Anti-TAUC3 antibodies and their applications

Abstract

Anti-TauC3 antibodies that are at least several orders of magnitude more specific for TauC3 than for full-length tau (2N4R) are described. Methods of using the anti-TauC3 antibodies are also described.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 829,774, filed on April 5, 2019, which is incorporated herein by reference. Background Art

[0002] Tau protein is a microtubule-associated protein that is mainly distributed in axons and regulates the assembly, spatial organization and behavior of microtubules (MT) in neurons. Tau protein is encoded by a single gene located on chromosome 17.

[0003] There are six known isoforms of tau protein. These isoforms differ in the presence or absence of an insert of 29 or 58 amino acids in the amino-terminal region and in the addition or deletion of tandem repeats (which can be repeated 3 or 4 times) in the carboxyl-terminal region of tau, which is called the microtubule-binding domain. The microtubule-binding domain consists of imperfect repeats of 31 or 32 amino acid residues. The longest tau protein isoform (2N4R) is 441 amino acids in length and contains four repeats (R1, R2, R3 and R4) and two inserts. The smallest tau isoform contains 352 amino acid residues, has three tandem repeats (R1, R3 and R4) in the microtubule-binding domain, and has no amino-terminal insertion. The amino acid sequences corresponding to human tau protein isoforms are provided in SEQ ID NOs: 1-6.

[0004] SEQ ID NO: 1 is the longest tau isoform htau40, containing two N-terminal insertion fragments and four microtubule binding domains (2N4R), as follows:

[0005]

[0006] SEQ ID NO: 2 comprises two N-terminal inserts and three microtubule binding domains (2N3R) as follows:

[0007]

[0008]

[0009] SEQ ID NO: 3 comprises an N-terminal insert and four microtubule binding domains (IN4R), as follows:

[0010]

[0011] SEQ ID NO: 4 contains zero N-terminal inserts and four microtubule binding domains (0N4R) as follows:

[0012]

[0013] SEQ ID NO: 5 comprises an N-terminal insert and three microtubule binding domains (1N3R) as follows:

[0014]

[0015] SEQ ID NO:6 contains zero N-terminal inserts and three microtubule binding domains (ON3R), as follows:

[0016]

[0017] TauC3 is an extremely deleterious, nucleating, pre-tangled, intracellular and preferentially secreted, C-terminally truncated tau fragment ending in aspartic acid 421. Compared to full-length Tau (FLT)(2N4R), TauC3 is present in low abundance but has been shown to produce disproportionately large pathological effects. TauC3 may contribute, for example, to the seeding and spread of pathological tau aggregates.

[0018] Pathological aggregation and pathological spread of tau in the brain are associated with more than 20 neurodegenerative diseases, including, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc. (collectively referred to as "tauopathies"). Summary of the invention

[0019] One object of the present invention is to provide a chimeric antibody that can be used to diagnose and treat neurodegenerative diseases associated with the pathological activity of TauC3 in the brain.

[0020] Another object of the present invention is to provide a humanized antibody that can be used to diagnose and treat neurodegenerative diseases associated with the pathological activity of TauC3 in the brain.

[0021] Another object of the present invention is to provide a human antibody that can be used to diagnose and treat neurodegenerative diseases associated with the pathological activity of TauC3 in the brain.

[0022] Another object of the present invention is to provide a chimeric antibody that is specific to the C-terminus of TauC3.

[0023] Another object of the present invention is to provide a chimeric antibody which is specific to the C-terminus of TauC3 and has a 1x10 -3 s -1 The following dissociation rates (K d ).

[0024] Another object of the present invention is to provide a humanized antibody that is specific to the C-terminus of TauC3.

[0025] Another object of the present invention is to provide a humanized antibody which is specific to the C-terminus of TauC3 and has a 1x10 -3 s -1 The following dissociation rates (K d ).

[0026] To facilitate the above and other purposes, the present invention relates to chimeric antibodies, humanized antibodies and human antibodies ("anti-TauC3 antibodies") that are specific for the C-terminus of TauC3. The binding affinity (KD) of the anti-TauC3 antibodies to TauC3 is 1x10 -10 Up to 1x10 -12 , with a binding affinity (KD) of 1x10 -4 Up to 1x10 -8 M. For example, the binding affinity (KD) of the anti-TauC3 antibody to TauC3 may be about 5x10 -12 M to about 1.2x10 -10 M, about 1x10 -11 M to about 1x10 -10 M, about 1x10 -11 M is about 9x10 -11 M, about 1x10 -11 M is about 8x10 -11 M, about 1x10 -11 M is about 7x10 -11 M, about 1x10 -11 M to about 6x10 -11 M, about 1x10 -11 M to about 5x10 -11 M, or about 1x10 -11 M to about 4x10 -11 M; and the binding affinity (KD) for FLT is 1x10-4 to 1x10- 8M. In a preferred embodiment, the antibody can still maintain its binding ability after being subjected to a temperature of about 40°C to about 67°C for 10 minutes, and can still maintain its binding ability after incubation in serum (e.g., mice) at 37°C for 21 days. The high performance of the anti-TauC3 antibody allows the antibody to target TauC3 without affecting the normal physiological function of FLT. In some embodiments, the specificity of the antibody allows only the most harmful tau species to be targeted. Compared with antibodies that are not specific and do not distinguish between different types of tau, this can allow, for example, to potentially reduce the effective therapeutic dose. Anti-TauC3 antibodies and antigen-binding fragments thereof can be used, for example, to diagnose and treat neurodegenerative diseases associated with the pathological activity of TauC3 in the brain, including, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), cortical basal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc. The anti-TauC3 antibody may have a water solubility of 50 mg / ml or more (e.g., about 50 mg / ml to about 200 mg / ml, about 55 mg / ml to about 180 mg / ml, about 55 mg / ml to about 170 mg / ml, about 55 mg / ml to about 150 mg / ml, about 55 mg / ml to about 140 mg / ml, about 55 mg / ml to about 130 mg / ml; or about 60 mg / ml to about 130 mg / ml).

[0027] The present invention further relates to chimeric, humanized and human anti-TauC3 antibodies having a higher binding affinity (KD) for TauC3 than a murine anti-TauC3 antibody. In some embodiments, the binding affinity (KD) of the chimeric, humanized and human anti-TauC3 antibodies to TauC3 is at least 2-fold, 3-fold or 4-fold higher than the binding affinity (KD) of the murine anti-TauC3 antibody to TauC3. In some embodiments, the binding affinity KD of the murine anti-TauC3 antibody to TauC3 is about 4.9x10 -11 M, the binding affinity KD of chimeric, humanized and human anti-TauC3 antibodies to TauC3 is about 1x10 -11 M to about 2.5x10 -11 M. The binding affinity KD of chimeric, humanized and human anti-TauC3 antibodies to TauC3 can be, for example, about 1.1×10 -11 M, about 1.3x10 -11 M, about 1.5x10 -11 M, about 1.7x10 -11 M, about 1.9x10 -11 M, about 2.1x10 -11 M or about 2.3x10 -11M. The water solubility of the anti-TauC3 antibody can be 50 mg / ml or higher (e.g., about 50 mg / ml to about 200 mg / ml, about 55 mg / ml to about 180 mg / ml, about 55 mg / ml to about 170 mg / ml, about 55 mg / ml to about 150 mg / ml, about 55 mg / ml to about 140 mg / ml, about 55 mg / ml to about 130 mg / ml; or about 100 mg / ml to about 200 mg / ml, about 100 mg / ml to about 180 mg / ml, about 100 mg / ml to about 170 mg / ml, about 100 mg / ml to about 150 mg / ml, about 100 mg / ml to about 140 mg / ml, or about 100 mg / ml to about 130 mg / ml).

[0028] The present invention also relates to chimeric, humanized and human anti-TauC3 antibodies having a binding affinity (KD) of 1x10 -10 To lx10 -12 , the dissociation rate (Kd) is 1x10 -3 s -1 The binding affinity (KD) for FLT is 1x10 -4 Up to 1x10 -8 M.

[0029] The anti-TauC3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR1 represented by the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 represented by IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 represented by VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising a CDR1 represented by the sequence QEISVY (SEQ ID NO: 10), a CDR2 represented by the sequence GAF (SEQ ID NO: 11), and a CDR3 represented by the sequence LQYVRYPWT (SEQ ID NO: 12); and has a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 The following dissociation rates (K d ); and the binding affinity (KD) for FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8 M, or no detectable binding to FLT (SEQ ID NO: 1).

[0030] In certain embodiments, an anti-TauC3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR1 homologous to the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 homologous to IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 homologous to VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising a CDR1 homologous to the sequence QEISVY (SEQ ID NO: 10), a CDR2 homologous to the sequence GAF (SEQ ID NO: 11), and a CDR3 homologous to the sequence LQYVRYPWT (SEQ ID NO: 12); and has a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 The dissociation rate (Kd) was below 10 and the binding affinity (KD) for FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8 M, or no detectable binding to FLT (SEQ ID NO: 1).

[0031] In certain embodiments, the anti-TauC3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR1 identical to the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 identical to IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 identical to VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising a CDR1 identical to the sequence QEISVY (SEQ ID NO: 10), a CDR2 identical to the sequence GAF (SEQ ID NO: 11), and a CDR3 identical to the sequence LQYVRYPWT (SEQ ID NO: 12); and has a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 The dissociation rate (Kd) was below 10 and the binding affinity (KD) for FLT (SEQ ID NO: 1) was 1x10 -4 to 1x10 -8 M, or no detectable binding to FLT (SEQ ID NO: 1).

[0032] In certain embodiments, the anti-TauC3 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR1 of the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 of the sequence IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 of the sequence VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising a CDR1 of the sequence QEISVY (SEQ ID NO: 10), a CDR2 of the sequence GAF (SEQ ID NO: 11), and a CDR3 of the sequence LQYVRYPWT (SEQ ID NO: 12); and has a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 The dissociation rate (Kd) is below 1×10 -4 Up to 1x10 -8 M, or has no detectable binding to SEQ ID NO: 1, and is used to treat Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD). The antibody can also be used to diagnose tauopathy, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD) or frontotemporal lobar degeneration (FTLD).

[0033] In one aspect, the present invention relates to an anti-TauC3 antibody, which is a humanized antibody comprising (a) a heavy chain variable region comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9; and (b) a light chain variable region comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11, and CDR3 represented by SEQ ID NO: 12; and having a 1x10 affinity for TauC3. -10 and 1x10 -12 The binding affinity (KD) and 1x1 0-3 s -1 The following dissociation rates (K d )(e.g. 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to FLT (SEQ ID NO: 1) was 1x10-4 Up to 1x10 -8 M, or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9; and (b) a light chain variable region comprising CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12; and has a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 The dissociation rate (Kd) below (e.g. 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to SEQ ID NO: 1 was 1x10 -4 To lx10 -8 M, or has no detectable binding to FLT, and is used to treat tauopathies such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc.

[0034] In one aspect, the present invention relates to an anti-TauC3 antibody, which is a humanized antibody comprising (a) a heavy chain variable region comprising a CDR1 identical to SEQ ID NO: 7, a CDR2 identical to SEQ ID NO: 8, and a CDR3 identical to SEQ ID NO: 9; and (b) a light chain variable region comprising a CDR1 identical to SEQ ID NO: 7, a CDR2 identical to SEQ ID NO: 11, and a CDR3 identical to SEQ ID NO: 12; and having a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 s -1 The following dissociation rates (K d )(For example, 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8M, or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region having CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9; and (b) a light chain variable region having CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12; and has a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , the dissociation rate (Kd) is 1x10 -3 s -1 The following (for example, 1x10 -4 to 1x10 -3 s -1 ), and the binding affinity (KD) to SEQ ID NO: 1 was 1x10 -4 Up to 1x10 -8 M, or has no detectable binding to FLT, and is used to treat tauopathies such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc.

[0035] In one aspect, the present invention relates to an anti-TauC3 antibody, which is a humanized antibody comprising (a) a heavy chain variable region comprising a CDR1 homologous to SEQ ID NO: 7, a CDR2 homologous to SEQ ID NO: 8, and a CDR3 homologous to SEQ ID NO: 9; and (b) a light chain variable region comprising a CDR1 homologous to SEQ ID NO: 11, a CDR2 homologous to SEQ ID NO: 12, and a CDR3 homologous to SEQ ID NO: 13; and has a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , the dissociation rate (Kd) is 1x10 -3 s -1 The following (for example, 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8M, or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region comprising CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 8, and CDR3 of SEQ ID NO: 9; and (b) a light chain variable region comprising CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12; and has a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 The following (for example, 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to SEQ ID NO: 1 was 1x10 -4 Up to 1x10 -8 M, or has no detectable binding to FLT, and is used to treat tauopathies such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc.

[0036] A humanized antibody may, for example, comprise:

[0037] (a) a heavy chain variable region comprising the sequence

[0038] LVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVARIRSKSNNYATYYAASVKGRFTISRDDSKSMAYLQMDSLKTEDTAVYYCVGGGDFWGQGTLVTVSS (SEQ ID NO: 13) or a sequence homologous to SEQ ID NO: 13; and

[0039] (b) a light chain variable region comprising a sequence selected from the group consisting of:

[0040] DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWFQQKPGKAPKRLIYGAFKLQSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 14) or a sequence homologous to SEQ ID NO: 14,

[0041] DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWYQQKPGKAPKRLIYGAFTLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 15) or a sequence homologous to SEQ ID NO: 15,

[0042] DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWYQQKPGKAPKRLIYGAFSLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 16) or a sequence homologous to SEQ ID NO: 16,

[0043] DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWFQQKPGKAPKRLIYGAFKLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 17) or a sequence homologous to SEQ ID NO: 17, and DIQMTQSPSSLSASVGDRVTITCRASQEISVYLSWFQQKPGKAIKRLIYGAFSLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 18) or a sequence homologous to SEQ ID NO: 18, and a binding affinity (KD) for TauC3 of 1x10 -10 and 9x10 -12 The binding affinity (KD) for FLT (SEQ ID NO: 1) was 1x10 -4 to 1x10 -8 M, or no detectable binding to FLT.

[0044] In certain embodiments, the humanized antibody comprises the variable heavy chain (V H ) polypeptide and the variable light chain (V L ) polypeptide.

[0045] In certain embodiments, a humanized antibody comprises (a) a variable heavy chain (V H) polypeptide, which comprises CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, the variable heavy chain (V H ) polypeptide having at least 70% sequence identity to SEQ ID NO: 13; and (b) a variable light chain (V L ) polypeptide, which comprises CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12, the variable light chain (V L ) The polypeptide has at least 70% sequence identity to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

[0046] In certain embodiments, the humanized antibody comprises a V sequence having at least 75% sequence identity to SEQ ID NO: 13. L A polypeptide having at least 75% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. H Chain polypeptide.

[0047] In certain embodiments, the humanized antibody comprises a V sequence having at least 80% sequence identity to SEQ ID NO: 13. L A polypeptide having at least 80% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 H Chain polypeptide.

[0048] In certain embodiments, the humanized antibody comprises a V sequence having at least 85% sequence identity to SEQ ID NO: 13. L A polypeptide having at least 85% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. H Chain polypeptide.

[0049] In certain embodiments, the humanized antibody comprises a V sequence having at least 90% sequence identity to SEQ ID NO: 13. LA polypeptide having at least 90% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. H Chain polypeptide.

[0050] In certain embodiments, the humanized antibody comprises a V sequence having at least 95% sequence identity to SEQ ID NO: 13. L A polypeptide having at least 95% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. H Chain polypeptide.

[0051] In certain embodiments, an anti-TauC3 antibody comprises a variable heavy chain (V H ) polypeptide and variable light chain (V L ) polypeptide, the variable heavy chain (V H ) polypeptide comprises SEQ ID NO: 13, the variable light chain (V L ) The polypeptide comprises a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0052] In certain embodiments, an anti-TauC3 antibody comprises (i) a variable heavy chain (V H ) polypeptide comprising CDR1, CDR2 and CDR3, wherein CDR1 is the polypeptide of SEQ ID NO: 7, CDR2 is the polypeptide of SEQ ID NO: 8, and CDR3 is the polypeptide of SEQ ID NO: 9; (ii) a light chain (V L ) polypeptide comprising CDR1, CDR2 and CDR3, wherein CDR1 is the polypeptide of SEQ ID NO: 10, CDR2 is the polypeptide of SEQ ID NO: 11, and CDR3 is the polypeptide of SEQ ID NO: 12.

[0053] In certain embodiments, an anti-TauC3 antibody comprises (i) a variable heavy chain (V H ) polypeptide; (ii) light chain (V L ) polypeptide, wherein the variable heavy chain (V H ) polypeptide is the polypeptide of SEQ ID NO: 13, the variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18.

[0054] The anti-TauC3 antibody can also be a chimeric antibody, comprising (a) a heavy chain variable region comprising a CDR1 represented by SEQ ID NO: 7 or a sequence homologous to SEQ ID NO: 7, a CDR2 represented by SEQ ID NO: 8 or a sequence homologous to SEQ ID NO: 8, and a CDR3 represented by SEQ ID NO: 9 or a sequence homologous to SEQ ID NO: 9; and (b) a light chain variable region comprising a CDR1 represented by SEQ ID NO: 10 or a sequence homologous to SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11 or a sequence homologous to SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12 or a sequence homologous to SEQ ID NO: 12; and the binding affinity (KD) for TauC3 is 1x10 -10 and 1x10 -12 , the dissociation rate (Kd) is 1x10 -3 s -1 The following (for example, 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8 M, or no detectable binding to FLT (SEQ ID NO: 1).

