2 mediates neuronal apoptosis induced by soluble oligomers of the amyloid-beta peptide. FASEB J. 19(l):85-7 (2005).

Garcia, P., Youssef, I, Utvik, J.K., et al.: Ciliary neurotrophic factor cell-based delivery prevents synaptic impairment and improves memory in mouse models of Alzheimer's disease. J Neurosci. 30(22): 7516-27 (2010).

Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods., 65, 55-63 (1983).

Example 14: Surface plasmon resonance (SPR) analysis of mouse antibody 9F5, mouse antibody 10C12, mouse antibody 14H3, mouse antibody 17C12, mouse antibody 2D11 and mouse antibody 12C4

Surface plasmon resonance (SPR) analysis using Biacore was performed by immobilizing anti-mouse antibody (GE Lifesciences) onto a CM3 chip using EDC/NHS. Antibodies were then captured onto the anti-mouse surface to equivalent levels. Regeneration was carried out through 2 injections of 10 mM glycine with pH 1.7 for 30 s. Various concentrations ranging from 500 to 0.122 nM of 3-fold dilutions of recombinant human tau 4R0N were passed through the sensor during a 150-second association phase and a 300-second dissociation phase. Blank values were subtracted from the data for both the irrelevant probe not containing the test antibody to account for irrelevant binding to the prepared probe surface and the mobile phase vehicle to account for the dissociation of the test antibody from the uptake of the anti-mouse antibody. The data were then analyzed using built-in scoring software using a 1:1 overall fit. The results are presented in table. 51. All antibodies showed similar binding kinetics with Kd values within 200 pM of each other; The kinetic profiles were characterized by phases of fast association and slow dissociation.

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Table 51. Kinetics parameters of mouse antibody 9F5, mouse antibody 10C12, mouse antibody 14H3, mouse antibody 17C12, mouse antibody 2D11 and mouse antibody 12C4

Antibody k _a (M'S* ¹ ) kdK ¹ ) _Kd (M) 9F5 6.39e+6 2.24e-3 3.50e-10 10С12 6.53e+6 2.45e-3 3.75e-10 14NZ 7.60e+6 1.53e-3 2.02e-10 17С12 4.03e+6 1.94e-3 4.82е-10 2D11 6.93е+6 2.21e-3 3.18e-10 12С4 5.69е+6 2.06e-3 3.63e-10

Example 15 Mouse 9F5, Mouse 10C12, Mouse 14H3, Mouse 17C12, Mouse 2D11, and Mouse 12C4 detect tau in samples from Alzheimer's disease patients: Western blot analysis

Fresh frozen frontal cortex samples were obtained from the brains of one healthy control (HC) and four patients with Alzheimer's disease (AD). Samples were homogenized in 10 volumes of RIPA buffer containing protease inhibitors and centrifuged for 15 min at 16,000 x g at 4°C. The supernatants were removed to obtain the soluble fraction. The pellets were resuspended in 1xPBS containing 1% sarkosyl and homogenized. Sarcosyl homogenates were centrifuged for 60 min at 100,000 × g at 4°C, and supernatants and pellets were separated. The beads were resuspended in one volume of 2% SDS to obtain the insoluble fraction. The resulting soluble and insoluble fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies at a concentration of 0.5 μg/ml, washed and probed with goat anti-mouse secondary antibodies conjugated to IRDye-800 (LiCor). After washing, the blots were scanned using a LiCor scanner. The results are presented in Fig. 18. All antibodies specifically detected tau contained in insoluble fractions obtained from AD patients and detected tau in soluble fractions to varying degrees.

Example 16 Immunoprecipitation assay of an insoluble extract obtained from a patient with Alzheimer's disease using mouse antibody 9F5, mouse antibody 10C12, mouse antibody 14H3, mouse antibody 17C12, mouse antibody 2D11 and mouse antibody 12C4

To test the ability of the antibodies to capture tau aggregates, a sarkosyl-insoluble fraction obtained from one patient with Alzheimer's disease (AD) and one normal control was incubated with the indicated antibodies. Goat anti-mouse antibody-conjugated magnetic beads (Life Technologies) were added to the antibody extract mixture to capture antibody/antigen complexes. The beads were washed thoroughly, boiled in SDS-PAGE buffer containing DTT, and the beads were then removed. The resulting fractions were separated by SDS-PAGE electrophoresis and immunoblotted using the polyclonal antibody K9JA to detect all immunocaptured tau species. The results are presented in Fig. 19. Alternating bands represent immunoprecipitates from normal and AD tissue extracts. Molecular mass markers are indicated on the left. All antibodies captured insoluble tau obtained from AD patients to varying degrees and demonstrated the ability to capture large, high-mass insoluble aggregates. Of the antibodies tested, 9F5, 2D11, 17C12, 10C12 and 12C4 showed similar profiles, with strong immunoreactivity present in the 50-60 kDa range as well as in the concentrating gel. Capture of 14H3 also revealed similar, although less abundant, tau species. These data indicate that all antibodies tested are able to bind a similar array of forms of tau.

Example 17 Ability of Humanized 9F5 Variants to Withstand Mixing Stress

The marketability characteristics of the humanized 9F5 variants were tested for their ability to resist aggregation caused by agitation stress.

Antibodies were replaced with a buffer in histidine buffer (25 mM histidine, pH 6) at a concentration of 1 mg/ml and stirred at 1500 rpm at room temperature for 48 hours. Samples were taken after 0, 24 and 48 hours, filtered through 0.22 -micron filter and analyzed by analytical size exclusion chromatography (SEC). For each antibody, the total major peak area for each time point was normalized to the major peak area at t=0 h, and antibodies were ranked based on loss of antibody to aggregation over time. The results are presented in Fig. 20 and at

- 166 047155 table. 52. Abbreviations of the names of the humanized variants of 9F5 used in FIG. 20 correspond to the description given in the second column of the table. 52. Although no single point mutation can cause sensitivity to agitation stress, some combinations of mutations result in a lack of resistance. It should be noted that the combinations L27cG/L37G/M51G/L54R/L92I (DIM11), L27cS/L37G/M51GL54T (DIM17) and L27cG/L37Q/M51G/L54R (DIM5) lead to increased sensitivity to stirring, while combinations for example, L27cS/L37Q/M51G/L54R (DIM2), L27cG/L37G/M51G/L54T (DIM14) and L27cG/L37G/M51G/L54R (DIM13) lead to resistance to agitation stress. These results suggest the creation of specific combinations of residues in humanized variants that are particularly likely to withstand the rigors of production, processing and transportation.

Table 52: Ability of Humanized 9F5 Variants to Withstand Mixing Stress

Rank Humanized variant 9F5 Monomer with shaking for 24 hours (% t=0) Monomer with shaking for 48 hours (% t=0) 1 DIM2 [hu9F5VHv9/hu9F5VLv8_DIM2 SEQ ID NO: 127/ SEQ ID NO: 133] 100.6 103.3 2 DIMM [hu9F5VHv9/hu9F5VLv8_DIM14 SEQ ID NO: 127/ SEQ ID NO: 145] 100.1 107.1 3 DIM13 [hu9F5VHv9/hu9F5VLv8_DIM13 SEQ ID NO: 127/ SEQ ID NO: 144] 105.8 105.2 4 DIM7 [hu9F5VHv9/hu9F5VLv8_DIM7 102.3 105

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SEQ ID NO: 127/ SEQ Ш NO: 138] 5 DIM6 [hu9F5VHv9/hu9F5VLv8_DIM6 SEQ ID NO: 127/ SEQ Ш NO: 137] 98.8 104.6 6 DIM12 [hu9F5VHv9/hu9F5VLv8_DIM12 SEQ ID NO: 127/ SEQ Ш NO: 143] 98.2 103.8 7 DIM18 [hu9F5VHv9/hu9F5VLv8_DIM18 SEQ ID NO: 127 / SEQ ID NO: 149] 100.3 101.3 8 DIM30 [hu9F5VHv9/hu9F5VLv8_DIM30 SEQ ID NO: 127/ SEQ Ш NO: 161] 100 101.2 9 DIM8 [hu9F5VHv9/hu9F5VLv8_DIM8 SEQ ID NO: 127/ SEQ Ш NO: 139] 98.8 100.1 10 DIM28 [hu9F5VHv9/hu9F5VLv8_DIM28 SEQ ID NO: 127/ SEQ Ш NO: 159] 97.1 99.5 AND DIM29 [hu9F5VHv9/hu9F5VLv8_DIM29 SEQ ID NO: 127/ SEQ Ш NO: 160] 97.2 98.2 12 DIM27 [hu9F5VHv9/hu9F5VLv8_DIM27 SEQ ID NO: 127/ SEQ Ш NO: 158] 97.1 95.2 13 DIM5 [hu9F5VHv9/hu9F5VLv8_DIM5 SEQ ID NO: 127/ SEQ Ш NO: 136] 93.1 93.9 14 DIM17 [hu9F5VHv9/hu9F5VLv8_DIM17 SEQ ID NO: 127/ SEQ Ш NO: 148] 97.1 91.6 15 H10L9-DIM11 [hu9F5VHvlO/hu9F5VLv9_DIMl 1 SEQ ID NO: 128/ SEQ Ш NO: 168] 96.6 85.8 16 DIM11 [hu9F5VHv9/hu9F5VLv8_DIMl 1 SEQ ID NO: 127 / SEQ ID NO: 142] 97.4 82.4

Example 18 Ability of Humanized 9F5 Variants to Withstand Low pH Stress

The marketability characteristics of the humanized 9F5 variants were tested for their ability to withstand exposure to low pH levels as occurs during the viral inactivation steps of the manufacturing process. Antibodies were buffer exchanged in acetate buffer (10 mM acetate, pH 3) at a concentration of 1 mg/ml and incubated at room temperature for 6 h. Samples were collected at 0, 2, and 6 h, filtered through a 0.22-μm filter, and analyzed by analytical size exclusion chromatography (EC). For each antibody, the total major peak area for each time point was normalized to the major peak area at t=0 h, and antibodies were ranked based on loss of antibody to aggregation over time. The results are presented in Fig. 21 and in table. 53. Abbreviations for the names of the 9F5 humanized variants used in FIG. 21 correspond to the description given in the second column of the table. 53. Combinations L27cG/L37G/M51G/L54R/L92I (DIM11), L27cG/L37Q/M51G/L54R (DIM5) and L27cG/L37G/M51G/L54T (DIM14) lead to increased sensitivity to low pH stress, o as evidenced by a >5% reduction in the monomer peak at 6 h, while combinations such as L27cS/L37Q/M51G/L54R (DIM2), L27cD/L37Q/M51G/L54R (DIM6) and L27cG/L37G/M51G/L54R (DIM13) lead to resistance to stress caused by low pH levels. It is noteworthy that inclusion of the L27cD mutation resulted in the emergence of low pH-resistant variants DIM18, DIM6 and DIM7. These results suggest the creation of specific combinations of residues in humanized variants that are particularly likely to withstand the rigors of production, particularly the inclusion of low pH incubation typically performed during purification processes.