[0055] The present invention also relates to an antigen-binding fragment of an antibody, which comprises (a) a heavy chain variable region comprising a CDR1 represented by SEQ ID NO: 7, a CDR2 represented by SEQ ID NO: 8, and a CDR3 represented by SEQ ID NO: 9; and (b) a light chain variable region comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12; and has a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , the dissociation rate (Kd) is 1x10 -3 s -1 The following (for example, 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) to FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8M, or has no detectable binding to FLT (SEQ ID NO: 1). The antigen-binding fragment of an antibody may be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a scFv fragment.

[0056] The present invention also relates to an antigen-binding fragment of an antibody, comprising (a) a heavy chain variable region comprising a CDR1 homologous to SEQ ID NO: 7, a CDR2 homologous to SEQ ID NO: 8, and a CDR3 homologous to SEQ ID NO: 9; (b) a light chain variable region comprising a CDR1 homologous to SEQ ID NO: 10, a CDR2 homologous to SEQ ID NO: 11, and a CDR3 homologous to SEQ ID NO: 12; and having a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , the dissociation rate (Kd) is 1x10 -3 s -1 The following (for example, 1x10 -4 Up to 1x10 -3 s -1 ), and the binding affinity (KD) for FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8 M, or has no detectable binding to FLT (SEQ ID NO: 1). The antigen-binding fragment of an antibody may be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a scFv fragment.

[0057] The present invention also relates to an antigen-binding fragment of an antibody, comprising (a) a heavy chain variable region comprising a CDR1 identical to SEQ ID NO: 7, a CDR2 identical to SEQ ID NO: 8, and a CDR3 identical to SEQ ID NO: 9; and (b) a light chain variable region comprising a CDR1 identical to SEQ ID NO: 10, a CDR2 identical to SEQ ID NO: 11, and a CDR3 identical to SEQ ID NO: 12; and having a 1x10 -10 and 1x10 -12 The binding affinity (KD) and 1x10 -3 s -1 The following dissociation rates (K d )(For example, 1x10 -4 To lx10 -3 s -1 ), and the binding affinity (KD) to FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8M, or has no detectable binding to FLT (SEQ ID NO: 1). The antigen-binding fragment of an antibody can be, for example, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a scFv fragment.

[0058] The present invention also relates to methods for blocking pathological tau uptake, methods for blocking pathological tau seeding activity, methods for inhibiting pathological tau protein aggregation, and methods for blocking pathological tau, tau fibrils, and tau aggregates from spreading from one neuron to another or from one part of the brain to another. The method comprises administering an effective amount of an anti-TauC3 antibody to a subject in need thereof. In some of these embodiments, the anti-TauC3 comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9; and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0059] Once administered, anti-TauC3 antibodies can block or slow the spread of pathological tau from one neuron to another or from one part of the brain to another by, for example, blocking or slowing TauC3 seeding activity (e.g., by substantially blocking or slowing the intracellular uptake of TauC3). This mechanism occurs extracellularly and does not require the presence of anti-TauC3 antibodies inside neurons. Anti-TauC3 antibodies are capable of blocking or slowing the spread of TauC3 tau, fibrils comprising TauC3, and aggregates comprising TauC3 from one neuron to another and from one part of the brain to another. Aggregates can contain heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated). In addition to blocking the intracellular uptake of TauC3 and fibrils comprising TauC3, anti-TauC3 antibodies can also block or slow pathological tau aggregation within cells (e.g., neurons). Since the antibody has substantially no affinity for full-length tau (e.g., 2N4R), the antibody does not interfere with the normal non-pathological function of full-length tau. In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0060] Anti-TauC3 antibodies can also slow down the spread of TauC3-containing fibrils and aggregates by binding to extracellular TauC3 and aggregates containing TauC3 released from cells, thereby preventing TauC3 and aggregates containing TauC3 from entering neighboring cells and slowing down the spread of tau aggregates from one neuron to another and from one part of the brain to another. Therefore, anti-TauC3 antibodies can be used as a means to prevent TauC3 or aggregates containing TauC3 from entering cells (e.g., neurons). In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0061] Anti-TauC3 antibodies can also be used to slow down and / or reduce the neuron-to-neuron spread of tau aggregates. For example, anti-TauC3 antibodies can promote the disaggregation of protein fibrils comprising TauC3, block the intracellular conversion of monomeric TauC3 to fibrils and / or aggregates comprising TauC3, and promote the intracellular degradation of fibrils comprising TauC3 and / or aggregates comprising TauC3. In addition to TauC3, fibrils and aggregates can also contain heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated). In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0062] Anti-TauC3 antibodies can reduce brain atrophy in subjects with tauopathy. In some embodiments, the anti-TauC3 antibodies comprise (a) a variable heavy chain (VH ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0063] Anti-TauC3 antibodies can also inhibit the formation of insoluble aggregates comprising a heterogeneous population of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated), such as reducing the amount of pathological tau in the brain (e.g., TauC3, fibrils comprising TauC3, and aggregates comprising TauC3). In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0064] In certain embodiments, the anti-TauC3 antibody inhibits pathological aggregation of full-length Tau (e.g., 2N4R). In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0065] In certain embodiments, administration of an anti-TauC3 antibody can immunize a subject against developing tauopathy. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0066] Administration of anti-TauC3 antibodies can alleviate the symptoms of tauopathy in a subject and / or slow the progression of tauopathy in a subject. For example, in certain embodiments, administration of anti-TauC3 antibodies can improve cognitive function and / or motor / sensory motor function in a subject with tauopathy. In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0067] The anti-TauC3 antibodies of the present invention can be used to treat tauopathy in human subjects. Administration of anti-TauC3 antibodies is particularly useful for treating Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD). In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9, and (b) a variable light chain (V L ) polypeptide comprising a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12.

[0068] In certain embodiments, the present invention relates to a method for reducing the spread of tau aggregation in a subject's brain, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the antibody binds to TauC3 but does not bind to full-length Tau. In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0069] The present invention further relates to a method for treating tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody sufficient to block TauC3 seeding activity, wherein the anti-TauC3 antibody is a humanized antibody. In some embodiments, the humanized anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0070] The present invention also relates to a method for treating tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody sufficient to block neuronal reuptake of TauC3, wherein the anti-TauC3 antibody is a chimeric antibody. In some embodiments, the anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0071] The present invention also relates to a method for treating Alzheimer's disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT is 1x10 -4 Up to 1x10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0072] The present invention also relates to a method for treating progressive supranuclear palsy (PSP) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 Up to 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 The binding affinity (KD) for FLT (SEQ ID NO: 1) was 1x10 -4 Up to 1x10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0073] The present invention also relates to a method for treating frontotemporal dementia (FTD) in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 Up to 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10 -4 Up to 1x10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0074] The present invention also relates to a method for treating traumatic brain injury (TBI) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 Up to 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10 -4 Up to 1x10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0075] The present invention also relates to a method for treating Pick's disease (PiD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 Up to 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10 -4 Up to 1x10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0076] The present invention also relates to a method for treating corticobasal degeneration (CBD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10 -4 Up to 1x10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0077] The present invention also relates to a method for treating frontotemporal lobar degeneration (FTLD) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC3 antibody, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT is 1x10 -4 To lx10 -8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L ) polypeptide comprising CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11 and CDR3 represented by SEQ ID NO: 12.

[0078] The present invention also relates to therapeutic agents and compositions for blocking the intracellular uptake of pathological tau; therapeutic agents and compositions for blocking tau seeding activity; therapeutic agents and compositions for blocking tau aggregation; and therapeutic agents and compositions for blocking the pathological spread of tau, tau fibrils, tau aggregates, and fragments of any of the foregoing substances from one part of the brain to another, which is induced or regulated by TauC3. The therapeutic agents and compositions include the anti-TauC3 antibodies described above and below. In addition to the anti-TauC3 antibodies, the compositions of the present invention may include one or more pharmaceutically acceptable excipients. The therapeutic agents or compositions can also be used for passive immunization and treatment of tau diseases, such as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), cortical basal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc. In certain embodiments, the composition may further include an agent that prevents the production of TauC3 (eg, a caspase inhibitor) or an agent that promotes clearance (eg, a small molecule TauC3 aggregation inhibitor).

[0079] The present invention further relates to a composition comprising an anti-TauC3 antibody and one or more pharmaceutically acceptable excipients, wherein the anti-TauC3 antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , dissociation rate (K d ) is 1x10 -3 s -1 Below, the binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10 -4 Up to 1x10 - 8 M. In some embodiments, an anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide comprising CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8 and CDR3 represented by SEQ ID NO: 9, (b) a variable light chain (V L) polypeptide, which comprises a CDR1 represented by SEQ ID NO: 10, a CDR2 represented by SEQ ID NO: 11, and a CDR3 represented by SEQ ID NO: 12. The composition can be, for example, a liquid composition. The composition comprises an effective amount of an anti-TauC3 antibody to treat tauopathy, including, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc. In certain preferred embodiments, the composition is stable (i.e., at least 90% of the anti-TauC3 antibodies in the composition retain their binding ability after storing the composition at 37°C for 21 days). BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 The protein and DNA sequences of the MoTau01 kappa light chain variable region.

[0081] Figure 2 The protein and DNA sequences of the MoTau01κ heavy chain variable region.

[0082] Figure 3 It is the pHuKLIC vector.

[0083] Figure 4 It is the pHuG4 LIC vector.

[0084] Figure 5 The protein and DNA sequences of the chimeric MoTau01 VK.

[0085] Figure 6 The protein and DNA sequences of the chimeric MoTau01 VH.

[0086] Figure 7 The binding ability of chimeric Tau01 to TauC3 and FL Tau was tested using an enzyme-linked immunosorbent assay.

[0087] Figure 8 Binding of murine and chimeric Tau01 antibodies to TauC3 and FL Tau was tested using Octet.

[0088] Fig. 9 are the protein and DNA sequences of Tau01 HA.

[0089] Fig.10 are the protein and DNA sequences of Tau01 HB.

[0090] Fig.11 are the protein and DNA sequences of Tau01 HC.

[0091] Fig.12 The protein and DNA sequences of Tau01 KA.

[0092] Fig.13 The protein and DNA sequences of Tau01 KB.

[0093] Fig.14 The protein and DNA sequences of Tau01 KC.

[0094] Fig.15 Humanized and chimeric Tau01 combined with TauC3: A and B forms.

[0095] Fig.16 ELISA of binding of humanized and chimeric Tau01 to TauC3: HA to HL variants.

[0096] Fig.17 Octet screen of humanized Tau01 antibodies against TauC3: HB, HC, and HF with KA-KC.

[0097] Fig.18 ELISA of the binding of humanized Tau01 antibody to TauC3: KA and KJ variants.

[0098] Fig.19 is an Octet screen of humanized Tau01 antibodies against TauC3:HB with KA to KJ variants.

[0099] Fig. 20 It is the second round of ELISA of binding of humanized Tau01 antibody to TauC3: HM, HN and HO variants.

[0100] Fig.21 This is the second round of Octet screening of humanized Tau01 antibodies against TauC3: HM, HN and HO variants.

[0101] Fig. 22 It is an ELISA containing the binding of humanized antibodies HC and HM to TauC3.

[0102] Fig.23 This is the second round of Octet screening of humanized Tau01 antibodies against TauC3: HM variants.

[0103] Fig.24 Second round of Octet screening of humanized Tau01 antibodies against TauC3 and FL Tau: Lead variants.

[0104] Fig.25is the off-rate ranking of lead humanized candidates with TauC3 using Biacore.

[0105] Fig.26 It is a Biacore binding test for FL Tau.

[0106] Fig. 27 is the thermal stability of chimeric and humanized antibody candidates.

[0107] Fig.28 is the SEC-MALS clustering analysis of purified Mo Tau01HuG4K and Tau01HCKB HuG4K antibodies.

[0108] Fig.29 is DLS analysis of purified Tau01 HMKN, HMKO, HMKP, HMKE and HMKM HuG4K antibodies.

[0109] Fig.30 are mass spectra of purified chimeric and humanized candidate antibodies.

[0110] Fig.31 is the Biacore kinetics of the humanized candidate antibody.

[0111] Fig.32 Thermal shift analysis of humanized antibody candidates.

[0112] Fig.33 Non-specific protein-protein interactions (cross-interaction chromatography) of humanized candidate antibodies.

[0113] Fig.34 Solubility assessment of purified humanized antibody candidates.

[0114] Fig.35 Humanized candidate antibodies were subjected to freeze / thaw and heat stress analysis by circular dichroism.

[0115] Fig.36 is capillary isoelectric focusing to determine the isoelectric point of the humanized candidates.

[0116] Fig.37 It is the serum stability assessment of humanized candidate antibodies.

[0117] definition

[0118] As used herein, "antibody" is intended to include intact molecules (i.e., full-length antibodies (IgM, IgG, IgA, IgE)) and fragments thereof, as well as synthetic and biological derivatives thereof, such as Fab, F(ab')2, and Fv fragments, which are free or expressed on the surface of filamentous phage, such as pill or pVIII or other surface proteins, or on the surface of bacteria, which are capable of binding antigen. C Fab, F(ab')2, and Fv fragments of the fragment are cleared from the circulation more quickly and may have less nonspecific tissue binding to the antibody. The antibody may be a monoclonal antibody. Recombinant antibodies include the term "antibody". The term "antibody" includes chimeric antibodies and humanized antibodies. The antibody may also be a fully human antibody (e.g., from a transgenic mouse or phage).

[0119] As used herein, the term "humanized antibody" refers to an antibody in which the complementarity determining region (CDR) of a mouse or other non-human antibody is grafted onto a human antibody framework. A human antibody framework refers to an entire human antibody excluding the CDR.

[0120] As used herein, the term "human antibody" refers to antibodies in which the entire sequence is derived from human genetic pools (eg, from transgenic mice or phages).

[0121] As used herein, the term "homologous" means that the sequence has at least 80% identity with its homologous sequence, and a polymeric peptide (e.g., an antibody) comprising the homologous sequence has substantially the same biological activity as a polymeric peptide comprising its homologous sequence. For example, a humanized antibody comprises (a) a heavy chain variable region comprising a CDR1 represented by the sequence GFTFNTYA (SEQ ID NO: 7), a CDR2 represented by IRSKSNNYAT (SEQ ID NO: 8), and a CDR3 represented by VGGGDF (SEQ ID NO: 9); (b) a light chain variable region comprising a CDR1 represented by the sequence QEISVY (SEQ ID NO: 10), a CDR2 represented by the sequence GAF (SEQ ID NO: 11), and a CDR3 represented by the sequence LQYVRYPWT (SEQ ID NO: 12); and the antibody in which one or more CDR sequences are replaced by homologous sequences has a binding affinity (KD) for TauC3 of 1x10 -10 and 1x10 -12 , and the binding affinity (KD) for FLT is 1x10 -4 Up to 1x10 -8 M, or no detectable binding to FLT. By definition, homologous antibodies have substantially similar three-dimensional shapes.

[0122] As used herein, "CDR" means "complementarity determining region". A CDR may also be referred to as a hypervariable region. Unless otherwise indicated, the CDR sequences disclosed herein are defined by the IMGT numbering system.

[0123] As used herein, "represented by SEQ ID NO:" with respect to a CDR sequence means that the sequence of the CDR is identical or homologous to the stated SEQ ID NO.

[0124] As used herein, the term "chimeric antibody" refers to an antibody in which the entire variable region of a mouse or rat antibody is expressed together with a human constant region.

[0125] The term "mouse anti-TauC3 antibody" as used herein refers to the "TauC3 antibody" in Nicholls, SB, SL De Vos, C. Commins, C. Nobuhara, RE Bennett, DL Corjuc, E. Maury et al. 2017. "Characterization of TauC3 antibody and demonstration of its potential to block tau propagation." PLoS ONE 12(5): e0177914.

[0126] As used herein, a "light chain" is a small polypeptide subunit of an antibody. A typical antibody comprises two light chains and two heavy chains.

[0127] As used herein, a "heavy chain" is a large polypeptide subunit of an antibody. The heavy chain of an antibody comprises a series of immunoglobulin domains, having at least one variable domain and at least one constant domain.

[0128] As used herein, the term "affinity" refers to the strength with which an antibody molecule binds to its epitope. Affinity was determined by surface plasmon resonance (SPR) using Biacore kinetics.

[0129] The term "KD" as used herein refers to the equilibrium dissociation constant (KD = Kd / Ka, wherein Kd is the dissociation rate constant and Ka is the association rate constant).

[0130] As used herein, the term "immunodepletion" refers to the removal of proteins by the use of antibodies. The term "immunodepletion" is used interchangeably with the term "immunoprecipitation". The term refers to the ability of an antibody to reduce or immunoprecipitate (IP) a target antigen from a sample, which results in immunodepletion.

[0131] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to the amount of a therapeutic agent (e.g., an anti-TauC3 antibody) or composition that results in a measurable clinical effect in a subject. The effective amount of the therapeutic agent is determined by the circumstances surrounding the case, including the compound administered, the route of administration, the state of the symptoms being treated, and other considerations such as similar subjects and administration circumstances. An "effective amount" generally includes about 0.0001 mg / kg to about 100 mg / kg, preferably 0.5 mg / kg to 20 mg / kg of an anti-TauC3 antibody described herein. In certain embodiments, an amount of 1 mg / kg, 3 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, or 10 mg / kg is used.