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Table 53. Ability of humanized 9F5 variants to withstand low pH stress

Rank Option h9F5 Monomer after 2h exposure pH 3 (% t=0) Monomer after 6 hours of exposure to pH 3 (% t=0) 1 DIM6 [hu9F5VHv9/hu9F5VLv8_DIM6 SEQ ID NO: 127/ SEQ ID NO: 137] 100.8 100.6 2 DIM2 [hu9F5VHv9/hu9F5VLv8_DIM2 100.6 99.6 SEQ ID NO: 127/ SEQ ID NO: 133] 3 DIM13 [hu9F5VHv9/hu9F5VLv8_D]IM13 SEQ ID NO: 127/ SEQ ID NO: 144 97.4 99.3 4 DIM8 [hu9F5VHv9/hu9F5VLv8_DIM8 SEQ ID NO: 127/ SEQ ID NO: 139] 99.2 99.2 5 DIM7 [hu9F5VHv9/hu9F5VLv8_DIM7 SEQ ID NO: 127/ SEQ ID NO: 138] 99.6 98.9 6 DIM28 [hu9F5VHv9/hu9F5VLv8_DIM28 SEQ ID NO: 127/ SEQ ID NO: 159] 99.1 98.9 7 DIM17 [hu9F5VHv9/hu9F5VLv8_DIM17 SEQ ID NO: 127/ SEQ ID NO: 148] 96.5 97 8 DIM18 [hu9F5VHv9/hu9F5VLv8_DIM18 SEQ ID NO: 127 / SEQ ID NO: 149] 97.2 96.5 9 DIM30 [hu9F5VHv9/hu9F5VLv8_DIM30 SEQ ID NO: 127/ SEQ ID NO: 161] 96.5 96.3 10 DIM11 [hu9F5VHv9/hu9F5VLv8_DIMl 1 SEQ ID NO: 127 / SEQ ID NO: 142] 93.6 94.5 AND DIM29 [hu9F5VHv9/hu9F5VLv8_DIM29 SEQ ID NO: 127/ SEQ ID NO: 160] 94.0 93.6 12 DIM12 [hu9F5VHv9/hu9F5VLv8_DIM12 SEQ ID NO: 127/ SEQ ID NO: 143] 93.9 93.3 13 DIMM [hu9F5VHv9/hu9F5VLv8_DIM14 SEQ ID NO: 127/ SEQ ID NO: 145] 93.5 89.6 14 DIM5 [hu9F5VHv9/hu9F5VLv8_DIM5 SEQ ID NO: 127/ SEQ ID NO: 136] 93.6 89.2 15 H10L9-DIM11 [hu9F5VHvlO/hu9F5VLv9_DIMl 1 SEQ ID NO: 128/ SEQ ID NO: 168] 93.2 83.3 16 DIM27 [hu9F5VHv9/hu9F5VLv8_DIM27 91.3 68.4 SEQ ID NO: 127/ SEQ ID NO: 158]

Example 19: PEG Precipitation with 9F5 Variants

PEG precipitation was used to determine the relative propensity of different humanized 9F5 variants to aggregate under simulated high concentration conditions. Antibodies were buffer exchanged in histidine buffer (25 mM histidine, pH 6) and added to a microplate containing the indicated amount of PEG6000 to a final protein concentration of 1 mg/ml and incubated at room temperature.

- 169 047155 temperature for 1 hour. After incubation, turbidity was measured at absorbance of 350 nm. The results are presented in table. 59-60 and in Fig. 22.

Table 59. PEG deposition of 9F5 variants DIM2, DIM13, DIM7, DIM6, DIM18, DIM8, DIM17 and DIM28

PEG6000 % DIM2 DIM13 DIM7 DIM6 DIM18 DIM8 DIM17 DIM28 25 0.7784 0.7944 0.8912 0.7688 0.901 0.8085 0.811 0.733 20 0.7904 0.8737 0.7454 0.8063 0.8743 0.9733 1.001 0.842 15 0.4729 0.5612 0.3895 0.5658 0.6362 0.6438 0.7885 0.4952 12 0.1578 0.1952 0.1563 0.2206 0.2022 0.1592 0.3472 0.1586 10 0.1554 0.1582 0.1512 0.1541 0.1541 0.1538 0.1582 0.1583 8 0.1607 0.1546 0.15 0.15 0.1508 0.1506 0.1556 0.1591 6 0.1538 0.1519 0.1486 0.1564 0.1494 0.1502 0.1541 0.157 4 0.1526 0.1516 0.1526 0.1479 0.1462 0.1504 0.1513 0.1587 2 0.1495 0.1511 0.1523 0.148 0.1467 0.1478 0.1497 0.1518 0 0.1464 0.1481 0.1501 0.1458 0.1451 0.1471 0.1477 0.1496

Table 60. PEG deposition of 9F5 variants DIM29, DIM14, DIM30, DIM27, DIM11, H10L9DIM11, DIM5 and DIM12

PEG6000 % DIM29 DIM14 DIM30 DIM27 DIM11 H10L9DIM11 DIM5 DIM12 25 0.7422 0.8867 0.7759 0.8138 0.8785 0.7122 0.7042 0.7078 20 0.8623 1.0284 0.8549 0.732 0.8999 0.8663 0.8869 0.8767 15 0.4642 1.0377 0.598 0.1843 0.6288 0.5383 0.5228 0.6414 12 0.1564 0.8123 0.1752 0.1536 0.1802 0.1755 0.1652 0.2092 10 0.1539 0.3975 0.1545 0.1525 0.1522 0.1575 0.1548 0.159 8 0.1542 0.1564 0.1475 0.1532 0.1514 0.1535 0.1549 0.1572 6 0.1518 0.1505 0.1492 0.1497 0.1486 0.1503 0.157 0.1559 4 0.1494 0.1496 0.1483 0.1469 0.1484 0.149 0.1523 0.153 2 0.1475 0.1485 0.1473 0.1469 0.1485 0.1482 0.1507 0.1526 0 0.1482 0.1537 0.1499 0.148 0.1484 0.1465 0.1487 0.149

Abbreviations for the names of humanized variants of 9F5 used in Table. 59-60 and in Fig. 22 correspond to the description given in the second column of the table. 53. Antibodies were qualitatively ranked according to the concentration of PEG6000 that caused the onset of turbidity. DIM14 [hu9F5VHv9/hu9F5VLv8_DIM14; SEQ ID NO: 127/ SEQ ID NO: 145] and DIM17

[hu9F5VHv9/hu9F5VLv8_DIM17; SEQ ID NO: 127/ SEQ ID NO: 148] exhibited aggregation at a lower concentration of PEG6000 compared to most antibodies, and DIM27

[hu9F5VHv9/hu9F5VLv8_DIM27; SEQ ID NO: 127/ SEQ ID NO: 158] exhibited aggregation at a higher concentration of PEG6000 compared to most antibodies. The combinations L27cG/L37G/M51G/L54T/L92I (DIM14) and L27cS/L37G/M51G/L54T (DIM17) resulted in decreased solubility in the presence of increasing amounts of PEG6000, as evidenced by earlier onset of haze. In contrast, the L37Q/M51G/L54R combination in combination with the parent leucine at position L27c(DIM27) results in increased solubility in the presence of increasing amounts of PEG6000. These results suggest the creation of specific residue combinations in humanized variants that are particularly likely to withstand formulation at the high antibody concentrations typically required for clinical use.

Example 20 Immunohistochemical analysis of human brain samples using control, mouse 2D11, mouse 9F5, mouse 12C4, mouse 14H3 and mouse 17C12.

Fresh frozen human brain samples were placed in OCT and sectioned using a cryostat to obtain 10-μm sections. Sections were fixed on glass slides using 10% neutral buffered formalin for 10 min at 4°C and treated with glucose oxidase to block endogenous peroxidases. Sections were stained with the indicated primary antibodies using a BOND Polymer Refine Detection system (Leica) containing DAB chromagen and hematoxylin contrast. Sections were then dehydrated in an increasing series of ethanol and xylene concentrations before coating. The results are shown in Fig. 23A-F. As expected, non-immune

- 170 047155 control antibody had virtually no binding to either normal controls or samples obtained from patients with Alzheimer's disease at both concentrations tested. In contrast, antibodies to MTBR showed abundant binding to pathological features present in Alzheimer's disease, including dystrophic neurites and neurofibrillary tangles. This binding showed a dose dependence, with stronger binding observed at higher antibody concentrations. A subset of antibodies showed little binding to normal control sections, particularly 9F5, 12C4 and 17C12. 14H3 and 2D11 showed binding to normal control tissue, indicating subtle differences in specificity for tau present in these samples. For 14H3, the lower selectivity for AD compared to controls reflects the difference in immunoreactivity found in immunoprecipitation experiments, indicating that this antibody binds normal forms of tau to a greater extent than 2D11, 9F5, 12C4, and 17C12.

Example 21. CDR Examples

Examples of CDR antibodies according to the invention are given in table. 54.

Table 54. Examples of CDRs

CDR and definition Amino acid CDR sequences SEQ ID NO: Examples of VH or VL where CDR is present CDR-H1 according to composite definition according to Kabat/Chothia GFNIKDDYMN 8 VH murine 9F5 CDR-H2 by Kabat WIDPENGDTEYASKFQG 9 VH murine 9F5 CDR-H3 by Kabat SNG 10 VH murine 9F5 CDR-L1 by Kabat RS SKSLLHSNGIT YLY 12 VL mouse 9F5 CDR-L2 by Kabat QMSNLAS 13 VL mouse 9F 5 CDR-L3 by Kabat AQNLELPLT 14 VL mouse 9F 5 CDR-H1 Kabat DDYMN 40 VH murine 9F5 CDR-H1 Chothia GFNIKDD 41 VH mouse 9F 5 CDR-H2 by Chothia DPENGD 42 VH mouse 9F 5 CDR-H2 by ABM WIDPENGDTE 43 VH murine 9F5 CDR-H1 Contact KDDYMN 44 VH murine 9F5 CDR-H2 by Contact WIGWIDPENGDTE 45 VH mouse 9F 5 CDR-H3 by Contact TTSN 46 VH mouse 9F 5 CDR-L1 by Contact ITYLYWY 47 VL mouse 9F5 CDR-L2 by Contact LLIYQMSNLA 48 VL mouse 9F 5 CDR-L3 by Contact AQNLELPL 49 VL mouse 9F 5

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CDR-H1 according to composite GFTIKDDYMN 50 hu9F5VHv4, hu9F5VHv5 hu9F5VHv6 And definition Kabat-Chothia no CDR-H2 Kabat no WVDPEDGETEYASKFQG 51 hu9F5VHv5, hu9F5VHv6 hu9F5VHv7 And CDR-H2 Kabat no WVDPENGDTEYASKFQG 52 hu9F5VHv8 CDR-L1 Kabat no RS SKSLLHSNGYTYL Y 53 hu9F5VLv5 and hu9F5VLv6 CDR-L1 Kabat no RS SKSLLHSNGINYL Y 54 hu9F5VLv7 CDR-L2 Kabat no QGSNRAS 55 hu9F5VLv4, hu9F5VLv5, hu9F5VLv6, hu9F5VLv7, SEQ ID NO:133, 135, 136, 137, 142, 143, 144, 149, 158, 159, 163, 164, 165, 168, 169 CDR-L1 Kabat no RSSKSDLHSNGITYLY 172 hu9F5VLv2_L27bD, SEQ NO:71 ID CDR-L1 Kabat no RSSKSTLHSNGITYLY 173 hu9F5VLv2_L27bT, SEQ ID NO:79 CDR-L1 Kabat no RSSKSQLHSNGITYLY 174 hu9F5VLv2_L27bQ, SEQ NO:81 ID CDR-L1 Kabat no RSSKSLDHSNGITYLY 175 hu9F5VLv2_L27cD, SEQ ID NO:63, hu9F5VLv8_DIM6, SEQ ID NO: 137, hu9F5VLv8_DIM7, SEQ ID NO: 138 and hu9F5VLv8_DIM18, SEQ ID NO: 149