[0132] The term "pathological tau" includes TauC3, fibrils comprising TauC3, and aggregates comprising TauC3 (e.g., a heterogeneous population comprising full-length tau, tau oligomers, and / or post-translationally modified tau (truncated or phosphorylated)). In addition to TauC3, pathological tau may also include a heterogeneous population of full-length tau (e.g., 2N4R), tau oligomers, and / or post-translationally modified tau (truncated or hyperphosphorylated).

[0133] The term "TauC3" refers to a C-terminally truncated tau fragment ending at aspartic acid 421 of htau40 (SEQ ID NO: 1).

[0134] As used herein, "FLT" is an abbreviation for full-length Tau (e.g., htau40 (SEQ ID NO: 1).

[0135] The terms "treating" or "treatment" include the attenuation, reversal or amelioration of at least one symptom or symptom associated with tauopathy.

[0136] The term "seeding" refers to the extracellular activity that precedes the intracellular aggregation of TauC3, TauC3-containing fibrils and / or TauC3-containing aggregates as part of a heterogeneous population of full-length tau (e.g., 2N4R), tau oligomers and / or post-translationally modified tau (truncated or hyperphosphorylated).

[0137] The term "aggregation" refers to the activities that occur within cells after TauC3 and / or fibrils comprising TauC3 and / or aggregates comprising TauC3 are taken up by cells.

[0138] "ExpiCHO" is the abbreviation for Chinese Hamster Ovary (CHO High Density / Serum-free) cells.

[0139] "A" is the abbreviation for adenine.

[0140] "bp" is the abbreviation for base pair.

[0141] "℃" is the abbreviation for degrees Celsius.

[0142] "C" is the abbreviation for cytosine.

[0143] "MEM" is the abbreviation for Minimum Essential Medium.

[0144] "DNA" is the abbreviation for deoxyribonucleic acid.

[0145] "ELISA" is the abbreviation for enzyme-linked immunosorbent assay.

[0146] "EC50" is an abbreviation for the concentration of an antibody that elicits 50% of the maximal response.

[0147] "EC80" is an abbreviation for the concentration of antibody that elicits 80% of the maximal response.

[0148] "ECD" is the abbreviation for extracellular domain.

[0149] "g" is the abbreviation for gram.

[0150] "G" is the abbreviation for guanine.

[0151] "HRP" is the abbreviation for horseradish peroxidase.

[0152] "IgG" is the abbreviation for immunoglobulin-G.

[0153] "K" is an abbreviation for G or T (IUPAC convention).

[0154] "LIC" is an abbreviation for ligase-independent cloning.

[0155] "Min" is the abbreviation for minute.

[0156] "M" is an abbreviation for A or C (IUPAC convention).

[0157] "Nm" is the abbreviation for nanometer.

[0158] "OD" is the abbreviation for optical density.

[0159] "PBS" is the abbreviation for phosphate buffered saline.

[0160] "PCR" is the abbreviation for polymerase chain reaction.

[0161] "R" is an abbreviation for A or G (IUPAC convention).

[0162] "RT" is the abbreviation for room temperature.

[0163] "s" is the abbreviation for seconds.

[0164] "S" is an abbreviation for C or G (IUPAC convention).

[0165] "T" is the abbreviation for thymine.

[0166] "TBS" is the abbreviation for Tris-buffered saline.

[0167] "UV" is the abbreviation for ultraviolet light.

[0168] "V" is an abbreviation for A or C or G (IUPAC convention).

[0169] "VCI" is an abbreviation for vernier residue, canonical residue and interface residue.

[0170] "VH" is the abbreviation for immunoglobulin heavy chain variable region.

[0171] "VK" is the abbreviation for the variable region of immunoglobulin kappa light chain

[0172] "W" is an abbreviation for A or T (IUPAC convention).

[0173] "Y" is an abbreviation for C or T (IUPAC convention). DETAILED DESCRIPTION

[0174] TauC3 is one of the many existing high molecular weight species responsible for tau aggregation and seeding activity. In addition to TauC3, tau aggregates may also contain heterogeneous populations of full-length (normal tau), tau oligomers and / or post-translationally modified tau (truncated or hyperphosphorylated). It has been shown that other neurodegenerative diseases besides sporadic AD also have increased TauC3 levels.

[0175] TauC3 is neurotoxic and may cause microtubule dysfunction. TauC3 may also be responsible for the spread of Tau fibrils from one part of the brain to another.

[0176] Anti-TauC3 Antibody

[0177] The anti-TauC3 antibodies of the present invention recognize aggregated, extracellular forms of pathological TauC3. The anti-TauC3 antibodies of the present invention can be, for example, chimeric, humanized or human anti-TauC3 antibodies.

[0178] When tested against recombinant TauC3 protein, the anti-TauC3 antibodies showed very tight binding specificity for the target caspase-cleaved Tau protein. In certain embodiments, the anti-TauC3 antibodies blocked seeding in biosensor assays and effectively blocked entry into neurons of species responsible for inducing intracellular tau aggregation (i.e., effectively blocked TauC3 and TauC3 fibrils from entering cells).

[0179] Anti-TauC3 antibodies typically have subnanomolar specificity for TauC3, and have a specificity for TauC3 that is at least 100 times greater than that for full-length Tau (2N4R) (e.g., 100 times or more greater than that for full-length Tau). For example, an anti-TauC3 antibody may have a specificity for TauC3 that is 150 to 5000 times greater than that for full-length Tau (2N4R). In certain embodiments, an anti-TauC3 antibody has a specificity for TauC3 that is 500 to 2500 times greater than that for full-length Tau (2N4R). In certain embodiments, an anti-TauC3 antibody has a specificity for TauC3 that is 750 to 2000 times greater than that for full-length Tau (2N4R). In certain embodiments, an anti-TauC3 antibody has a specificity for TauC3 that is 1000 to 1500 times greater than that for full-length Tau (2N4R). In all of these embodiments, the anti-TauC3 antibodies may have no detectable binding to full-length Tau (2N4R).

[0180] In certain embodiments, the antibodies of the invention are chimeric, humanized or human anti-TauC3 antibodies that have a higher binding affinity (KD) for TauC3 than a murine anti-TauC3 antibody. In some embodiments, the binding affinity (KD) of chimeric, humanized and human anti-TauC3 antibodies for TauC3 is at least 2-fold, 3-fold or 4-fold higher than the binding affinity (KD) of a murine anti-TauC3 antibody for TauC3. In some embodiments, the binding affinity KD of a murine anti-TauC3 antibody for TauC3 is about 4.9x10 -11 M, the binding affinity KD of chimeric, humanized and human anti-TauC3 antibodies to TauC3 is about 1x10 -11 M to about 2.5x10 -11 M. The chimeric, humanized and human anti-TauC3 antibodies of the present invention may have a binding affinity KD for TauC3 of, for example, about 1.1 x 10 -11 M, about 1.3x10 -11 M, about 1.5x10 -11 M, about 1.7x10 -11 M, about 1.9x10 -11 M, about 2.1x10 -11M or about 2.3x10 -11 M. In some embodiments, the binding affinity KD of the murine anti-TauC3 antibody to TauC3 is about 3.9x10 -11 M, the binding affinity KD of chimeric, humanized and human anti-TauC3 antibodies to TauC3 is about 1x10 -11 M to about 2.5x10 -11 M. The chimeric, humanized and human anti-TauC3 antibodies of the present invention may have a binding affinity KD for TauC3 of, for example, about 1.1 x 10 -11 M, about 1.3x10 -11 M, about 1.5x10 -11 M, about 1.7x10 -11 M, about 1.9x10 -11 M, about 2.1x10 -11 M, or about 2.3x10 -11 M.

[0181] In certain embodiments, the antibody is present at 1×10 -10 M to 1x10 -11 The equilibrium constant KD of M binds to TauC3; and the equilibrium constant KD of M with 2N4R is 1x10 -4 M to 1x10 -8 M or shows no detectable binding to full-length Tau (e.g., 2N4R). In a preferred embodiment, the anti-TauC3 antibody is used at 1x10 -9 M to 1x10 -12 The equilibrium constant KD of M binds to TauC3 and is 1x10 -8 M to 9x10 -8 The antibody has an equilibrium constant KD of 1×10 M for binding to the full length (e.g., 2N4R) or shows no detectable binding to 2N4R. In some of these embodiments, the antibody has a very slow off-rate for TauC3 (i.e., an off-rate (Kd) of 1×10 -4 Up to 1x10 -3 s -1 ) and has essentially no affinity for 2N4R (i.e., ka is less than 100,0001 / MS).

[0182] In certain embodiments, the anti-TauC3 antibody is a chimeric or humanized antibody having a KD value for TauC3 of about 5 pM to about 90 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 to about 40 pM, or about 10 pM to about 35 pM.

[0183] In certain embodiments, the anti-TauC3 antibody is a chimeric or humanized antibody having a KD value for TauC3 of about 10 to about 90 pM and a very slow dissociation rate, such as less than 2×10 -3 s -1 In other words, these antibodies are highly specific for TauC3 (TauC3 is the target protein produced in diseased states) and have slow off rates, both of which are ideal for antibodies used in immunization strategies.

[0184] Anti-TauC3 antibodies include, but are not limited to, monoclonal, chimeric, humanized, single chain, Fab fragments, and Fab expression libraries. Anti-TauC3 antibodies may be natural or recombinant, immobilized, free in solution, or displayed on the surface of various molecules or bacteria, viruses, or other surfaces.

[0185] In certain embodiments, the anti-TauC3 antibody recognizes the sequence SSTGSIDMVD (SEQ ID. No. 23) located at the C-terminus of TauC3, but does not recognize the same sequence when it is present within FLT.

[0186] Anti-TauC3 antibodies (eg, humanized antibodies) useful according to the invention can be administered to a subject who may be susceptible to or suffering from a tauopathy to block seeding and / or aggregation of TauC3 and thereby treat one or more symptoms of the tauopathy.

[0187] In another embodiment of the present invention, the anti-TauC3 antibody of the present invention can be conjugated with a cytoprotectant or an agent that promotes and / or improves the ability of the antibody to cross the blood-brain barrier ("BBB"). The cytoprotectant can be an antioxidant (e.g., melatonin); the agent that promotes or improves the ability of the antibody to cross the blood-brain barrier is a hydrophobic substance that can cross the blood-brain barrier and is generally recognized as sage (GRAS) by the U.S. Food and Drug Administration ("FDA"). The cytoprotectant or the agent that promotes or improves the ability of the antibody to cross the BBB can be coupled to the antibody directly or through a linker. The linker can be selected from: a hydrazine linker, a disulfite linker, a thioether linker, and a peptide linker. In certain embodiments, the equilibrium constant KD of the antibody for TauC3 is 2-3 orders of magnitude higher than the equilibrium constant KD of the antibody for 2N4R, and the cytoprotectant is melatonin.

[0188] How to use

[0189] In one aspect, the present invention provides an anti-TauC3 antibody for a living person suffering from or at risk of developing a tauopathy. Tauopathy includes, for example, Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), traumatic brain injury (TBI), Pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal lobar degeneration (FTLD), etc.

[0190] Methods for blocking the proliferation of pathological Tau aggregates

[0191] In one aspect, the invention relates to a method of blocking the spread of pathological tau from one neuron to another or from one part of the brain to another.

[0192] In one aspect, the invention relates to a method of blocking TauC3 seeding activity in the brain of a subject.

[0193] In another aspect, the invention relates to a method of reducing the spread of pathological Tau aggregates in the brain of a subject.

[0194] The present invention further relates to methods of reducing the spread of aggregates comprising TauC3 in the brain of a subject.

[0195] The present invention further relates to methods of reducing the spread of fibrils comprising TauC3 in the brain of a subject.

[0196] In another aspect of the invention, the present invention relates to a method of reducing the intracellular aggregation of tau induced by the intracellular uptake of TauC3 and TauC3 fibrils.

[0197] In each aspect, the method includes administering a therapeutically effective amount of an anti-TauC3 antibody to a human. The anti-TauC3 antibody is capable of uniquely recognizing aggregated extracellular forms of pathological tau without binding to physiological tau. In a preferred embodiment, a key portion of the anti-TauC3 antibody epitope is a carboxyl group that forms a new epitope at the C-terminal residue of a peptide corresponding to the last ten C-terminal residues of TauC3 (e.g., TauC3 or SEQ ID: 23). The equilibrium constant KD of the anti-TauC3 antibody for TauC3 is 2-3 orders of magnitude higher than the equilibrium constant KD of the antibody for 2N4R and one or more pharmaceutically acceptable excipients. The anti-TauC3 antibody is 1x10 -10 M to 1x10 -11 The equilibrium constant KD of M binds to TauC3, but the equilibrium constant KD of full-length tau (e.g., 2N4R) is 1x10 -4 M to 1x10 -8 M, or no detectable binding to full-length tau (e.g., 2N4R). In a preferred embodiment, the anti-TauC3 antibody is used at 1x10-11 M to 9x10 -11 The equilibrium constant KD of M for binding to TauC3 is 1x10 -8 M to 9x10 -8 The antibody preferably has a very slow dissociation rate from TauC3 and has substantially no affinity for 2N4R (i.e., ka is less than 100,0001 / MS). The antibody can be, for example, selected from a humanized antibody, a chimeric antibody, or an immune fragment of any of the foregoing antibodies. In a preferred embodiment, the antibody is an antibody selected from the humanized anti-TauC3 antibodies described herein.

[0198] Prior to administering a therapeutically effective amount of an anti-TauC3 antibody, a person may or may not have symptoms associated with tau aggregation. In other words, a person may or may not experience symptoms associated with tau seeding and / or aggregation. One of ordinary skill in the art will appreciate that pathological tau seeding and aggregation may begin before diagnosis or the onset of symptoms associated with tau aggregation. In some embodiments, a person has symptoms associated with tau seeding and / or aggregation. In other embodiments, a person does not have symptoms associated with tau seeding and / or aggregation. In other embodiments, a person has detectable tau pathology but does not have any other symptoms associated with tau symptoms and / or aggregation. Reducing the spread of tau aggregates in the human brain by administering therapeutic agents and pharmaceutical compositions according to the present invention can reduce the development and / or progression of symptoms associated with pathological seeding and / or aggregation of tau.

[0199] Therefore, prevention, inhibition or slowing down of the diffusion of pathological tau aggregates can be used to treat the pathology associated with the generation and diffusion of tau aggregates.A definition of symptoms associated with tau inoculation and / or aggregation refers to any symptom caused by the formation of tau aggregates composed in part of tau fibrils.Exemplary diseases with symptoms associated with tau aggregation include, but are not limited to, progressive supranuclear palsy, pugilistic dementia (chronic traumatic encephalopathy), frontotemporal dementia and Parkinson's disease associated with chromosome 17, Lytico-Bodig disease (Guam Parkinson's dementia), tangled dominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, pallidonivarius degeneration (Hallervorden-Spatz disease), lipofuscinosis, Pick's disease, cortical basal ganglia degeneration, argyrophilic granulopathy (AGD), frontotemporal lobar degeneration, Alzheimer's disease and frontotemporal lobar dementia.The method for diagnosing these diseases is known in the art.

[0200] Exemplary symptoms associated with tau inoculation or aggregation may include, for example, impaired cognitive function, behavioral changes, mood disorders, epileptic seizures, and impaired nervous system structure or function. Impaired cognitive function includes, but is not limited to, difficulties with memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and organization. Altered behavior includes, but is not limited to, physical or verbal aggression, impulsivity, decreased inhibition, apathy, decreased initiative, personality changes, abuse of alcohol, tobacco, or drugs, and other addiction-related behaviors. Emotional disorders include, but are not limited to, depression, anxiety, mania, irritability, and emotional instability. Epileptic seizures include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic psychogenic seizures. Impaired nervous system structure or function includes, but is not limited to, hydrocephalus, Parkinson's disease, sleep disorders, psychosis, balance and coordination disorders. This includes movement disorders such as monoparesis, hemiplegia, quadriplegia, ataxia, convulsions, and tremors. This also includes sensory loss or dysfunction, including smell, touch, taste, vision, and hearing. In addition, this includes autonomic nervous system impairment, such as bowel and bladder dysfunction, sexual dysfunction, blood pressure and temperature dysregulation. Finally, this includes hormonal impairment attributable to hypothalamic and pituitary dysfunction, such as deficiency and dysregulation of growth hormone, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, gonadotropin-releasing hormone, prolactin and many other hormones and regulators. Methods for detecting and evaluating symptoms associated with tau aggregation are known in the art.

[0201] In some embodiments, the symptoms associated with tau aggregation refer to dementia. Dementia itself is not a specific disease, but an overall term that describes a wide range of symptoms associated with a decline in memory or other thinking abilities, the severity of which is sufficient to reduce a person's ability to perform daily activities. Dementia is also a common clinical feature of many diseases associated with tau protein aggregation. Experienced practitioners will be familiar with a variety of methods that can be used to diagnose the severity of dementia. For example, several cognitive tests and screening questionnaires for dementia are known in the art, all of which have varying degrees of sensitivity and specificity. Non-limiting examples include the Mini-Mental State Examination (MMSE), the Mini-Mental State Examination (AMTS), the Modified Mini-Mental State Examination (3MS), the Cognitive Dysfunction Screening Form (CASI), the Trail-making test, the Clock Drawing Test, the Questionnaire for Cognitive Decline in the Elderly, the General Practitioner Cognitive Assessment, the Clinical Dementia Rating (CDR), and the 8-item Dementia Screening Form (AD8).