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CDR-L1 by Kabat RSSKSLGHSNGITYLY 176 hu9F5VLv2_L27cG, SEQ ID NO:64, hu9F5VLv8_DIM5, SEQ ID NO: 136, hu9F5VLv8_DIM8, SEQ ID NO: 139, hu9F5VLv8_DIM9, SEQ ID NO: 140, hu9F5VLv8_DIMll, SEQ ID NO: 142, hu9F5VLv8_DIM12, SEQ ID NO: 143, hu9F5VLv8_DIM13, SEQ ID NO: 144, hu9F5VLv8_DIM14, SEQ ID NO: 145, hu9F5VLv8_DIM15, SEQ ID NO: 146, hu9F5VLv8_DIM16, SEQ ID NO: 147, hu9F5VLv8_DIM23, SEQ ID NO: 154, hu9F5VLv8_DIM24, SEQ ID NO: 155, hu9F5VLv8_DIM25, SEQ ID NO: 156, hu9F5VLv9_DIM5, SEQ ID NO: 165, hu9F5VLv9_DIM8, SEQ ID NO: 166, hu9F5VLv9_DIMl 1, SEQ ID NO: 168 and hu9F5VLv9_DIM13, SEQ ID NO: 169

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CDR-L1 by Kabat RSSKSLSHSNGITYLY 177 hu9F5VLv2_L27cS, SEQ ID NO:65, hu9F5VLv8_DIMl, SEQ ID NO: 132, hu9F5VLv8_DIM2, SEQ ID NO: 133, hu9F5VLv8_DIM3, SEQ ID NO: 134, hu9F5VLv8_DIM4, SEQ ID NO: 135, hu9F5VLv8_DIM10, SEQ ID NO: 141, hu9F5VLv8_DIM17, SEQ ID NO: 148, hu9F5VLv8_DIM19, SEQ ID NO: 150, hu9F5VLv8_DIM20, SEQ ID NO: 151, hu9F5VLv8_DIM21, SEQ ID NO: 152, hu9F5VLv8_DIM22, SEQ ID NO: 153, hu9F5VLv8_DIM28, SEQ ID NO: 159, hu9F5VLv8_DIM29, SEQ ID NO: 160, hu9F5VLv8_DIM30, SEQ I NO: 161, hu9F5VLv9_DIMl, SEQ ID NO: 162, hu9F5VLv9_DIM2, SEQ ID NO: 163, hu9F5VLv9_DIM4, SEQ ID NO: 164, hu9F5VLv9_DIM10, SEQ ID NO: 167, hu9F5VLv9_DIM19, SEQ ID NO: 170 and hu9F5VLv9_DIM20, SEQ ID NO:171 CDR-L1 no Kabat RS SKSLEHSNGITYLY 178 hu9F5VLv2_L27cE, SEQ ID NO:66 CDR-L1 no Kabat RSSKSLTHSNGITYLY 179 hu9F5VLv2_L27cT, SEQ ID NO:69

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CDR-L1 Kabat no RS SKSLNHSNGITYL Y 180 hu9F5VLv2_L27cN, SEQ ID NO :70 CDR-L1 Kabat no RS SKSLAHSNGITYL Y 181 hu9F5VLv2_L27cA, SEQ ID NO:74 CDR-L1 Kabat no RSSKSLPHSNGITYLY 182 hu9F5VLv2_L27cP, SEQ IDNO:83 CDR-L1 Kabat no RSSKSLIHSNGITYLY 183 hu9F5VLv8_DIM26, SEQ ID NO:157 CDR-L1 Kabat no RSSKSLLHSNGETYLY 184 hu9F5VLv2_I30E, SEQ ID NO:67 CDR-L1 Kabat no RSSKSLLHSNGKTYLY 185 hu9F5VLv2_I30K, SEQ IDNO:68 CDR-L1 Kabat no RSSKSLLHSNGGTYLY 186 hu9F5VLv2_130G, SEQ ID NO:72 CDR-L1 Kabat no RSSKSLLHSNGQTYLY 187 hu9F5VLv2_I30Q, SEQ IDNO:78 CDR-L1 Kabat no RS SKSLLHSNGIGYL Y 188 hu9F5VLv2_T31G, SEQ IDNO:80 CDR-L1 Kabat no RS SKSLLHSNGITYNY 189 hu9F5VLv2_L33N, SEQ IDNO:73 CDR-L1 Kabat no RSSKSLLHSNGITYTY 190 hu9F5VLv2_L33T, SEQ ID NO:75 CDR-L1 Kabat no RSSKSLLHSNGITYSY 191 hu9F5VLv2_L33S, SEQ ID NO:76 CDR-L1 Kabat no RS SKSLLHSNGITYRY 192 hu9F5VLv2_L33R, SEQ ID NO:77 CDR-L1 Kabat no RSSKSLLHSNGITYGY 193 hu9F5VLv2_L33G, SEQ IDNO:82 CDR-L2 Kabat no QESNLAS 194 hu9F5VLv2_M51E, SEQ ID NO:61

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CDR-L2 Kabat no QDSNLAS 195 hu9F5VLv2_M51D, SEQ IDNO:62 CDR-L2 Kabat no QGSNLAS 196 hu9F5VLv8_DIM30, SEQ ID NO:161 CDR-L2 Kabat no QMSNRAS 197 hu9F5VLv8_DIM29, SEQ ID NO: 160 CDR-L2 Kabat no QGSNGAS 198 hu9F5VLv8_DIMl, SEQ ID NO: 132 and hu9F5VLv9_DIMl, SEQ ID NO: 162 CDR-L2 Kabat no QGSNTAS 199 hu9F5VLv8_DIM3,SEQ ID NO: 134, hu9F5VLv8_DIM14, SEQ ID NO: 145, hu9F5VLv8_DIM15, SEQ ID NO: 146, hu9F5VLv8_DIM16, SEQ ID NO: 147 and hu9F5VLv8_DIM17, SEQ ID NO: 148 CDR-L2 Kabat no QKSNRAS 200 hu9F5VLv8_DIM7, SEQ ID NO: 138, hu9F5VLv8_DIM8, SEQ ID NO: 139 and hu9F5VLv9_DIM8, SEQ ID NO: 166 CDR-L2 Kabat no QKSNGAS 201 hu9F5VLv8_DIM9, SEQ ID NO: 140, hu9F5VLv8_DIM10, SEQ ID NO: 141 and hu9F5VLv9_DIM10, SEQ ID NO: 167 CDR-L2 Kabat no QISNRAS 202 hu9F5VLv8_DIM19, SEQ ID NO: 150 and hu9F5VLv9_DIM19, SEQ ID NO: 170 CDR-L2 Kabat no QISNGAS 203 hu9F5VLv8_DIM20, SEQ ID NO: 151, hu9F5VLv8_DIM21. SEQ ID NO: 152 and hu9F5VLv9_DIM20, SEQ ID NO: 171

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CDR-L2 Kabat no QESNRAS 204 hu9F5VLv8_DIM22, SEQ ID NO: 153, hu9F5VLv8_DIM24, SEQ ID NO: 155, hu9F5VLv8_DIM25, SEQ ID NO: 156 and hu9F5VLv8_DIM26, SEQ ID NO:157 CDR-L2 Kabat no QESNGAS 205 hu9F5VLv8_DIM23, SEQ NO:154 ID CDR-L3 Kabat no GQNLELPLT 206 hu9F5VLv2_A89G, SEQ NO: 108 ID CDR-L3 Kabat no AQNDELPLT 207 hu9F5VLv2_L92D, SEQ NO: 100 ID CDR-L3 Kabat no AQNEELPLT 208 hu9F5VLv2_L92E, SEQ ID NO:102 CDR-L3 Kabat no AQNGELPLT 209 hu9F5VLv2_L92G, SEQ ID NO: 103, hu9F5VLv8_DIM4, SEQ ID NO: 135, hu9F5VLv8_DIM12, SEQ ID NO: 143, hu9F5VLv8_DIM15, SEQ ID NO: 146, hu9F5VLv8_DIM25, SEQ ID NO: 156 and hu9F5VLv9_DIM4, SEQ ID NO: 164 CDR-L3 Kabat no AQNQELPLT 210 hu9F5VLv2_L92Q, SEQ NO: 104 ID CDR-L3 Kabat no AQNTELPLT 211 hu9F5VLv2_L92T, SEQ ID NO: 107

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CDR-L3 no AQNIELPLT 212 hu9F5VLv8_DIMl, SEQ ID Kabat NO: 132, hu9F5VLv8_DIM2, SEQ

ID NO: 133, hu9F5VLv8_DIM3, SEQ ID NO: 134, hu9F5VLv8_DIM5, SEQ ID NO: 136, hu9F5VLv8_DIM6, SEQ ID NO: 137, hu9F5VLv8_DIM7, SEQ ID NO: 138, hu9F5VLv8_DIM8, SEQ ID NO: 139, hu9F5VLv8_DIM9, SEQ ID NO 140, hu9F5VLv8_DIM10, SEQ ID NO: 141, hu9F5VLv8_DIMll, SEQ ID NO: 142, hu9F5VLv8_DIM14, SEQ ID NO: 145, hu9F5VLv8_DIM17, SEQ ID NO: 148, hu9F5VLv8_DIM18, SEQ ID NO : 149, hu9F5VLv8_DIM19, SEQ ID NO: 150, hu9F5VLv8_DIM20, SEQ ID NO:151, hu9F5VLv8_DIM22, SEQ ID NO: 153, hu9F5VLv8_DIM23, SEQ ID NO: 154, hu9F5VLv8_DIM24, SEQ ID NO: 155, hu9F5VLv8_DIM27, SEQ ID NO: 158 , hu9F5VLv8_DIM28, SEQ ID NO: 159, hu9F5VLv8_DIM29, SEQ ID NO: 160, hu9F5VLv8_DIM30, SEQ ID NO: 161, hu9F5VLv9_DIMl, SEQ ID NO: 162, hu9F5VLv9_DIM2, SEQ ID NO: 163, hu9F5VLv9_DIM5, SEQ ID NO: 165, hu9F5VLv9_DIM8, ID NO: 166, hu9F5VLv9 DIM10,

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CDR-L3 Kabat no AQNLGLPLT 213 hu9F5VLv2_L93G, SEQ NO: 105 ID CDR-H2 Kabat no WIDPENGDTAYASKFQG 220 VH murine 12C4 CDR-H1 according to composite definition of Kabat-Chothia no AFNIKDDYMN 226 VH murine 17C12 CDR-H2 Kabat no WIDPENGDTKYASKFQG 227 VH murine 17C12 CDR-L1 Kabat no TSSQSLLHSNRKTYLH 229 VL murine 17C12 CDR-L2 Kabat no LVSKLES 230 VL murine 17C12 CDR-L3 Kabat no LQTTHFPRT 231 VL murine 17C12 CDR-H1 according to composite definition of Kabat-Chothia no GFSLSTYGMGVG 241 VH murine 14H3 CDR-H2 Kabat no NIWWDDIKYYNAALKS 242 VH murine 14H3 CDR-H3 Kabat no NVDY 243 VH murine 14H3 CDR-L1 Kabat no RSSQSLVHSNGNTFLH 245 VL murine 14H3 CDR-L2 Kabat no KVSNRFS 246 VL murine 14H3

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CDR-L3 by Kabat SQSTLVPWT 247 VL murine 14H3 CDR-H2 by Contact WIGWIDPNGDTA 258 VH murine 12C4 CDR-H1 Chothia AFNIKDD 259 VH murine 17C12 CDR-H2 by AbM WIDPENGDTK 260 VH murine 17C12 CDR-H2 by Contact WIGWIDPENGDTK 261 VH murine 17C12 CDR-L1 no Contact KTYLHWL 262 VL murine 17C12 CDR-L2 no Contact LLIYLVSKLE 263 VL murine 17C12 CDR-L3 no Contact LQTTHFPR 264 VL murine 17C12 CDR-H1 no Kabat TYGMGVG 265 VH murine 14H3 CDR-H1 no Chothia GFSLSTYGM 266 VH murine 14H3 CDR-H2 no Chothia WWDDI 267 VH murine 14H3 CDR-H2 no AbM NIWWDDIKY 268 VH murine 14H3 CDR-H1 no Contact STYGMGVG 269 VH murine 14H3 CDR-H2 no Contact WLANIWWDDIKY 270 VH murine 14H3 CDR-H3 no Contact ARNVD 271 VH murine 14H3 CDR-L1 no Contact NTFLHWY 272 VL murine 14H3 CDR-L2 no Contact LLIYKVSNRF 273 VL murine 14H3 CDR-L3 no Contact SQSTLVPW 274 VL murine 14H3 CDR-H1 according to composite by definition Kabat-Chothia GFSLSTYGMGVS 275 hul4H3VHvl and hul4H3VHv2