[0202] In some embodiments, a clinical dementia score is used to quantify the severity of dementia symptoms. Using the clinical dementia score, 0 points represent no symptoms, 0.5 points represent very mild symptoms, 1 point represents mild symptoms, 2 points represent moderate symptoms, and 3 points represent severe symptoms. Therefore, any increase in a person's clinical dementia score indicates cognitive deterioration and increased dementia. In addition, a change in the clinical dementia score from 0 to greater than 0 indicates the development or onset of dementia.

[0203] In some embodiments, symptoms associated with tau seeding or aggregation refer to tau pathology or tauopathy. The term "tau pathology" or "tauopathy" refers to pathological seeding or aggregation of tau. In some embodiments, tau pathology refers to neurofibrillary tangles. In other embodiments, tau pathology refers to hyperphosphorylated tau. In other embodiments, tau pathology refers to high levels of tau aggregates detectable in blood, plasma, serum, CSF or ISF, 2 to about 40 times higher than the levels detected in individuals without the disease.

[0204] Application

[0205] Administration of the anti-TauC3 antibodies described herein can be used as a treatment or immunotherapy for tauopathy.

[0206] Preferably, the therapeutically effective amount of the antibody (including immunoreactive fragment) of pharmaceutical grade can be administered to humans. Standard effective techniques are used for administration, including peripheral administration (i.e., not administration to the central nervous system) or topical administration to the central nervous system. Peripheral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, oral, sublingual, or suppository administration. Topical administration includes direct access to the central nervous system (CNS), including, but not limited to, controlled release formulations implanted via lumbar, intraventricular, or intraparenchymal catheters or using surgery.

[0207] Suitable people for treatment include individuals who are at risk of disease but do not show symptoms, as well as subjects who currently show symptoms. As far as Alzheimer's disease is concerned, almost anyone is at risk of suffering from Alzheimer's disease. Therefore, the present method can be applied to the general population prophylactically without any assessment of the risk of the subject. This preventive administration can start, for example, at the age of 50 or older. The present method is particularly useful for individuals who do have a known genetic risk of tau protein disease (e.g., Alzheimer's disease). Such individuals include those who have relatives who have suffered from this disease, and individuals whose risks are determined by genetic or biochemical marker analysis. For example, genetic markers of Alzheimer's disease risk include mutations in the APP gene, particularly mutations at position 717 and mutations at positions 670 and 671, respectively referred to as Hardy mutations and Swedish mutations. Other risk markers are mutations in presenilin genes (PS1 and PS2) and ApoE4, AD family history, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be identified from characteristic dementia by the presence of the above-mentioned risk factors. In addition, a number of diagnostic tests are available to identify individuals with AD. These include imaging and / or measuring CSF tau and Aβ42 levels. Elevated tau protein and decreased Aβ42 levels indicate the presence of AD. Individuals with Alzheimer's disease can also be diagnosed by the Alzheimer's Disease and Related Disorders Association criteria.

[0208] In asymptomatic subjects, treatment can start at any age (e.g., 10, 20, 30, 40, 50, or 60). However, treatment does not need to be started before the subject reaches 40, 50, 60, 70, 75, or 80 years old. Treatment usually requires multiple administrations over a period of time. Treatment can be monitored by analyzing the reaction of antibodies, or activated T cells or B cells to therapeutic agents over time. If the reaction decreases, a booster dose is indicated. In the case of potential Down syndrome subjects, treatment can start antenatally by administering therapeutic agents to the mother or administering therapeutic agents shortly after birth.

[0209] In preventive applications, pharmaceutical compositions or medicines are applied to subjects who are susceptible to or have a risk of suffering from tau disease in an amount that is enough to eliminate or reduce risk, mitigate severity or delay the onset of disease, including biochemical, histological and / or behavioral symptoms, its complications and intermediate pathological phenotypes that appear in the disease process. In therapeutic applications, compositions or medicines are applied to subjects who suspect or have suffered from such diseases in an amount that is enough to cure or at least partially prevent the biochemical, histological and / or behavioral symptoms of the disease, including complications and intermediate pathological phenotypes in the disease process. In some methods, the use of medicaments reduces or eliminates mild cognitive impairment. An amount that is enough to complete treatment or preventive treatment is defined as a treatment or prevention effective dose or amount. In preventive and therapeutic schemes, medicaments are usually applied with several dosages until enough immune responses are obtained. Usually, immune responses are monitored and repeated doses are given when immune responses begin to weaken.

[0210] The effective dose of the compositions of the present invention for treating the above-mentioned conditions depends on many different factors, including the mode of administration, the target site, the physiological state of the subject, other drugs administered, and whether the treatment is preventive or therapeutic. The therapeutic dose needs to be tested case by case in clinical trials and titrated frequently to optimize safety and effectiveness. Another advantage of the anti-TauC3 antibodies of the present invention in certain embodiments may be that, for equal mass doses, the anti-TauC3 antibody doses of the present invention contain higher molar doses of effective clearance and / or "inactivation" antibodies compared to antibody compositions containing anti-TauC3 antibodies with lower specificity for TauC3 than the anti-TauC3 antibodies according to the present invention. Typically, the anti-TauC3 antibodies of the present invention will be administered by intravenous infusion or subcutaneous injection. The amount of anti-TauC3 antibody administered by intravenous infusion can vary from 0.5 to 10 mg per subject. Subcutaneous injections generally require higher doses to reach the brain in sufficient amounts. Antibodies (e.g., complete IgG molecules) can be administered once a month.

[0211] In some methods, two or more antibodies (e.g., recombinant, monoclonal, chimeric and / or humanized) with the same or different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the range shown. In this case, both or more antibodies can be directed against, for example, truncated tau. Alternatively, one or more antibodies can be directed against, for example, truncated tau, and one or more additional antibodies can be directed against the amyloid-β (Aβ) peptide associated with Alzheimer's disease. Antibodies are typically administered multiple times. The intervals between single doses can be hourly, daily, weekly, monthly, or yearly. In some methods, the dose is adjusted to achieve a plasma antibody concentration of 1-1000 μg / ml, and in some methods 25-300 μg / ml. Alternatively, the antibody can be administered as a sustained-release formulation, in which case less frequent administration is required.

[0212] The dosage and frequency vary according to the half-life of the antibody in the subject. In general, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some subjects will continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to use relatively high doses at relatively short intervals until the progression of the disease slows or terminates, preferably until the subject shows partial or complete improvement in symptoms of the disease. Thereafter, the patient can be treated prophylactically.

[0213] The dose of anti-TauC3 antibody that blocks TauC3 seeding is not necessarily the same as the dose of anti-TauC3 antibody that inhibits TauC3 aggregation. Based on the information provided in this specification, the specific dose can be determined by routine experiments.

[0214] The efficacy of administration / treatment can be assessed by measuring the levels of pathogenic tau or phosphorylated tau in plasma and / or CSF. Based on this assessment, the dosage and / or frequency of administration can be adjusted accordingly.

[0215] In certain embodiments, cognitive effects may also be assessed.

[0216] The effect can also be assessed by the degree of brain atrophy determined by MRI

[0217] The safety of dosing / treatment can be assessed by the number of participants experiencing adverse events (AEs), serious AEs, and abnormalities in clinical laboratory tests, vital signs, ECG, MRI, and physical and neurological examinations, and cognitive deterioration. Based on this assessment, the dosing dose and / or frequency can be adjusted accordingly.

[0218] Anti-TauC3 antibodies and immunogens can be administered intranasally, subcutaneously, intramuscularly, by IV infusion, transdermally, orally, or as described in more detail below.

[0219] Pharmaceutical composition

[0220] The pharmaceutical composition according to the present invention comprises the anti-TauC3 antibody or fragment thereof described herein, and one or more pharmaceutically acceptable excipients. The anti-TauC3 antibody is 1x10 -10 M to 1x10 -11 The equilibrium constant KD of M for binding to TauC3 is 1x10 -4 M to 1x10 -8M, or no detectable binding to full-length tau (e.g., 2N4R). In a preferred embodiment, the anti-TauC3 antibody is used at 1x10 -11 M to 9x10 -11 The equilibrium constant KD of M for binding to TauC3 is 1x10 -8 M to 9x10 -8 The equilibrium constant KD of M binds to 2N4R or shows no detectable binding to 4RTau. Preferably, the anti-TauC3 antibody has a very slow dissociation rate from TauC3 (i.e., a dissociation rate of 1×10 -4 Up to 1x10 -3 s -1 ) and have substantially no affinity for 4RTau (ie, ka less than 100,0001 / MS). The antibody can be, for example, a humanized, chimeric or human (eg, from a tg mouse) antibody.

[0221] The pharmaceutical composition is designed to be suitable for the selected mode of administration and appropriately uses pharmaceutically acceptable excipients, such as compatible dispersants, buffers, surfactants, preservatives, solubilizers, isotonic agents, stabilizers, and the like.

[0222] Effective peripheral systemic delivery by intravenous or subcutaneous injection is the preferred method of administration to living subjects. Suitable carriers for such injections are readily appreciated.

[0223] The concentration of the humanized antibody in the formulation to be administered is an effective amount and ranges from as low as about 0.1% by weight to as high as about 95 or about 99.9% by weight, and will be selected primarily based on fluid volumes, viscosities, etc., as needed to select a particular mode of administration. In certain embodiments, the antibody may comprise about 15% or about 20% by weight of the composition.

[0224] The composition for injection into the subject can be prepared as any one or combination of 1-250 ml of sterile buffered water containing phosphate buffered saline and about 1-5000 mg of the anti-TauC3 antibody of the present invention. The preparation can be sterile filtered after preparation, or prepared in other ways that are acceptable to microbiology. The volume of a typical composition for intravenous infusion can be 1-250 ml of fluid (e.g., sterile Ringer's solution), and the anti-tau antibody concentration can be 1-100 mg / ml or higher. The discovered therapeutic agent can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier before use. Lyophilization and reconstitution may result in varying degrees of antibody activity loss (e.g., when conventional immunoglobulins are used, the activity loss of IgM antibodies is often greater than that of IgG antibodies).

[0225] The dosage is an effective amount for the specified purpose and may have to be adjusted to compensate. The pH of the formulation, which is generally of pharmaceutical quality, will be selected to balance antibody stability (chemical and physical) and the comfort of the subject during administration. Typically, a pH between 4 and 8 can be tolerated. Dosages will vary from individual to individual based on the size, weight, and other physiological and biological characteristics of the individual receiving successful administration.

[0226] In one aspect, a typical dose comprises about 0.1 mg to about 10 mg of an anti-TauC3 antibody described herein. In certain embodiments, a typical dose comprises about 0.5 mg to about 10 mg of an anti-TauC3 antibody. The dose may be in the range of about 0.55 mg / kg to about 10 mg / kg. The dosing frequency of a complete IgG antibody is typically once a month, while antibody fragments require more frequent dosing due to their shorter half-life to effectively treat symptoms.

[0227] The timing of treatment administration relative to the disease itself and the duration of treatment will depend on the circumstances surrounding the case. Treatment can be initiated after the disease associated with tau aggregation is diagnosed. Alternatively, treatment can be initiated after clinical confirmation of symptoms associated with tau aggregation. In addition, treatment can be initiated after tau pathology is detected. Treatment can be initiated immediately in a hospital or clinic, or after discharge from the hospital or after an outpatient visit. The duration of treatment can range from a single dose one-time administration to a lifelong course of treatment.

[0228] Although the above methods appear to be the most convenient, appropriate and effective for the administration of proteins such as humanized antibodies, other effective administration techniques such as intracerebroventricular administration, transdermal administration and oral administration may also be employed by appropriate adaptation, provided that appropriate formulations are used therein.

[0229] Based on the information provided herein and knowledge available in the art, a typical effective amount or dosage can be determined and optimized using standard clinical techniques, and will depend on the mode of administration.

[0230] Example 1: (Determination of mouse MoTau01 antibody sequence)

[0231] Sequence determination of MoTau01 antibody

[0232] Preparation of RNA from hybridoma cells.

[0233] Cryoprecipitates of mouse hybridoma cells (MoTau01) stored at -80°C were provided by Genscript on behalf of Tau-Biologic and processed using the Qiagen RNeasy kit to isolate RNA according to the manufacturer's protocol.

[0234] First-strand cDNA synthesis

[0235] MoTau01 RNA (~21 μg) was reverse transcribed to produce cDNA using the GE Life Sciences First Strand cDNA Synthesis Kit according to the manufacturer's protocol and purified as described in Example 5. This was repeated twice to generate three independent cDNA products (rounds 1, 2, and 3) to detect and avoid reverse transcriptase-induced cDNA mutations.

[0236] cDNA sequence determination

[0237] MoTau01 cDNA was amplified by PCR as described in Example 5. Immunoglobulin cDNA was PCR amplified using Phusion Flash High Fidelity PCR Master Mix with either kappa light chain primers plus MKC (Table 1) or heavy chain primers (1-12 and 14) plus MHC mix (Table 2). The MoTau01 VH PCR primer set failed to generate any product.

[0238] Therefore, additional primers were designed based on known sequences in the leader and terminal regions to facilitate cloning of VH domains from hybridoma cells. Additional primer sequences are included in the primer table as MHV13, in the "Additional Primers" section (Table 2).

[0239] Table 1. PCR primers used to amplify mouse VK

[0240]

[0241]

[0242]

[0243] Fuzzy base code: W = A or T; Y = C or T; K = G or T

[0244] MKV represents a primer that hybridizes with the leader sequence of the mouse kappa light chain variable region gene; MKC represents a primer that hybridizes with the mouse kappa constant region gene. The bold underlined portion represents the M13 forward or M13 reverse sequencing primer. Wobble base pairs are defined in the definition section.

[0245] Table 2. PCR primers for amplifying mouse VH

[0246]

[0247]

[0248]

[0249] Fuzzy base code: R = A or G; K = G or T; M = A or C.

[0250] MHV represents a primer that hybridizes to the leader sequence of the mouse heavy chain variable region gene. MHCG represents a primer that hybridizes to the mouse constant region gene. The bold underlined portion represents the M13 forward sequencing primer or the M13 reverse sequencing primer. The primer MHC mixture consists of an equimolar mixture of primers MHCG1, MHCG2a, MHCG2b and MHCG3. "Wobble" base pairs are defined in the definition section.

[0251] The result of each PCR reaction was a single amplification product, which was purified using the QIAquick PCR Purification Kit and sequenced (by Eurofins / GATCGenomics) in both directions using M13-forward and M13-reverse primers (Table 3) to obtain three independent sets of sequence information for each immunoglobulin chain.

[0252] Table 3. Universal PCR and sequencing primers

[0253] name Sequence (5′→3′) HCMVi promoter TGTTCCTTTCCATGGGTCTT (SEQ ID NO: 59) HuG4_LIC_Reverse CTCTCGGAGGTGCTCCTGGAG (SEQ ID NO: 60) HuK LIC reverse GCATTCCAGATTTCAACTG (SEQ ID NO: 61) M13-Forward TGTAAAACGACGGCCAGT (SEQ ID NO: 62) M13-Reverse CAGGAAACAGCTATGACC (SEQ ID NO: 63)

[0254] VK and VH MoTau01 DNA sequences

[0255] The consensus DNA sequences of the MoTau01 VK PCR product and the MoTau01 VH PCR product are shown in Figure 1 and 2 The variable region DNA sequence obtained was identical to the sequence determined by Genscript. Germline analysis of the MoTau01 sequence showed that the kappa light chain was murine VK1 IGKV9-124*01 and the heavy chain was murine VH1 IGHV10-1*02.

[0256] Example 2: (Generation of chimeric MoTau01 antibody)

[0257] Construction of chimeric MoTau01 expression vector

[0258] The genes for MoTau01 VH and VK were synthesized by GenScript. The sequences of MoTau01 VH and VK were optimized by silent mutagenesis using GenScript's proprietary software algorithm to use codons that are preferentially utilized and synthesized by human cells.

[0259] The construction of chimeric expression vectors required cloning the synthetic variable regions into IgG / κ vectors (pHuK and pHuG4, respectively) using ligase-independent cloning (LIC). Figure 3 and 4 The vector (pCMV modified) was digested with BfuA1 (BspM1) and then T4 DNA polymerase 3′-5′ exonuclease activity (+dATP) was used to generate compatible overhangs.

[0260] Antibody sequence ( Figure 5 and 6 ) are generated as follows: first, the synthetic variable region is amplified by PCR using a primer containing the 3′ end of the leader sequence (most of the sequence is present in the vector) - forward primer - or the beginning of the constant region (IgG4 or κ) - reverse primer, followed by the beginning of the variable region (in each direction), Table 4.

[0261] Table 4. Cloning and mutagenesis primers

[0262]

[0263]

[0264] Complementary overhangs were generated in the PCR products by T4 DNA polymerase + dTTP treatment (protocol provided in Example 5). The vector and insert were incubated at room temperature, transformed into chemically competent TOP10 bacteria and plated on kanamycin plates. Several clones were isolated and screened by PCR using the forward primer HCMVi promoter and the HuG4 LIC reverse primer (for VH) or the HuK LIC reverse primer (for VK) (Table 3).

[0265] Clones producing PCR products of the correct size were selected, miniprepped using a QIAGEN kit and sequenced using the same primers.

[0266] Production of chimeric antibodies

[0267] ExpiCHO suspension cells grown in ExpiCHO transfection medium and antibiotics were co-transfected with MoTau01_VH.pHuG4 and MoTau01_VK.pHuK (1 μg DNA each) using ExpiFectamine CHO reagent. Cells were grown in 1 mL growth medium for 7 days. MoTau01 HuG4k antibody was quantitatively measured in the conditioned medium by Octet at up to 160 μg / mL (Table 14A).

[0268] TauC3 binding activity of chimeric antibodies

[0269] TauC3 and FL Tau antigens were produced and purified by Genscript and provided at concentrations of 2.54 mg / ml or 0.24 mg / ml, respectively. The binding of the chimeric antibody to TauC3 and FL Tau was determined by binding ELISA. The chimeric antibody was able to bind to TauC3 with an EC50 of 0.7 nM ( Figure 7 A), but no binding to FL Tau was observed ( Figure 7B). No non-specific binding to either antigen was observed, and the isotype confirmed that the observed binding was specific. Binding of mouse and chimeric Tau01 antibodies to purified TauC3 was measured using biolayer interferometry (OctetRed96, ForteBio Section 8.12). Mouse and chimeric antibodies were assayed against a concentration series of 20 nM-0.31 nM TauC3 ( Figure 8 A and Figure 8 B). Both mouse and chimeric antibodies were able to bind to TauC3 in a concentration-dependent manner. Mouse and chimeric antibodies were also tested for binding to FL Tau, but low signal or no binding was observed ( Figure 8 C and Figure 8 D), confirming that the antibody binding is specific for TauC3.

[0270] Example 3: Design of Tau01 humanized antibody variants

[0271] Human VH and VK cDNA database

[0272] The protein sequences of human and mouse immunoglobulins from the International Immunogenetics Database 2009 (Lefranc, 2015) and the Kabat database of immunological protein sequences, version 5 (last updated 1999-11-17) (Kabat et al., 1991) were used to compile a database of aligned human immunoglobulin sequences. The database contains 10,406 VH and 2,894 VK sequences.

[0273] Molecular model of MoTau01

[0274] The MoTau01 VH and VK sequences were used to design humanized versions of the MoTau01 antibody. Homology models of the variable regions of the MoTau01 antibody have been generated using the Antibody Prediction Panel in Maestro 11.5. The selected human frameworks were used to generate 10 loop models, which were prepared using the One-Step Protein Preparation Wizard procedure. Protein reliability reports were generated for all 10 models and no major differences were found in terms of model quality. All 10 models were used to determine the CDR loop The consistency of the residues within the CDRs is captured in order to capture the different orientations of the CDRs.

[0275] People frame selection

[0276] Humanization requires identification of appropriate human V regions. The MoTau01 VH and VK protein sequences were queried against human VH and VK databases using the sequence analysis program Gibbs using various selection criteria. Software that identifies CDR residues in the mouse Tau01 antibody structure (defined by IMGT) The FW residues within the Neighboring residues". The human VH sequence alignment with the highest identity to MoTau01 VH in the neighboring residues is shown in Table 5. Table 6 lists these envelope residues and VCI, as well as FW, VCI or The number of residues in the proximity residues that are identical to the mouse equivalent position in the sequence of Table 5.

[0277] Humanized and incomplete sequences were removed from the analysis. Sequence DQ840895.1 was selected as a human heavy chain donor candidate. This sequence scored high in terms of sequence identity and similarity and had only 2 somatic mutations from its IGHV3-73*01 VH germline. It has eight Adjacent residues and one VCI residue were changed, but this was the minimum change that could be achieved (Table 8).

[0278]

[0279]

[0280]

[0281] Similarly, sequence L33034 was selected as a human kappa light chain donor candidate. This sequence has high sequence identity and similarity scores with Tau01 VK and has only one somatic mutation from the IGKV1-17*01 germline. It has five potential Neighboring residues and one VCI residue were changed.

[0282] The sequences of the kappa light chain humanization strategy are shown in Table 12.

[0283]

[0284]

[0285]

[0286] Design of Tau01 humanized heavy chain variants

[0287] Once a suitable human framework is identified, synthetic protein and DNA sequences can be designed. The initial design of the humanized version of Tau01 was to graft CDR1, 2, and 3 of MoTau01 VH onto the acceptor FW of DQ840895.1, thereby generating the variant Tau01HA. In the humanized version Tau01 HB, the 8 Adjacent residues and 1 VCI residue (at positions 1, 4, 35, 49, 58, 61 and 76-78) were backmutated to the mouse equivalent residues, one at a time in the following variants: The sequences were assembled in silico and designated TauOl HD to TauOl HL. Table 8 compares the murine and humanized versions of the TauOl VH protein sequence.

[0288] Design of Tau01 humanized light chain variants

[0289] The framework from L33034 was used to design the DNA and protein of the humanized constructs. CDR1, 2, and 3 from Tau01 VK are shown grafted into the acceptor FW of L33034 to generate the initial form of humanized Tau01. There are five unmatched residues at positions 34, 36, 44, 53, 69, and 71 of Tau01 KA. Adjacent residues and one VCI residue were backmutated to the mouse equivalent residues in variant Tau01 KB (Table 12).

[0290] These residues were mutated one at a time in the following variants: The sequences were assembled in silico and designated Tau01 KD to Tau01 KI. In the Tau01 KG form, residue K, backmutated to mouse residue T, was also mutated to human germline residue S. This additional variant was designated Tau01 KJ.

[0291] Design of heavy and light chain C forms

[0292] Following the design of the initial humanized variants, a homology model of Tau01 HAKA was built and evaluated. The latter model was overlaid with the mouse antibody model. Each position identified for backmutation and the surrounding regions of these positions were highlighted and examined in the model. Based on this data, it was predicted which residues were most important for backmutation and these residues were incorporated to form the HC form of the heavy chain (Table 8) and the KC form of the light chain (Table 12).

[0293] Example 4: (Production and properties of humanized antibodies)

[0294] Generation of humanized antibodies against Tau01

[0295] The sequences of Tau01 HA / B / C and KA / B / C were codon optimized to use codons preferentially used by human cells and synthesized by Genscript. The KA / B / C and HA / B / C constructs were PCR amplified and cloned into pHuK and pHuG4 in a ligase-independent cloning reaction for transformation of TOP10 bacteria. The HA or KA forms were subsequently modified by PCR mutagenesis using the primers in Table 4 to obtain the other humanized variants annotated in Tables 8 or 12, respectively.

[0296] The clones were sequenced and plasmid DNA was prepared using the QIAGEN Plasmid Miniprep Kit or the Qiagen Plasmid Maxiprep Kit. The expression construct sequences (HA, HB, HC, KA, KB, and KC) were as follows: Figures 9 to 14 shown.

[0297] Antibody expression

[0298] Expression plasmid preparations encoding (humanized or chimeric) VH and VK were used to transfect ExpiCHO cells, cultured in serum-free medium for 7 days, and then the conditioned medium containing secreted antibodies was harvested. The concentration of IgG4κ antibodies in the conditioned medium of ExpiCHO cells was measured by octet and is shown in Table 14A-C. Most antibodies were produced at good expression levels.

[0299] Table 14. IgG levels in conditioned medium from transfected ExpiCHO cells

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] * Expression of the transfected control Hu1210 HuG1K was also reduced (expected level -100 μg / ml).

[0307] Antigen binding of the initial form of humanized antibodies

[0308] Binding of the humanized variants to the provided TauC3 antigen was tested by binding ELISA as described in Example 5. Fig.15The data shown in show ELISA binding of TauC3 to humanized antibodies consisting of HA / HB heavy chains and KA / KB light chains. The HAKA and HAKB humanized antibodies did not bind to TauC3. The MoTau01HuG4K chimera bound to TauC3 with an EC50 value of 0.65 nM, and the HBKA and HBKB variants bound with similar EC50 values, with the HBKB form being the closest (0.78 nM).

[0309] In view of this data, additional versions of humanized heavy and light chains were expressed, each with a single back mutation (Tables 8 and 12). Fig.16 Results are shown for heavy chain single mutants and HC forms in combination with KA-KC, tested by ELISA for binding to TauC3. The data show that the heavy chain single mutants are unable to bind TauC3 as well as the chimeric antibodies. The HB and HC forms in combination with KA-KC bind to TauC3 with the highest EC50 values, with HBKB and HBKC showing values ​​closest to the chimeric antibodies (0.81 nM and 0.84 nM, respectively). These results were confirmed in screening experiments using one concentration of TauC3 on the Octet instrument as described in Section 8.12 ( Fig.17 ). The Octet data were not optimal as two-way binding and dissociation events could be observed, which may be due to the nature of the TauC3 protein. However, the Octet data were sufficient to screen and rank the candidates for humanization.

[0310] Since HB / HC is the best heavy chain form, by combining ELISA ( Fig.18 ) and Octet screening test ( Fig.19 A and Fig.19 B) They were tested in combination with all light chain forms (KA-KJ) for binding to TauC3. Many light chain single mutants were able to maintain binding to TauC3, but light chain forms KE, KG, KI and KJ ranked highest in both assays.

[0311] Second round of humanized antibody design and generation

[0312] Based on the suboptimal binding results of the initial humanized variants, a second round of variants was designed by incorporating additional back mutations into the HC and KC forms (HM-HO and KL and KM, respectively) or two back mutations in the combined KA form (KN-KP) (Tables 8 and 12). Mutagenesis, DNA preparation, expression, and quantification were performed. The expression levels obtained are shown in Tables 14D and 14E. Most antibodies were produced at good expression levels.

[0313] Table 14. IgG levels in conditioned medium from transfected ExpiCHO cells

[0314]

[0315]

[0316]

[0317]

[0318] Antigen binding of the second round of humanized Tau01 antibodies

[0319] To evaluate the heavy chain, ELISA ( Fig. 20 ) and Octet( Fig.21 ) Antibody variants comprising HM, HN and HO in combination with light chains KA, KB, KG, KI, KJ were tested for binding activity against TauC3. The HM and HO variants ranked highest in both assays. The only difference between the HO and HM variants was an additional back mutation of LV in position 4, which was also present in the HN variant. Since the binding of the HN form was not as good as that of the HM form, the LV back mutation was not considered necessary, so HM was selected as the head heavy chain from the second round of design.

[0320] The lead heavy chain HM was expressed with several light chains; KA-KC, previously highlighted single mutants (KE, KG, KI, KJ) and second round designs KL, KN, KO, KP. For comparison, these light chains were also expressed in combination with the earlier preferred heavy chain HC. These humanized antibody variants were tested for binding to TauC3 by binding ELISA ( Fig. 22 ). Overall, the HM variant bound with a higher EC50 than the HC variant, so the former variant was evaluated in the Octet screening experiment ( Fig.23 ). Antibody variants HMKE, HMKN, HMKO, and HMKP ranked highest in both assays. The KM light chain was also screened in combination with HC / HM and compared to the lead antibody variants by Octet ( Fig.24 A). The HMKM form ranked very high in the Octet screening experiment, while HCKM ranked lowest. The humanized lead was also tested for binding to FL Tau using Octet to determine how much selectivity was retained ( Fig.24 B). The signal obtained using 500 nM FL Tau was very low (0.03-0.07 nm), which is in the range of non-specific binding.

[0321] To further evaluate the binding data observed on Octet, dissociation rate ranking experiments were performed on a Biacore200 instrument. Biacore offers higher selectivity than Octet and facilitates the immobilization of TauC3 to the CM5 chip through amine coupling of the stabilized antigen. This will produce higher quality data with a stable baseline and good reproducible responses. Once TauC3 is immobilized, a concentration (5 nM) of antibody is added, followed by a dissociation and regeneration step (Section 8.20). The dissociation rate is fitted using a two-phase decay model ( Fig.25 B) or use a one-phase decay model to fit only the second dissociation rate ( Fig.25 A). Candidates were then ranked based on the off-rates obtained using fit and comparison. The data are consistent with the Octet results (although the ranking order is different), highlighting Tau01 HMKM, HMKO, HMKP, HMKN, and HMKE as humanized variants with the slowest off-rates. The lack of binding of FL Tau to the chimeric and humanized candidates was also confirmed by Biacore by loading the antibody onto a Protein G chip and adding 250 nM of FL Tau. Fig.26 It was shown that no binding of MoTau01HuG4k or Tau01 HCKB was observed.

[0322] Thermal stability of humanized Tau01 candidate antibodies

[0323] The purpose of this experiment was to test the thermal stability of chimeric and some humanized antibodies (Tau01 HCKG, HCKN, HMKE, HMKN, HMKO, HMKP) when subjected to higher temperatures, from 35°C to 95°C for 10 minutes, cooled to 4°C, and used in binding ELISA (Section 8.15) at the EC80 concentration of each candidate. All humanized candidate antibodies were more stable than the chimeric antibody and maintained their binding ability to TauC3 until 67-68°C, after which binding to TauC3 decreased ( Fig. 27 ). The humanized variants exhibited higher thermal stability compared to the chimeric antibody MoTau01 HuG4k, which only maintained binding up to about 55°C. Variants containing the HM heavy chain maintained binding at slightly higher temperatures than the HC heavy chain variants tested.

[0324] Selection of lead humanized Tau01 candidate antibodies

[0325] Putting all of these results together, lead humanized antibody variants Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP were selected for scale-up and purification using affinity chromatography and size exclusion chromatography as described in Section 8.16. The purified antibodies were further characterized in a series of biophysical assays. HCKB humanized candidates that bind weaker to TauC3 were also scaled up for expression and purification to be used as comparator antibodies for ranking in the seeding assay. This will allow us to further test the correlation between in vitro antibody affinity and potency in cellular assays.

[0326] Aggregation of humanized Tau01 candidate antibodies

[0327] As part of the post-purification QC process, antibody samples were subjected to SEC-MALS / DLS followed by mass spectrometry. To determine the absolute molar mass and check for aggregation, purified antibody samples were injected onto a size exclusion column in an HPLC system and analyzed by multi-angle light scattering. The plots for MoTau01 HuG4k and Tau01 HMKM showed no signs of aggregation and had average molecular weights of approximately 147-8 kDa, which is the expected range for IgG monomers in this analytical setup ( Fig.28 A). The Tau01 HCKB antibody spectrum has a broad peak that biases the data, resulting in a molecular weight of 179.6 kDa ( Fig.28 B). All three antibodies were monodisperse (Mw / Mn < 1.05) and showed no signs of aggregation.

[0328] Dynamic light scattering is a complementary technique to static light scattering (SEC-MALS) for the detection of soluble aggregates and was used to QC humanized variants of Tau01 HMKN, HMKO, HMKP and HMKE. The Z-Ave or hydrodynamic diameter of the antibody is expected to be approximately 10 and the polydispersity index (PdI) should be < 0.1 if the sample is monodisperse. Fig.29 As shown, all antibody samples contained one major population that was monodisperse and consistent with the size of the monoclonal antibodies.

[0329] To confirm the exact molecular weight of the antibody, perform mass spectrometry analysis on intact and reduced antibody samples, e.g. Fig.30 A to Fig.30 G. The molecular weights matched the predicted molecular weights of all antibodies tested and the amino acid sequences were confirmed. No other liabilities were marked. Overall, the purified chimeric and humanized antibodies passed QC.

[0330] Kinetic study of humanized Tau01 candidate antibody against TauC3

[0331] To determine the affinity of the binding interaction, a biacore kinetic assay was developed, which involves immobilizing TauC3 on a CM5 chip via amine coupling and injecting a series of concentrations of the corresponding antibodies onto it. The chimeric antibody binds to TauC3 with a KD of 57 pM ( Fig.31 A).

[0332] Fig.31 B shows that Tau01 HCKB is the weakest TauC3 binder (1.2 nM), followed by HMKM, which binds TauC3 with an affinity of 110 pM ( Fig.31 G). Fig.31 Tau01 HMKO, HMKN, HMKP, and HMKE shown in CF have KD values ​​comparable or tighter than the chimeric antibodies. However, all humanized candidates have slightly faster dissociation rates compared to the chimeras (0.001-0.004 for humanized and 0.0007 for chimeras). It should be noted that the ka is at the limit of the instrument, so absolute values ​​should be compared with caution. It can be concluded that antibodies HMKN, HMKP, and HMKE bind in the picomolar range and have the slowest dissociation rates of all humanized variants tested.

[0333] Determination of melting temperature (Tm) of humanized Tau01 candidate antibodies

[0334] To determine the melting temperature of the lead antibodies Tau01HMKE, HMKM, HMKN, HMKO, HMKP, a thermal shift assay was performed. The samples were incubated with a fluorescent dye (Sypro Orange) for 71 cycles in a qPCR thermal cycler (section 8.21) with an increase of 1°C per cycle. The Tm of the humanized antibody was calculated to be 68-69°C. Fig.32 ).

[0335] Non-specific protein-protein interactions (CIC) of humanized Tau01 candidate antibodies

[0336] Cross-interaction chromatography using large amounts of purified human polyclonal IgG is a technique used to monitor non-specific protein-protein interactions and can provide an indication of any solubility issues that can cause downstream manufacturing problems, as explained in Example 5. An elevated retention index (k') indicates a tendency for self-interaction and low solubility. Humanized Tau01 HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies showed a retention index below 0.038, indicating a low tendency for non-specific interactions and good solubility ( Fig.33 ).