Claims (36)

1. An isolated monoclonal antibody that binds to human tau, comprising three light chain CDRs and three heavy chain CDRs of a murine antibody, characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 11; or three heavy chain CDRs and three light chain CDRs of a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 219 and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 11; or three heavy chain CDRs and three light chain CDRs of a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 225 and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 228. 2. The antibody of claim 1, wherein said antibody comprises three heavy chain CDRs and three light chain CDRs of a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 7, and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 11. 3. The antibody of claim 2, wherein the three heavy chain CDRs CDR-H1, CDR-H2 and CDR-H3 as defined by the Kabat/Chothia composite definition are, respectively, SEQ ID NO: 8, 9 and 10, except that position H28 may be occupied by N or T, position H51 may be occupied by I or V, position H54 may be occupied by N or D, and position H56 may be occupied by D or E, and the three light chain CDRs CDR-L1, CDR- L2 and CDR-L3 as defined by the Kabat/Chothia composite definition are, respectively, SEQ ID NOs: 12, 13 and 14, except that position L27b - 216 047155 occupied by L, D, T or Q, position L27c occupied by L, D, G, S, E, T, N, A, P or I, position L30 can be occupied by I, Y, E, K, G or Q, position L31 can be occupied by T, N or G, position L33 can be occupied by L N, T, S, R or G, position L51 can be occupied by M, G, E, D, K or I, position L54 can be occupied by L, R, G or T, position L89 is occupied by A or G, position L92 is occupied by L, D, E, G, Q, T or I, and position L93 is occupied by E or G. 4. The antibody of claim 3, wherein CDR-H1 has an amino acid sequence comprising SEQ ID NO: 50, wherein CDR-H2 has an amino acid sequence comprising SEQ ID NO: 51, or SEQ ID NO: 52, wherein CDR- L1 has an amino acid sequence including any of SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 172-193, wherein CDR-L2 has an amino acid sequence including any of SEQ ID NO: 55 and SEQ ID NO: 194-205, and wherein CDR-L3 has an amino acid sequence comprising any of SEQ ID NOs: 206-213. 5. An antibody according to any one of claims 1 to 4, wherein the antibody is a humanized antibody comprising a humanized mature heavy chain variable region and a humanized mature light chain variable region. 6. The antibody of claim 5, wherein the humanized mature heavy chain variable region contains three Kabat heavy chain CDRs having the sequences SEQ ID NO: 40, SEQ ID NO: 9 and SEQ ID NO: 10, and the humanized mature light variable region chain contains three Kabat light chain CDRs having the sequences SEQ ID NO: 12-14. 7. Antibody according to any one of claims 1-6, in which at least one of the following positions is occupied by the specified amino acid: H1 is occupied by Q or E, H5 is occupied by Q or V, H11 is occupied by L or V, H12 is occupied by V or K, H17 occupied by S or T, H20 occupied by L or I, H23 occupied by T or K, H28 occupied by N or T, H38 occupied by K, R or Q, H40 occupied by R or A, H42 occupied by E or G, H43 occupied by Q or K, H48 occupied I or M, H51 occupied I or V, H54 occupied N or D, H56 occupied D or E, H66 occupied K or R, H69 occupied I or M, H75 occupied S or T, H76 occupied N or D, H79 occupied Y , Q, D, N or G, H80 occupied by L, M, P, D, G or E, H81 occupied by Q or E, H82 occupied by L, P, K, R, E or N, H82a occupied by S or G, H82c occupied by L, G, D or S, H83 occupied by T or R, H93 occupied by A or T, H94 occupied by S or T, H108 occupied by T or L, H109 occupied by L or V, L3 is V or Q, L7 is A or S, L8 is A or P, L9 is F or L, L11 is N or L, L15 is L or P, L17 is T or E, L18 is S or P, L27b is L, D, T or Q, L27c is L, D, G, S, E, T, N, A, P or I, L30 is I, Y, E, K or G, L31 is T, N or G , L33 is L, N, T, S, R or G, L37 is L, Q, G or I, L39 is R or K, L51 is M, G, E, D, K or I, L54 is R, G or T, L60 is N or D, L64 is G or S, L66 is E or G, L73 is L, P or G, L74 is R or K, L75 is I, D, P, Q or G, L76 is S, P or G, L77 is R or D, L78 is V, R, D, E, P, K, G or Q, L85 is V or G, L86 is Y or T, L89 is A or G, L92 is L, D, E, G, Q, T or I, L93 is E or G, L100 is G or Q. 8. The antibody of claim 7, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 128. 9. The antibody according to any one of claims 1 to 6, wherein the mature heavy chain variable region has any amino acid sequence of SEQ ID NO: 15-22 and SEQ ID NO: 109-129, and the mature light chain variable region has any amino acid sequence from SEQ ID NO: 23-29, SEQ ID NO: 61-108 and SEQ ID NO: 130-171. 10. The antibody of claim 9, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 149; or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 142, or the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence SEQ ID NO: 127 chain has the amino acid sequence of SEQ ID NO: 159, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 148, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 137, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: - 217 047155 145, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 136, or the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 127 and the mature variable region the light chain region has the amino acid sequence of SEQ ID NO: 138, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 158, or the mature heavy chain variable region has the amino acid sequence the sequence is SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 143, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 144, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 133, or the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 127 and the mature variable region the light chain region has the amino acid sequence of SEQ ID NO: 160, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 127, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 161, or the mature heavy chain variable region has the amino acid sequence the sequence is SEQ ID NO: 127 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 139, or the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 128 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 168. 