[0337] Solubility of humanized Tau01 antibody candidates

[0338] Humanized Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP candidate antibodies were concentrated using a solvent absorption concentrator (MWCO 7500 kDa) and the concentrations were measured at regular intervals. Tau01 HMKP was concentrated to 123 mg / ml, and Tau01 HMKN, HMKM, and MoTau01HuG4K antibodies were concentrated to 87-88 mg / mL ( Fig.34 ). Tau01 HMKO HuG4K was concentrated to 59 mg / ml with no obvious precipitation, and Tau01 HMKE HuG4K was concentrated to 57 mg / ml. The data showed that the antibody was not easily precipitated at a concentration as high as 57 mg / mL.

[0339] Freeze-thaw and heat stress analysis of humanized Tau01 candidate antibodies by circular dichroism

[0340] Circular dichroism (CD) is a spectroscopic technique that allows us to observe the overall secondary structure of purified protein samples.

[0341] The freeze-thaw (FT) stress experiment involved subjecting the purified candidate antibody samples to 10 cycles of 15 minutes at -80°C, followed by thawing at room temperature for 15 minutes. For the heat stress experiment, the purified candidate antibody samples were exposed to a) 4°C, b) room temperature (RT), c) 37°C, and d) 50°C for 25 days.

[0342] The samples were then analyzed by circular dichroism to check whether the secondary structure was preserved ( Fig.35 ). All humanized variants tested passed our internal thresholds. Overall, the data suggest that heat stress and freeze-thaw cycles do not affect the secondary structure of the humanized Tau01 HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies.

[0343] Isoelectric point analysis of humanized Tau01 candidate antibodies

[0344] Capillary isoelectric focusing (cIEF) was used to perform pi analysis of humanized candidate antibodies. This technique allows the separation of antibodies according to their isoelectric point (pi) using a pH gradient running through a capillary. Fig.36 Chromatograms are shown and the major pi isoform (defined as greater than 10% of the peak area) and the pi range for each antibody are shown in Table 15. The major isoelectric points of humanized TauOl HMKE, HMKM, HMKN, HMKO, HMKP candidates were -8.86-8.81.

[0345] Serum stability assessment of humanized Tau01 candidate antibodies

[0346] Purified samples of chimeric and humanized antibodies were incubated in mouse, human, and cynomolgus monkey sera for 21 days. The binding of Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP candidate antibodies incubated in the three different sera was compared with antibodies incubated in PBS and a 4°C positive control sample by binding ELISA to TauC3 ( Fig.37 ). Tau01 HMKE, HMKM, HMKN, HMKO, and HMKP candidate antibodies retained their binding ability after incubation in mouse, human, and cynomolgus monkey sera.

[0347] Data summary of lead Tau01 HMKEHMKN, HMKO, HMKP, HMKM humanized candidates compared to MoTau01HuG4k

[0348] Table 16 shows a summary of the binding, kinetic affinity, and biophysical properties of the lead humanized Tau01 HMKE, HMKN, HMKO, HMKP, HMKM antibody candidates compared to the chimeric antibody MoTau01 HuG4k. The binding of all humanized candidates was in the picomolar range, but among them, Tau01 HMKN, HMKO, and HMKP had the highest affinity and slowest dissociation rate and also passed all of our biophysical assays. Considering all the data, Tau01 HMKP was selected as the lead humanized candidate because it had the highest affinity, slow dissociation rate, and did not show potential redundancy in the biophysical analysis. Tau01 HMKN and Tau01 HMKO and KMKE are all good backup humanized lead candidates because they also have excellent properties.

[0349] Table 16

[0350] Summary of final humanized antibody candidates

[0351]

[0352]

[0353] in conclusion

[0354] The aim of this research project was to humanize the MoTau01 antibody and ensure that the resulting antibody binds TauC3 with comparable affinity compared to the chimeric antibody. The MoTau01 antibody has been designed and expressed as a humanized antibody without significant loss of binding affinity. Tau01 HMKE, HMKM, HMKN, HMKO, HMKP humanized antibodies showed high affinity in binding ELISA, Octet ranking and kinetic studies using Biacore, in the picomolar range ( Fig.31), and also passed all our biophysical assays.

[0355] The Tau01 HMKP antibody showed the best drug-like characteristics as well as excellent binding kinetics and was therefore selected as the lead candidate (Table 16). In our opinion, the combination of excellent binding, expression, thermal stability, affinity, and biophysical properties makes Tau01 HMKP a suitable candidate for further development. Tau01HMKN, HMKO, and HMKE also showed excellent properties and are very good backup humanized lead candidates.

[0356] Example 5 (Scheme)

[0357] The following protocol / procedure was used in Examples 1-4.

[0358] RNeasy Mini protocol for isolation of total RNA (Qiagen)

[0359] 1. Disrupt cells by adding Buffer RLT. For pelleted cells, thoroughly loosen the cell pellet by flicking the tube. Add Buffer RLT (600 μl) and proceed to step 2. Note: Incomplete loosening of the cell pellet may result in inefficient lysis and reduced yield.

[0360] 2. Homogenize the cells by passing the lysis buffer at least 5 times through an 18-20 gauge needle mounted on an RNase-free syringe.

[0361] 3. Add 1 volume of 70% ethanol to the homogenized lysate and mix thoroughly by pipetting. Do not centrifuge. The volume of the lysate may be less than 600 μl due to losses during the homogenization process.

[0362] 4. Transfer up to 700 μl of sample (including any precipitate that may have formed) to an RNeasy spin column placed in a 2 mL collection tube. Gently close the cap and centrifuge at ≥8000 x g for 15 seconds. Discard the flow-through. Reuse the centrifuge tube from step 5.

[0363] 5. Add 700 μl of Buffer RW1 to the RNeasy column. Gently close the cap and centrifuge at ≥8000 x g for 15 seconds to wash the column membrane. Discard the flow-through. Reuse the centrifuge tube in step 6.

[0364] 6. Add 500 μl of Buffer RPE to the RNeasy column. Gently close the cap and centrifuge at ≥8000 x g for 15 seconds to wash the column membrane. Discard the flow-through. Reuse the centrifuge tube in step 7.

[0365] 6. Add 500 μl of Buffer RPE to the RNeasy column. Gently close the lid and centrifuge at ≥8000 x g for 2 minutes to dry the RNeasy spin column membrane.

[0366] 7. Place the RNeasy spin column in a new 2 mL collection tube and discard the old collection tube with the flow-through. Gently close the lid and centrifuge at full speed for 1 minute.

[0367] 8. To perform the elution, transfer the RNeasy column to a new 1.5 mL collection tube. Add 30 μl of RNase-free water directly onto the RNeasy spin column membrane. Gently close the tube. Let stand for 1 minute, then centrifuge at ≥8000 x g for 1 minute.

[0368] First-Strand cDNA Synthesis Protocol (GE Life Sciences)

[0369] 1. Place the RNA sample in a microcentrifuge tube and add RNase-free water to bring the RNA to an appropriate volume (20 μL - 12-fold dilution, see Table A).

[0370] 2. Heat the RNA solution to 65°C for 10 minutes and then cool on ice. Gently pipette a large amount of the first-strand cDNA reaction mixture to obtain a uniform suspension. (BSA may precipitate in the mixture during storage; this precipitate will dissolve during incubation).

[0371] 3. Add a large amount of the first-strand cDNA reaction mixture (11 μL) to a sterile 1.5 or 0.5 mL microcentrifuge tube. Add 1 μL of DTT solution, 1 μL (0.2 μg, 1:25 dilution) of NotI-d(T)18 primer, and heat-denatured RNA to the tube. Pipette up and down several times to mix.

[0372] 4. Incubate at 37°C for 1 hour and heat at 95°C to inactivate the transcriptase for 5 minutes.

[0373] Table A. Volumes of components in the first chain reaction

[0374] Large-scale first-strand reaction mixture Primers DTT RNA Final volume of first chain reaction 11μL 1μL 1μL 20μL 33μL

[0375] cDNA purification

[0376] 1. A simple protocol designed to remove contaminating first-strand cDNA primers that may interfere with subsequent PCR reactions.

[0377] 2. Add 99 μl of Buffer QG (from Qiagen Gel Extraction Kit, Cat. No. 28704) and 33 μl of IPA. Mix and add to QiaQuick Gel Extraction Column. Spin and discard the flow-through.

[0378] 3. Wash the column once with 500 μl of Buffer QG. Discard the flow-through.

[0379] 4. Wash the column once with 750 μl of buffer PE. Discard the flow-through.

[0380] 5. Spin the column to remove any residual alcohol and allow the column to dry.

[0381] 6. Elute cDNA with 50 μl of distilled water preheated to 65°C.

[0382] PCR cloning of mouse variable regions

[0383] 1. Perform PCR reactions on the purified cDNA using the primers in Tables 1 and 2. Use a different forward primer (MHV1-12 and 14 and MKV1-11) in each reaction as follows:

[0384]

[0385] 2. Loop:

[0386]

[0387]

[0388] 3. Run 5 μl of sample from each PCR reaction on a 2% (w / v) agarose gel to determine which lead primer produces the PCR product. The size of the positive PCR clones is approximately 420-500 bp.

[0389] 4. For positive clones, perform PCR purification on the remaining samples using QIAGEN PCR purification kit and elute into 40 μL nuclease-free water. Send to an external contractor (e.g. GATC) and perform PCR fragment sequencing using M13 forward primer and M13 reverse primer.

[0390] QIAquick PCR Purification Microcentrifugation and Vacuum Protocol (QIAGEN)

[0391] 1. All centrifugation steps were performed at 17,900 x g (13,000 rpm) in a conventional benchtop microcentrifuge.

[0392] 2. Add 5 volumes of Buffer PB to 1 volume of PCR reaction and mix. If the color of the mixture is orange or purple, add 10 μl of 3M Sodium Acetate, pH 5.0, and mix. The color of the mixture will turn yellow.

[0393] 3. Place the QIAquick column into the provided 2 mL collection tube or vacuum manifold.

[0394] 4. To bind DNA, add the sample to the QIAquick column and centrifuge for 30-60 seconds or apply vacuum to the manifold until all the sample has passed through the column. Discard the flow-through and return the QIAquick column to the same tube.

[0395] 5. To wash, add 0.75 mL of Buffer PE to the QIAquick column and centrifuge for 30-60 seconds or apply vacuum. Discard the flow-through and return the QIAquick column to the same tube.

[0396] 6. Centrifuge the column in a 2 mL collection tube (provided) for 1 minute.

[0397] 7. Place each QIAquick column into a clean 1.5 mL microcentrifuge tube.

[0398] 8. To elute the DNA, add 40-50 μl of buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick membrane and centrifuge the column for 1 minute.

[0399] Generation of mAb expression vectors by LIC

[0400] Insertion preparation

[0401] 1. Generate LIC primers using the sequence.

[0402] 2. LIC PCR was performed on the codon-optimized synthetic gene (Genscript) using LIC primers (Table 4).

[0403] 3. Set up PCR reaction:

[0404]

[0405]

[0406] Note: A polymerase that produces blunt-ended PCR products must be used in this step. Other polymerases that produce T-overhangs will not work.

[0407] 4. Loop:

[0408]

[0409] 5. Run 5 μL of PCR product on a gel to ensure the product is the correct size - it should be around 370 bp.

[0410] 6. PCR purification products were purified using Qiagen PCR purification kit to remove nucleotides and primers and eluted into 40 μL of nuclease-free water.

[0411] 7. Treat the insert fragment with T4 DNA polymerase:

[0412]

[0413] 8. Incubate at room temperature for 30 minutes, then inactivate the enzyme at 70°C for 20 minutes.

[0414] Vector preparation

[0415] 9. Digest the LlC vector with BfuAI by incubating at 50°C for 3 hours or overnight. Figure 3 and 4 Vector diagram shown in ):

[0416]

[0417]

[0418] 10. After BfuAI digestion, add 2 μL Bam HI and incubate at 37°C for 2 hours.

[0419] 11. Run the digested vector on a 1% (w / v) agarose / 1x TAE gel containing 1x SYBR Safe DNA stain. Two bands may be visible - cut out the higher MW band and extract using a gel extraction kit and elute in 50 μL of EB.

[0420] 12. Treat the vector with T4 DNA polymerase as follows:

[0421]

[0422] 13. Incubate at room temperature for 30 minutes, then inactivate the enzyme at 70°C for 20 minutes.

[0423] clone

[0424] 14. Mix 1 μL of insert with 0.5 μL of vector in a total of 10 μL of nuclease-free water for 20 minutes at room temperature. Always perform vector-alone transformation.

[0425] 15. Use the ligation mixture to transform 25-50 μL of chemically competent Invitrogen TOP 10 bacteria according to the manufacturer's instructions and spread on 90 mm diameter LB agar plates containing kanamycin (50 μg / mL). Incubate overnight at 37°C.

[0426] Select clones from transformants

[0427] 16. PCR confirmation using Phusion PCR master mix:

[0428] Reagents Volume (for 20 μl final reaction volume) 2x Phusion PCR Master Mix 25μL HCMVi primers 1μL HuG4 / HuK LIC Primers 1μL <![CDATA[dH20]]> Up to 23 μL DNA Colony dip (daily culture of the same colony)

[0429]

[0430]

[0431] 17. Run each PCR reaction on a 2% agarose E-Gel gel cassette and run for 15 minutes to determine the size of any PCR product bands on the gel.

[0432] 18. Grow a starter culture overnight using LB supplemented with kanamycin to miniprep the construct and sequence the DNA of at least two independent positive clones for the variable gene (using the same primers) to identify any possible errors due to the PCR reaction itself.

[0433] TOP10 TM Transformation of E. coli (Invitrogen protocol)

[0434] 1. Briefly centrifuge the vial containing the ligation reaction and place on ice.

[0435] 2. Thaw a 50 μL vial of One Shot Cells on ice for one or two ligations / transformations.

[0436] 3. Use a pipette to add 1 to 2 μL of each ligation reaction directly to the vial of competent cells and gently tap to mix. Do not mix by pipetting up and down. The remaining ligation mixture can be stored at -20°C.

[0437] 4. Incubate the vial on ice for 15-30 minutes.

[0438] 5. Incubate in a 42°C water bath for exactly 30 seconds and then place on ice for 2 minutes.

[0439] 6. Add 250 μL of pre-warmed SOC medium to each vial.

[0440] 7. Shake the vials in a shaking incubator at 225 rpm at 37°C for exactly 1 hour.

[0441] 8. Spread 200 μL from each transformation flask onto a separate, labeled LB agar plate containing 500 μg / mL kanamycin.

[0442] 9. Invert the plate and incubate overnight at 37°C.

[0443] use Miniprep isolation of plasmid DNA (Oiagen protocol)

[0444] 1. Resuspend the pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Make sure RNase A has been added to Buffer P1.

[0445] 2. Add 250 μL of Buffer P2 and gently invert the tube 4-6 times to mix.

[0446] 3. Add 350 μL of Buffer N3 and immediately gently invert the tube 4-6 times. The solution should become cloudy.

[0447] 4. Centrifuge at 13,000 rpm (-17,900 x g) for 10 minutes in a benchtop microcentrifuge. A compact white pellet will form.

[0448] 5. Add the supernatant from step 4 to the QIAprep spin column by pipetting.

[0449] 6. Centrifuge for 30-60 seconds and discard the flow-through.

[0450] 7. Wash the column by adding 0.5 mL of Buffer PB and centrifuging for 30-60 s.

[0451] 8. Wash the column by adding 0.75 mL of Buffer PE and centrifuging for 30-60 s.

[0452] 9. Discard the flow-through and centrifuge for another minute.

[0453] 10. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute the DNA, add 50 μL of nuclease-free water to the center of the QIAprep spin column, let stand for 1 minute, and then centrifuge for 1 minute.

[0454] ExpiCHO transfection in 24-well plates 1 ml transfection (ExpiCHO TM Expression System Kit-Invitrogen)

[0455] 1. Passage and expand expiCHO cells until the cell density reaches approximately 4-6x10 6 viable cells / mL.

[0456] 2. One day before transfection (Day -1), split the expiCHO culture into 3-4x10 6 The cells were grown overnight at a final density of 10 viable cells / mL.

[0457] 3. Dilute the cells to 6x10 6 viable cells / mL.

[0458] 4. Aliquot 0.9 mL of cells into each well of a 24-well plate for transfection.

[0459] 5. Preparation of ExpiFectamine / DNA complex.

[0460] 6. Dilute the plasmid DNA (1 μg plasmid DNA per mL culture volume to be transfected) by adding 1 μL DNA to each well to be transfected in a final volume of 50 μL OptiPro.

[0461] 7. For each well to be transfected, dilute 4 μL of ExpiFectamine CHO reagent in 46 μL of OptiPro medium (no incubation time required).

[0462] 8. Add the diluted ExpiFectamine CHO to the diluted DNA and gently pipette 3-4 times to mix (incubate for 1 to 5 minutes).

[0463] 9. Add 100 μL of the complex mixture to each well of the 24-well plate containing the culture.

[0464] 10. Cover the board with a breathable lid.

[0465] 11. Place the 24-well plate on an orbital shaker (recommended shaker speed is 225 rpm for a shaker with 19 mm orbital stroke) in a 37°C incubator with 8% CO2.

[0466] 12. Add ExpiFectamine Enhancer (6ul ExpiCHO Enhancer) and ExpiCHO Feed (190ul ExpiCHO Feed) 18-22 hours after transfection

[0467] 13. By day 7-8 post-transfection, protein expression is usually complete and the supernatant is ready for collection.

[0468] Quantification of IgG by Octet

[0469] 1. Prepare 100 μl of each concentration of standard curve and ExpiCHO supernatant as follows:

[0470] a. HuG4K isoform standards at concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.81, and 3.9 μg / ml, using ExpiCHO expression medium as the diluent;

[0471] b. Test (unknown) samples.

[0472] 2. Pre-soak (≥10 min) Protein G-coated biosensors (Pall ForteBio) in 200 μl ExpiCHO expression medium.

[0473] 3. Aliquot 45 μl of standards and test samples into 384-well slant bottom plates in duplicate, including a media only control. Seal the plate and spin in a benchtop centrifuge (1000 rpm, 1 minute).

[0474] 4. Remove the board seal and insert the board and pre-soaked sensor into the Octet.

[0475] 5. Quantification was performed as follows:

[0476] a. Regenerate the protein G coated sensor in 10 mM glycine (pH 1.5) for 5 seconds and neutralize in ExpiCHO expression medium for 5 seconds. Repeat three times.

[0477] b. Measure the standard or sample for 120 seconds.

[0478] c. Repeat the above regeneration and neutralization steps.

[0479] 6. Import the data into analysis software and fit the data to a 5PL weighted fit of the dose response to give the IgG concentration in μg / ml.

[0480] TauC3 binding ELISA

[0481] 1. Apply 50 / 30 μL aliquots of 1 μg / mL TauC3 in PBS to each well of a 94 / 384-well MaxiSorp plate, respectively. Incubate overnight at 4°C.

[0482] 2. Wash three times with PBS-T (0.1% Tween 20).

[0483] 3. In a 96 / 384-well plate, each well was blocked with 150 / 80 μL PBS+5% BSA+0.1% Tween 20, respectively.

[0484] 4. Incubate at 37°C for 1 hour. Wash three times with PBS-T (0.1% Tween 20).

[0485] 5. Add 50 / 30 μl of primary antibody serially diluted in PBS + 0.2% BSA + 0.1% Tween 20 to the assay plate (96 / 384 well plate, respectively). Use a 3-fold dilution series starting at ~4 μg / mL. Repeat the incubation and wash steps (step 4).

[0486] 6. Dilute 3ul anti-human kappa chain HRP (Sigma A7164-1mL) per 10ml in PBS + 0.2% BSA + 0.1% Tween 20 and add 50 / 30μL to each well of a 96 / 384 well plate, respectively. Repeat the incubation and washing steps (step 4).

[0487] 7. Add 75 / 20 μL of K-Blue substrate (Neogen) to each well and incubate at room temperature for 5-10 minutes.

[0488] 8. Stop the reaction by adding 50 / 10 μl of RED STOP solution (Neogen) to each well of a 96 / 384 well plate, respectively.

[0489] 9. Read the optical density at 650 nm using a Pherastar Plus.

[0490] Tau01 variant screening and affinity determination by Octet

[0491] 1. Immediately before use, place the protein G-coated sensor (Pall ForteBio) in HBS-P + Incubate in buffer for 10 minutes.

[0492] 2. HBS-P + 1 μg / mL antibody in was loaded onto the Protein G sensor for 600 seconds (immobilization level was 0.8-1 nm).

[0493] 3. Let the sensor in HBS-P + Equilibrate for 180 seconds.

[0494] 4. For screening experiments, the binding step was performed for 600 seconds using 10 nM TauC3. For kinetic analysis, the binding step was performed in HBS-P + Antibodies were loaded at ˜0.5 μg / mL for 10 min, and a binding step was performed for 10 min using TauC3 concentrations ranging from 20 nM to 0.31 nM.

[0495] 5. In HBS-P + The dissociation step was performed for 600 sec.

[0496] 6. Regenerate the sensor with 10 mM glycine (pH 1.5-2.0) for 5-30 seconds and then + Incubate in buffer for 30-60 seconds to neutralize. Repeat three times.

[0497] QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene)

[0498] 1. Prepare the reaction as follows:

[0499] a. 5 μL of 10× reaction buffer

[0500] b. 0.12 μL (25 ng) of RHA or RKA template

[0501] c.1.3 μL (125 ng) of oligonucleotide mutagenesis forward primer

[0502] d.1.3 μL (125 ng) of oligonucleotide mutagenesis reverse primer

[0503] e. 1 μL of dNTP mixture

[0504] f. 1.5 μL QuikSolution reagent

[0505] g. ddH2O to a final volume of 50 μL

[0506] h. 1 μL of QuikChange Lightning enzyme.

[0507] 2. Cycle each reaction using the cycling parameters listed in the table below:

[0508]

[0509] 3. Add 2 μL of Dpn I restriction enzyme.

[0510] 4. Gently and thoroughly mix each reaction, microcentrifuge briefly, and then immediately incubate at 37°C for 5 minutes to digest the parental dsDNA.

[0511] 5. Transform 2 μL of Dpn I-treated DNA from each reaction into separate 45 μL (+ 2 μL of β-ME) aliquots of XL10-Gold supercompetent cells (see TOP10 TM coli transformation).

[0512] 6. Using the Phusion method, miniprep and sequence screen colonies to check for correct mutations.

[0513] Qiagen HiSpeed ​​Maxiprep System Solution

[0514] 1. Pick from a freshly streaked selective plate or glycerol stock of the desired clone and inoculate a starter culture of 2-5 ml LB medium supplemented with kanamycin. Incubate at 37°C with shaking at 250-300 rpm for about 8 hours.

[0515] 2. Dilute the starter culture 1 / 1000 and inoculate 150-250 mL of LB medium supplemented with kanamycin from the starter culture and incubate overnight (12-16 hours) at 37°C with shaking at 250-300 rpm.

[0516] 3. Collect cells at 6,000 × g for 15 minutes. Discard supernatant.

[0517] 4. Completely resuspend the cell pellet in 10 mL of Buffer P1 by vortexing or pipetting.

[0518] 5. Add 10 mL of Buffer P2. Invert vigorously 4-6 times to mix. Incubate at room temperature for 5 minutes.

[0519] 6. Add 10 mL of cooled Buffer P3. Invert vigorously 4-6 times to mix.

[0520] 7. Pour the lysate into the bucket of the QIAfilter Cartridge and incubate at room temperature for 10 minutes.

[0521] 8. Add 10 ml of Buffer QBT to equilibrate the HiSpeed ​​Maxi Tip and drain by gravity flow.

[0522] 9. Using a QIAfilter, filter the lysate into an equilibrated HiSpeed ​​Maxi Tip. Allow the lysate to flow into the resin by gravity.

[0523] 10. Wash the HiSpeed ​​Maxi Tip with 60 ml of Buffer QC.

[0524] 11. Elute the DNA with 15 ml of Buffer QF.

[0525] 12. Add 10.5 ml of isopropanol to the eluted DNA to precipitate the DNA. Mix and incubate at room temperature for 5 minutes.

[0526] 13. Transfer the eluent / isopropanol mixture to a 30 ml syringe and filter through a QIAprecipitator module.

[0527] 14. Wash the DNA in the QIAprecipitator with 2 ml of 70% ethanol. Dry the membrane by passing air through the QIAprecipitator by pressing several times.

[0528] 15. Using a 5 ml syringe, elute the DNA in 1 ml of nuclease-free water. Transfer the eluate to a syringe and perform a second elution.

[0529] Thermal stability comparison

[0530] 1. Dilute the fully humanized antibody and chimeric control to 1 μg / mL in PBS / 0.2% Tween and aliquot into PCR tubes at appropriate volumes of EC80 concentration. Adjust the volume to 100 μl using the same buffer.

[0531] 2. Heat each tube at a temperature between 30°C and 85°C at 5°C intervals for 10 minutes and then cool to 4°C.

[0532] 3. Freeze the 1 μg / mL stock solution for 1 hour and then dilute to the EC80 concentration.

[0533] 4. Perform binding assays against TauC3 (Section 8.11) in 96-well plates using 100 μl of each antibody per well (tested in duplicate at each temperature).

[0534] Biacore off-rate ranking and kinetic study of Tau01 humanized antibody

[0535] Dissociation rate ranking

[0536] 1. Amine couple 0.5 μg / mL human TauC3 in acetate buffer pH 5 on 1 flow channel in a CM5 chip (GE Healthcare). Aim ~15 RU using the immobilization wizard with HBS-EP+ as running buffer.

[0537] 2. Load the antibody supernatant in 2.5 nM HBS-EP + buffer at a flow rate of 30 μL / min for 300 seconds, then dissociate for 600 seconds and regenerate for 30 seconds using 3M MgCl2. Export the raw data, subtract the buffer baseline and fit the data using a single-phase decay or a two-phase decay in GraphPadPrism.

[0538] dynamics

[0539] 1. On 1 flow channel of a CM5 chip, amine couple 0.5 μg / mL human TauC3 in acetate buffer pH 5. Use an immobilization guide to ~15RU with HBS-EP+ as running buffer.

[0540] 2. Each antibody was diluted to 5 nM and a 2-fold dilution series was generated in HBS-EP+ buffer to 0.08 nM. Each concentration was injected at 30 μL / min for 300 seconds, followed by 600 seconds of dissociation and 30 seconds of regeneration using 3M MgCl2, with a stabilization period of 600 seconds between cycles. The data were fitted using a 1:1 global fit.

[0541] Binding test FL Tau

[0542] 1. Load 0.25 μg / mL antibody in HBS-EP+ buffer onto a Protein G chip (GE Healthcare) at 10 μL / min for 30 seconds. Increase the flow rate to 30 μL / min and add 250 nM FLTau in HBS-EP+ buffer for 180 seconds. Regenerate with 10 mM glycine (pH 1.5) for 30 seconds

[0543] Purification of antibody candidates

[0544] Instrument: GE Healthcare Purification system

[0545] Software: UNICORN

[0546] Column: HiTrap MabSelect SuRe, 1mL; HiLoad 16 / 600Superdex 200pg

[0547] Mobile phase: IgG elution buffer; Dulbecco's 1x PBS

[0548] Sample preparation: Filter through 0.22 μm

[0549] Injection volume: 200 mL Expi293 conditioned medium (1:1) in DPBS

[0550] Flow rate: 0.5 mL / min for sample loading; 1.5 mL / min for gel filtration; 1 mL / min for elution

[0551] SEC-MALS

[0552] 1. Inject 10 μl of each sample (1 mg / mL) onto the SEC column (AdvanceBio SEC 4.6x150mm, 2.7μm, LC column, Agilent), followed by three detectors in series:

[0553] a.UV (Agilent 1260 Infinity HPLC system with constant temperature column oven)

[0554] b. Light scattering (Wyatt Technology DAWN HELEOS)

[0555] c. Differential refractometer (Wyatt Technology Optilab TRex)

[0556] 2. A mobile phase of Gibco PBS (Thermo Fisher) containing 0.05% sodium azide was applied at a constant flow rate of 0.4 mL / min. All experiments were performed at 25°C.

[0557] 3. Data were analyzed using Wyatt Technology ASTRA software (version 6.1.2.83) and the refractive index increment (dn / dc) was set to 0.185 (ie, for protein analysis).

[0558] 4. All samples were stored at 4 °C before SEC-MALS analysis.

[0559] Dynamic Light Scattering (DLS)

[0560] 1. Prepare 50 μl of 1.3 mg / ml sample (in Dulbecco's PBS; Sigma D8537) and aliquot into 384-well polypropylene plates (Greiner bio-one).

[0561] 2. Data were recorded on a Zetasizer APS (Malvern). All values ​​were recorded in triplicate and processed using the associated Zetasizer software (version 7.11).

[0562] 3. Cumulative analysis was performed to obtain the average particle size (z-average) and polydispersity index (PDI).

[0563] Mass spectrometry

[0564] Fig.30 Mass spectrometry analysis of purified chimeric and humanized candidate antibodies is described in .

[0565] Thermal displacement comparison

[0566] 1. Prepare samples directly into a 96-well white PCR plate in a final volume of 25 μL (final concentrations of purified antibodies are 1 μM and 2 μM).

[0567] 2. Sypro Orange - make a 1:100 stock solution in PBS buffer and then add 1:10 to the final sample (e.g. 2.5 μL of 25 μL)

[0568] 3. Load into qPCR machine and use MxPro software, SYBR Green method, (filter = FRROX, no reference dye). Thermal profile setting - 71 cycles, 1° increments

[0569] 4. Plot the results and determine Tm.

[0570] Cross-interaction chromatography (CIC)

[0571] 1. Samples were analyzed by two separate 20 μl injections (0.5 mg / mL); first onto 1 mL NHS-activated resin (GE Healthcare) coupled to 30 mg human polyclonal IgG (Sigma 14506), and then onto a blank coupled 1 mL NHS-activated resin as a control column.

[0572] 2. The mobile phase consisted of Dulbecco's PBS (Sigma D8537) containing 0.01% sodium azide (0.1 mL / min), and all experiments were performed at 25°C.

[0573] 3. The eluted samples were detected by UV absorbance (Agilent 1260 Infinity HPLC system with a thermostatic column oven), and the data were analyzed using Wyatt Technology ASTRA software (version 6.1.2.83) to determine the sample peak retention time. These were then used to calculate the retention factor k':

[0574]

[0575] Where T r is the retention time of the sample on the poly-IgG column, T m is the retention time on the mock (control) column.

[0576] Solubility

[0577] In a Vivapore solvent absorption concentrator 7500 kDa MWCO (VP0502 Satorius) 3.5-5.0 ml of a 1 mg / ml antibody solution in PBS was added.

[0578] 1. Monitor antibody concentration every 10 minutes by taking a small sample for measurement on a Nanodrop 2000 (ε=1.4) and continue until the concentrated volume reaches a dead volume of ~30-50 μl.

[0579] 2. Plot the concentration values ​​(mg / ml) against the corresponding time points to generate a concentration curve.

[0580] Circular dichroism

[0581] 1. Prepare 30 μL of sample at a concentration of 1 mg / ml (in Dulbecco's PBS; Sigma D8537).

[0582] 2. Dilute the 1 mg / mL sample to 0.15 times with 10 mM phosphate buffer.

[0583] 3. Readings were taken from 1 mM spectrally pure quartz cuvettes. Readings were taken at a DIT of 4 seconds and a scan speed of 20 nm / min with a step size of 1 nm.

[0584] 4. The average blank spectrum was subtracted from the sample spectrum and the spectra were then converted to Δε. The spectra were then zeroed relative to their 256-260nm values. Smoothing was performed by a Savitsky-Golay filter via a custom Excel function sgFilter() using a quadratic polynomial with a window size of 7 (-2, 3, 6, 7, 6, 3, -2). The spectra are shown with error bars, which are the mean of the standard deviation of the wavelength of + / - 2nm.

[0585] PI analysis using cIEF

[0586] 1. Concentrate the sample to >5 mg / ml and desalt to <50 mM NaCl level, then add 10 μl to 240 μl Pharmalyte / urea gel stock containing 4.5 / 5.1 / 9.5 and 10 isoelectric point markers.

[0587] 2. Mix the sample for at least 5 minutes and then add 200 μl to the PCR sample vial.

[0588] 3. Load the sample into the PA800 sample block along with the cIEF gel, catholyte, anolyte and chemical flow agent wash buffer. The PA800 is equipped with a neutral capillary and the default "Condition" method is run to prepare the capillary for sample analysis.

[0589] 4. Run each sample using the correct "Separation" method, which will depend on the level of urea present in the sample.

[0590] 5. Analyze the data using 32Karat software. The pI marker provides a standard curve to quantify the sample peak pI value.

[0591] Antibody serum stability assessment

[0592] 1. Prepare 600 μl of 0.4 mg / mL polishing antibody in PBS.

[0593] 2. Use mouse serum (SCD-808), human serum (S-123) and Cyno serum (S-118) from Seralab. Dispense 150ul of serum and PBS control in a round-bottom 96-well plate, add 50ul of 0.4mg / mL antibody solution (in PBS, final concentration is 100ug / mL) to each serum type in triplicate in a tissue culture cabinet (BSL-2). Store some at 4°C for use as a control.

[0594] Serum culture plate layout

[0595] PBS Mouse serum human serum Cyno Serum PBS Mouse serum human serum Cyno Serum PBS Mouse serum human serum Cyno Serum

[0596] 3. Seal the plate and incubate at 37°C.

[0597] 4. Under sterile conditions (BSL-2), take 20ul samples at specific time intervals (e.g., day 10, day 20) to avoid contamination. Freeze at -20°C until analysis.

[0598] 5. Analyze the longest incubation time first. Dilute the samples appropriately and measure antigen binding to TauC3 by generating an ELISA binding curve for each sample (3*dilution) (Section 8.11). Use unincubated antibody as a control (NI) to compare PBS / all sera for each mAb sample on the same plate.

[0599] All references, publications, and patent documents cited herein, as well as the text appearing in the Figures and Sequence Listing, are incorporated by reference in their entirety for all purposes to the same extent as if each were individually denoted.

[0600] In the foregoing description, the present invention has been described with reference to specific exemplary embodiments and examples thereof. However, it will be apparent that various modifications and changes may be made thereto without departing from the broader spirit and scope of the present invention as set forth in the appended claims. Therefore, the description and drawings are to be regarded as illustrative rather than restrictive.