11. The antibody according to claim 2, containing a humanized mature heavy chain variable region containing three Kabat heavy chain CDRs having the sequences SEQ ID NO: 40, SEQ ID NO: 9 and SEQ ID NO: 10, and a humanized mature light variable region a chain containing three Kabat light chain CDRs having the sequences SEQ ID NO: 12-14. 12. The antibody according to claim 11, in which at least one of the following positions is occupied by the specified amino acid: H1 is occupied by Q or E, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M, H93 is occupied by T, H94 is occupied by T , L64 represents S, L104 represents V or L. 13. The antibody of claim 11, wherein the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 214 or 215, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 216 or 217. 14. The antibody of claim 1, comprising three heavy chain CDRs and three light chain CDRs of a murine antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 219 and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 11. 15. The antibody of claim 14, wherein the antibody is a humanized antibody. 16. The antibody of claim 15, comprising a humanized mature heavy chain variable region comprising three Kabat heavy chain CDRs having the sequences SEQ ID NO: 40, SEQ ID NO: 220 and SEQ ID NO: 10, and a humanized mature light variable region a chain containing three Kabat light chain CDRs having the sequences SEQ ID NO: 12-14. 17. Antibody according to any one of claims 14-16, in which at least one of the following positions is occupied by the specified amino acid: H1 is occupied by Q or E, H48 is occupied by M or I, H93 is occupied by A or T, H94 is occupied by R or T, L64 represents G or S, L104 represents V or L. 18. The antibody according to any one of claims 14 to 16, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 221 or 222, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 223 or 224. 19. The antibody of claim 1, comprising three heavy chain CDRs and three light chain CDRs of a mouse antibody characterized by a heavy chain variable region having the amino acid sequence comprising SEQ ID NO: 225 and a light chain variable region having the amino acid sequence comprising SEQ ID NO: 228. 20. The antibody of claim 19, wherein the antibody is a humanized antibody. 21. The antibody according to claim 20, containing a humanized mature heavy chain variable region, - 218 047155 containing three Kabat heavy chain CDRs having the sequences SEQ ID NO: 40, SEQ ID NO: 227 and SEQ ID NO: 10, and a humanized mature light chain variable region containing three Kabat light chain CDRs having the sequences SEQ ID NO: 229-231. 22. Antibody according to any one of claims 19-21, in which at least one of the following positions is occupied by the specified amino acid: H1 is occupied by Q or E, H2 is occupied by I, H24 is occupied by A, H48 is occupied by I, H67 is occupied by A, H69 is occupied by M , H93 is occupied by T, H94 is occupied by T, H108 is occupied by T or L, H113 is occupied by R or S, L2 is V, L36 is L, L43 is P or S. 23. The antibody according to any one of claims 19 to 21, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 232 or 233, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 234 or 235. 24. An antibody according to any one of claims 1 to 23, wherein the antibody is an intact antibody. 25. An antibody according to any one of claims 1 to 23, wherein the antibody is a binding moiety. 26. An antibody according to any one of claims 1 to 25, wherein the isotype is human IgG1, human IgG2 or human IgG4. 27. The antibody according to any one of claims 5-10, 11-13, 15-18, 20-23 and 24-26, wherein the mature light chain variable region is fused to the light chain constant region, and the mature heavy chain variable region is fused to heavy chain constant region. 28. The antibody of claim 27, wherein the heavy chain constant region is a mutant form of the natural human antibody heavy chain constant region that has reduced binding to the Fcy receptor compared to the natural human antibody heavy chain constant region. 29. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 28 and a pharmaceutically acceptable carrier. 30. A nucleic acid encoding the heavy chain of an antibody described in any one of claims 1 to 28. 31. A nucleic acid encoding a light chain of an antibody described in any one of claims 1 to 28. 32. Nucleic acid encoding the heavy chain and light chain of the antibody described in any one of claims 1 to 28. 33. A method for humanizing a mouse antibody, comprising: (a) selecting one or more acceptor antibody sequences; (b) identifying the amino acid residues of the mouse antibody that should be conserved; (c) synthesizing a nucleic acid encoding a humanized heavy chain including a CDR of a mouse antibody heavy chain and a nucleic acid encoding a humanized light chain including a CDR of a mouse antibody light chain; and (d) expressing nucleic acids in a host cell to produce a humanized antibody; wherein the mouse antibody is characterized by a mature heavy chain variable region of SEQ ID NO: 7 and a mature light chain variable region of SEQ ID NO: 11, or the mouse antibody is characterized by a mature heavy chain variable region of SEQ ID NO: 219 and a mature light chain variable region of SEQ ID NO: 11, or the mouse antibody is characterized by a mature heavy chain variable region of SEQ ID NO: 225 and a mature light chain variable region of SEQ ID NO: 228. 34. A method of inhibiting or reducing tau aggregation in a subject having or at risk of developing tau-mediated amyloidosis, comprising administering to the subject an antibody according to any one of claims 1 to 28, thereby inhibiting or reducing tau aggregation in the subject. 35. A method of treating or preventing tau-associated disease in a subject, comprising administering an antibody according to any one of claims 1 to 28, thereby treating or preventing the disease. 36. The method of claim 35, wherein the tau-associated disease is 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 disease type C, supranuclear paralysis, frontotemporal dementia, frontotemporal lobar degeneration, argyrophil granule disease, globular glial tauopathy, amyotrophic lateral sclerosis/Guam parkinsonism-dementia complex, corticobasal degeneration (CBD), dementia with Lewy bodies, variant Alzheimer's disease with bodies Lewy body variant of Alzheimer disease, chronic traumatic encephalopathy (CTE), globular glial tauopathy (GGT), Parkinson's disease or progressive supranuclear palsy (PSP).