[0601] The citation of any document herein is not intended to be an admission that such document is relevant prior art or to be considered material for the patentability of any claim of the present application. Any statement as to the contents or date of any document is based on the information available to the applicant at the time of filing the application and does not constitute an admission as to the correctness of such statement. Sequence Listing <110> TAUC3 BIOLOGICS LIMITED <120> Anti-TAUC3 antibodies and their applications <130> 350011.1002PCT <150> 62 / 829,774 <151> 2019-04-05 <160> 141 <170> PatentIn version 3.5 <210> 1 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> htau ​​40 <400> 1 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205 Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln 275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly 290 295 300 Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335 Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350 Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala 370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser 405 410 415 Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 Ser Ala Ser Leu Ala Lys Gln Gly Leu 435 440 <210> 2 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> Tau 2N3R <400> 2 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95 Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120 125 Ser Lys Ser Leu Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205 Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220 Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230 235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr 275 280 285 Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly 290 295 300 Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln 305 310 315 320 Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly 325 330 335 Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys 340 345 350 Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val 355 360 365 Ser Gly Asp Thr Ser Pro Ala His Leu Ser Asn Val Ser Ser Thr Gly 370 375 380 Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu 385 390 395 400 Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 405 410 <210> 3 <211> 412 <212> PRT <213> Artificial Sequence <220> <223> Tau IN4R <400> 3 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Gly Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Ala Glu Glu Ala Gly Ile Gly 65 70 75 80 Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala 85 90 95 Arg Met Val Ser Lys Ser Leu Asp Gly Thr Gly Ser Asp Asp Lys Lys 100 105 110 Ala Lys Gly Ala Asp Gly Lys Thr Leu Ile Ala Thr Pro Arg Gly Ala 115 120 125 Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala 130 135 140 Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro 145 150 155 160 Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr 165 170 175 Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 180 185 190 Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 195 200 205 Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu 210 215 220 Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln 225 230 235 240 Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser 245 250 255 Asn Val Gln Ser Lys Cys Gly Ser Leu Asp Asn Ile Leu His Val Pro 260 265 270 Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys 275 280 285 Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly 290 295 300 Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg 305 310 315 320 Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly 325 330 335 Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn 340 345 350 Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro 355 360 365 Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser 370 375 380 Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala 385 390 395 400 Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 405 410 <210> 4 <211> 383 <212> PRT <213> Artificial Sequence <220> <223> Tau ON4R <400> 4 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Leu Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140 Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145 150 155 160 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Ala Thr Pro Pro Lys 165 170 175 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 Pro Asp Leu Lys Asn Val Lys Ser Leu Ile Gly Ser Thr Glu Asn Leu 195 200 205 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu 210 215 220 Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys 225 230 235 240 His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp 245 250 255 Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 260 265 270 Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe 275 280 285 Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His 290 295 300 Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe 305 310 315 320 Arg Glu Asn Ala Lys Ala Leu Thr Asp His Gly Ala Glu Ile Val Tyr 325 330 335 Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn 340 345 350 Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala 355 360 365 Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 5 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Tau 1N3R <400> 5 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Ala Glu Glu Ala Gly Ile Gly 65 70 75 80 Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala 85 90 95 Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys 100 105 110 Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala 115 120 125 Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala 130 135 140 Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro 145 150 155 160 Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr 165 170 175 Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 180 185 190 Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 195 200 205 Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu 210 215 220 Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln 225 230 235 240 Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser 245 250 255 Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro 260 265 270 Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp 275 280 285 Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro 290 295 300 Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu 305 310 315 320 Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser 325 330 335 Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser 340 345 350 Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu 355 360 365 Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 6 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Tau 0N3R <400> 6 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr Pro Thr Ala Glu Ala Glu Glu Ala Gly Ile Gly 65 70 75 80 Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala 85 90 95 Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys 100 105 110 Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala 115 120 125 Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala 130 135 140 Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro 145 150 155 160 Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr 165 170 175 Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 180 185 190 Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser 195 200 205 Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu 210 215 220 Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln 225 230 235 240 Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser 245 250 255 Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro 260 265 270 Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp 275 280 285 Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro 290 295 300 Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu 305 310 315 320 Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser 325 330 335 Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser 340 345 350 Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu 355 360 365 Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 370 375 380 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR1 <400> 7 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR2 <400> 8 Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr 1 5 10 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> VGGGDF <400> 9 Val Gly Gly Gly Asp Phe 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR1 <400> 10 Gln Glu Ile Ser Val Tyr 1 5 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR2 <400> 11 Gly Ala Phe 1 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LQVRYP <400> 12 Leu Gln Tyr Val Arg Tyr Pro Trp Thr 1 5 <210> 13 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain HM <400> 13 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain KE <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain KN <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain KO <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain KP <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain KM <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Immunopeptide <400> 19 Ser Ser Thr Gly Ser Ile Asp Met Val Asp 1 5 10 <210> 20 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 20 tgtaaaacga cggccagtat gaagttgcct gttaggctgt tggtgctg 48 <210> twenty one <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty one tgtaaaacga cggccagtat ggagwcagac acactcctgy tatgggtg 48 <210> twenty two <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty two tgtaaaacga cggccagtat gagtgtgctc actcaggtcc tggsgttg 48 <210> twenty three <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty three tgtaaaacga cggccagtat gaggrcccct gctcagwtty ttggmwtctt g 51 <210> twenty four <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> twenty four tgtaaaacga cggccagtat ggatttwagg tgcagattwt cagcttc 47 <210> 25 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 25 tgtaaaacga cggccagtat gaggtkckkt gktsagstsc tgrgg 45 <210> 26 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 26 tgtaaaacga cggccagtat gggcwtcaag atggagtcac akwyycwgg 49 <210> 27 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 27 tgtaaaacga cggccagtat gtggggayct ktttycmmtt tttcaattg 49 <210> 28 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 28 tgtaaaacga cggccagtat ggtrtccwca sctcagttcc ttg 43 <210> 29 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 29 tgtaaaacga cggccagtat gtatatatgt ttgttgtcta tttct 45 <210> 30 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 30 tgtaaaacga cggccagtat ggaagcccca gctcagcttc tcttcc 46 <210> 31 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 31 tgtaaaacga cggccagtat gragtywcag acccaggtct tyrt 44 <210> 32 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 32 tgtaaaacga cggccagtat ggagacacat tctcaggtct ttgt 44 <210> 33 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 33 tgtaaaacga cggccagtat ggattcacag gcccaggttc ttat 44 <210> 34 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 34 tgtaaaacga cggccagtat gatgagtcct gcccagttcc tgtt 44 <210> 35 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 35 tgtaaaacga cggccagtat gaatttgcct gttcatctct tggtgct 47 <210> 36 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 36 tgtaaaacga cggccagtat ggattttcaa ttggtcctca tctcctt 47 <210> 37 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 37 tgtaaaacga cggccagtat gaggtgccta rctsagttcc tgrg 44 <210> 38 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 38 tgtaaaacga cggccagtat gaagtactct gctcagtttc tagg 44 <210> 39 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 39 tgtaaaacga cggccagtat gaggcattct cttcaattct tggg 44 <210> 40 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 40 caggaaacag ctatgaccac tggatggtgg gaagatgg 38 <210> 41 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 41 tgtaaaacga cggccagtat gaaatgcagc tggggcatst tcttc 45 <210> 42 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 42 tgtaaaacga cggccagtat gggatggagc trtatcatsy tctt 44 <210> 43 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 43 tgtaaaacga cggccagtat gaagwtgtgg ttaaactggg ttttt 45 <210> 44 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 44 tgtaaaacga cggccagtat gractttggg ytcagcttgr ttt 43 <210> 45 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 45 tgtaaaacga cggccagtat ggactccagg ctcaatttag ttttcctt 48 <210> 46 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 46 tgtaaaacga cggccagtat ggctgtcytr gsgctrctct tctgc 45 <210> 47 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 47 tgtaaaacga cggccagtat ggratggagc kggrtctttm tctt 44 <210> 48 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 48 tgtaaaacga cggccagtat gagagtgctg attcttttgt g 41 <210> 49 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 49 tgtaaaacga cggccagtat ggmttgggtg tggamcttgc tattcctg 48 <210> 50 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 50 tgtaaaacga cggccagtat gggcagactt acattctcat tcctg 45 <210> 51 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 51 tgtaaaacga cggccagtat ggattttggg ctgatttttt ttattg 46 <210> 52 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 52 tgtaaaacga cggccagtat gatggtgtta agtcttctgt acctg 45 <210> 53 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 53 tgtaaaacga cggccagtat gaacaggctt acttcctcat tgctgctgc 49 <210> 54 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 54 caggaaacag ctatgaccca gtggatagac agatggggg 39 <210> 55 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 55 caggaaacag ctatgaccca gtggatagac cgatggggc 39 <210> 56 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 56 caggaaacag ctatgaccca gtggatagac tgatggggg 39 <210> 57 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 57 caggaaacag ctatgaccca agggatagac agatggggc 39 <210> 58 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 58 tgtaaaacga cggccagtga ggtgcaggtt gttgagtctg g 41 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 59 tgttcctttc catgggtctt 20 <210> 60 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 60 ctctcggagg tgctcctgga g 21 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 61 gcagttccag atttcaactg 20 <210> 62 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 62 tgtaaaacga cggccagt 18 <210> 63 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 63 caggaaacag ctatgacc 18 <210> 64 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 64 ctctggctcc ctgataccac cggagaggtg caggtggtgg agagc 45 <210> 65 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 65 ctctggctcc ctgataccac cggagatatc cagatgacac agtct 45 <210> 66 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 66 gggcccttgg tggaggcgga gctcactgtc agggcggt 38 <210> 67 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 67 cgcttggtgc tgccacagtt ctcttgatct ccagctttgt gccg 44 <210> 68 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 68 ctctggctcc ctgataccac cggactggtg cagctggtgg aaagcg 46 <210> 69 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 69 ctctggctcc ctgataccac cggagaggtg caggtggtgg aaagcg 46 <210> 70 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 70 gggcccttgg tggaggcgga gctcactgtc accagggtg 39 <210> 71 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 71 cctgatacca ccggagaggt gcagctggtg 30 <210> 72 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 72 caccagctgc acctctccgg tggtatcagg 30 <210> 73 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 73 cggactggtg caggtggtgg aaagcgg 27 <210> 74 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 74 ccgctttcca ccacctgcac cagtccg 27 <210> 75 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 75 tttaacacat atgcaatgaa ctgggtgcgg cagg 34 <210> 76 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 76 cctgccgcac ccagttcatt gcatatgtgt taaa 34 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 77 ctggagtggg tggcccggat cagatct 27 <210> 78 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 78 agatctgatc cgggccaccc actccag 27 <210> 79 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 79 ctaagagcaa caattatgca acatattatg cagcatctgt gaagggcag 49 <210> 80 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 80 ctgcccttca cagatgctgc ataatatgtt gcataattgt tgctcttag 49 <210> 81 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 81 tatgcaacag catatgcaga ttctgtgaag ggcaggttca 40 <210> 82 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 82 tgaacctgcc cttcacagaa tctgcatatg ctgttgcata 40 <210> 83 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 83 ccgcgacgat tctaagagta cagcctatct gcaga 35 <210> 84 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 84 tctgcagata ggctgtactc ttagaatcgt cgcgg 35 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 85 tctcccgcga cgattctaag aatatggcct atctgcagat 40 <210> 86 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 86 atctgcagat aggccatatt cttagaatcg tcgcgggaga 40 <210> 87 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 87 gacgattcta agaatacagt ctatctgcag atggactcc 39 <210> 88 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 88 ggagtccatc tgcagataga ctgtattctt agaatcgtc 39 <210> 89 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 89 ctctggctcc ctgataccac cggagacatc cagatgaccc agtctc 46 <210> 90 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 90 ctctggctcc ctgataccac cggagacatc cagatgacac agtctc 46 <210> 91 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 91 cgcttggtgc tgccacagtt ctcttgatct ccacctttgt gccg 44 <210> 92 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 92 ttctgctgat accagctcag gtacacggag atc 33 <210> 93 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 93 tgtacctggg ctggtttcag cagaagccc 29 <210> 94 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 94 gggcttctgc tgaaaccagc ccaggtaca 29 <210> 95 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 95 gaagcccggc aaggccatta agcggctgat ctac 34 <210> 96 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 96 gtagatcagc cgcttaatgg ccttgccggg cttc 34 <210> 97 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 97 atctacggcg ccttcacgct gcagtccg 28 <210> 98 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 98 cggactgcag cgtgaaggcg ccgtagat 28 <210> 99 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 99 ggatccagat ctggcagcga gtttaccctg a 31 <210> 100 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 100 tcagggtaaa ctcgctgcca gatctggatc c 31 <210> 101 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 101 cagatctggc accgagtata ccctgacaat ctcta 35 <210> 102 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 102 tagagattgt cagggtatac tcggtgccag atctg 35 <210> 103 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 103 ctacggcgcc ttcagcctgc agtccggagt 30 <210> 104 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 104 actccggact gcaggctgaa ggcgccgtag 30 <210> 105 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 105 Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Val Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Ala Leu Thr 100 105 110 Val Ser Ser 115 <210> 106 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 106 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Tyr Glu Gly Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 107 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 107 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 108 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 108 Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80 Val Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 109 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 109 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 110 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 110 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 111 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain <400> 111 Leu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 112 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 112 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 113 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 113 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 114 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain <400> 114 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 115 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 115 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 116 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 116 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 117 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 117 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Met 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 118 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 118 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 119 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 119 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 120 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 120 Leu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 121 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 121 Leu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Met 65 70 75 80 Ala Tyr Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Gly Gly Gly Asp Phe Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 122 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 122 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 123 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 123 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 124 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 124 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 125 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 125 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 126 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 126 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 127 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 127 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 128 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 128 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 129 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 129 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 130 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 130 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 131 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 131 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 132 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 132 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 133 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 133 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 134 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 134 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 135 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 135 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 136 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 136 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 137 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 137 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 138 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 138 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Val Tyr 20 25 30 Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Gly Ala Phe Lys Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Val Arg Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 139 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 139 tgtaaaacga cggccagtga ggtgcaggtt gttgagtctg g 41 <210> 140 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 140 tgtaaaacga cggccagtat gacattgaac atgctgttgg ggc 43 <210> 141 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primers <400> 141 gatctccgtg tacctgagct ggtatcagca gaa 33

Claims

1. An isolated anti-TauC3 antibody, wherein The anti-TauC3 antibody comprises (a) a variable heavy chain (V H ) polypeptide and (b) variable light chain (V L ) polypeptide, the variable heavy chain (V H ) polypeptide comprises a CDR1 consisting of SEQ ID NO: 7, a CDR2 consisting of SEQ ID NO: 8, and a CDR3 consisting of SEQ ID NO: 9, the variable light chain (V L ) The polypeptide comprises a CDR1 consisting of SEQ ID NO: 10, a CDR2 consisting of SEQ ID NO: 11, and a CDR3 consisting of SEQ ID NO:

12.

2. The anti-TauC3 antibody according to claim 1, wherein The anti-TauC3 antibody is a humanized antibody.

3. The anti-TauC3 antibody according to claim 1, wherein The anti-TauC3 antibody is a chimeric antibody.

4. The anti-TauC3 antibody according to claim 1, wherein The binding affinity (KD) of the anti-TauC3 antibody to TauC3 is 1×10 -10 Up to 1×10 -12 The binding affinity (KD) of the anti-TauC3 antibody to full-length tau is 1×10 -4 Up to 1×10 -8 M.

5. The anti-TauC3 antibody according to any one of claims 1 to 4, wherein The dissociation rate K of the anti-TauC3 antibody to TauC3 d 1x10 -4 Up to 1×10 -3 s -1 .

6. The anti-TauC3 antibody according to any one of claims 1 to 4, wherein The variable heavy chain (V H ) polypeptide is the polypeptide of SEQ ID NO: 13, the variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

18.

7. The anti-TauC3 antibody according to claim 6, wherein The variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:

17.

8. The anti-TauC3 antibody according to claim 6, wherein The variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:

14.

9. The anti-TauC3 antibody according to claim 6, wherein The variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:

15.

10. The anti-TauC3 antibody according to claim 6, wherein The variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:

16.

11. The anti-TauC3 antibody according to claim 6, wherein The variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:

18.

12. The anti-TauC3 antibody according to any one of claims 1 to 4, wherein The water solubility of the anti-TauC3 antibody is 50 mg / ml to 200 mg / ml.

13. The anti-TauC3 antibody according to any one of claims 1 to 4, wherein The anti-TauC3 antibody has a binding affinity (KD) for TauC3 of 10 to 35 pM.

14. The anti-TauC3 antibody according to any one of claims 1 to 4, wherein The anti-TauC3 antibody is used to treat Tau disease.

15. The anti-TauC3 antibody according to claim 14, wherein The tauopathy is selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy, frontotemporal dementia, traumatic brain injury, Pick's disease, and corticobasal degeneration.

16. The anti-TauC3 antibody according to claim 14, wherein The Tau disease is frontotemporal lobar degeneration.

17. The anti-TauC3 antibody according to claim 15, wherein The Tau disease is Alzheimer's disease.

18. A pharmaceutical composition comprising the anti-TauC3 antibody of claim 1 and one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition according to claim 18, wherein The variable heavy chain (V H ) polypeptide is the polypeptide of SEQ ID NO: 13, the variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

18.

20. The pharmaceutical composition according to claim 19, wherein The variable light chain (V L ) The polypeptide is the polypeptide of SEQ ID NO:

17.

21. The pharmaceutical composition according to claim 18, wherein The anti-TauC3 antibody has a binding affinity (KD) for TauC3 of 10 pM to 40 pM.

22. The pharmaceutical composition according to claim 21, wherein The anti-TauC3 antibody is a humanized antibody.

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