阅读说明：本技术 抗tauc3抗体及其应用 (anti-TAUC 3antibody and application thereof ) 是由 D·钱恩 P·巴克拉尼亚 S·帕特尔 于 2020-04-02 设计创作，主要内容包括：描述了对TauC3的特异性比对全长tau(2N4R)的特异性高至少几个数量级的抗TauC3抗体。还描述了使用抗TauC3抗体的方法。(anti-TauC 3 antibodies with specificity for TauC3 at least several orders of magnitude higher than that of full-length tau (2N4R) are described. Methods of using anti-TauC 3 antibodies are also described.)

1. An isolated anti-tauC 3antibody, wherein the anti-tauC 3antibody has a binding affinity (KD) for tauC3 of 1 × 10^-10To 1X10^-12The binding affinity (KD) of the anti-tauC 3antibody to full-length tau is 1X10^-4To 1X10^-8M, said antibody being a humanized, chimeric or fully humanized antibody.

2. The anti-tauC 3antibody of claim 1, wherein the anti-tauC 3antibody has an off-rate K of tauC3_dIs 1 × 10^-4To 1X10^-3s^-1。

3. The anti-TauC 3antibody of claim 1 or 2, wherein the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide and (b) a variable light chain (V)_L) Polypeptide, said variable heavy chain (V)_H) The polypeptide comprises SEQ ID NO: 7, CDR1, SEQ ID NO: 8 and CDR2 of SEQ ID NO: CDR shown in 93, the variable heavy chain (V)_H) Polypeptide and SEQ ID NO: 13 has at least 70% sequence identity; the variable light chain (V)_L) The polypeptide comprises SEQ ID NO: 10, CDR1, SEQ ID NO: 11 and CDR2 shown in SEQ ID NO: 12, the variable light chain (V) of CDR3_L) Polypeptide and SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18 have at least 70% sequence identity.

4. The anti-TauC 3antibody of claim 3, wherein variable heavy chain (V)_H) The polypeptide comprises SEQ ID NO: 13; variable light chain (V)_L) The polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

5. The anti-TauC 3antibody of claim 3, wherein in the variable heavy chain (V)_H) On the polypeptide, CDR1 is SEQ ID NO: 7, CDR2 is SEQ ID NO: 8, CDR3 is SEQ ID NO: 9; in the light chain (V)_L) On the polypeptide, CDR1 is SEQ ID NO: 10, CDR2 is SEQ ID NO: 11, CDR3 is SEQ ID NO: 12.

6. The anti-TauC 3antibody of claim 5, wherein variable heavy chain (V)_H) The polypeptide is SEQ ID NO: 13, variable light chain (V)_L) The polypeptide is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18.

7. The anti-TauC 3antibody of claim 3, wherein the anti-TauC 3antibody comprises an amino acid sequence that hybridizes to SEQ ID NO: 13V having at least 95% sequence identity_LChain polypeptide, and a polypeptide corresponding to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18V having at least 95% sequence identity_HA chain polypeptide.

8. The anti-TauC 3antibody of claim 1 or 2, wherein the water solubility of the anti-TauC 3antibody is about 50mg/ml to about 200 mg/ml.

9. The anti-TauC 3antibody of claim 11, wherein the anti-TauC 3antibody has a binding affinity (KD) for TauC3 of about 10 to about 35 pM.

10. The anti-tauC 3antibody of claim 1, wherein the anti-tauC 3antibody is used to treat a tauopathy.

11. An anti-TauC 3antibody according to claim 10, wherein the tauopathy is selected from alzheimer's disease, progressive supranuclear palsy, frontotemporal dementia, traumatic brain injury, pick's disease, corticobasal degeneration and frontotemporal degeneration.

12. The anti-TauC 3antibody of claim 10, wherein the tauopathy is alzheimer's disease.

13. A pharmaceutical composition comprising the anti-TauC 3antibody of claim 1 and one or more pharmaceutically acceptable excipients.

14. The pharmaceutical composition of claim 13, wherein the anti-TauC 3 comprises (a) a variable heavy chain (V)_H) A polypeptide and (b) a variable light chain (V)_L) Polypeptide, said variable heavy chain (V)_H) The polypeptide comprises SEQ ID NO: 7, CDR1, SEQ ID NO: 8 and CDR2 of SEQ ID NO: 9, CDR 3; the variable light chain (V)_L) The polypeptide comprises SEQ ID NO: 10, CDR1, SEQ ID NO: 11 and CDR2 shown in SEQ ID NO: 12, CDR 3.

15. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition has a binding affinity (KD) for TauC3 of 10pM to 40 pM.

Background

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

There are six known isoforms of Tau protein. These isoforms differ in the presence of a 29 or 58 amino acid insert in the amino-terminal region, and in the addition or deletion of a tandem repeat (which may be repeated 3 or 4 times) in the carboxy-terminal region of tau, which is known as the microtubule binding domain. The microtubule binding domain consists of an imperfect repeat sequence of 31 or 32 amino acid residues. The longest tau isoform (2N4R) is 441 amino acids in length and comprises four repeats (R1, R2, R3 and R4) and two inserts. The smallest tau isoform comprises 352 amino acid residues with three tandem repeats (R1, R3, and R4) in the microtubule binding domain, with no amino terminal insertions. In SEQ ID NO: 1-6 provide amino acid sequences corresponding to isoforms of human tau protein.

SEQ ID NO: 1 is the longest tau isomer htau40, comprising two N-terminal inserts and four microtubule binding domains (2N4R), as follows:

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

SEQ ID NO: 3 contains one N-terminal insert and four microtubule binding domains (IN4R), as follows:

SEQ ID NO: 4 contained zero N-terminal inserts and four microtubule binding domains (0N4R), as follows:

SEQ ID NO: 5 contains one N-terminal insert and three microtubule binding domains (1N3R) as follows:

SEQ ID NO: 6 contained zero N-terminal inserts and three microtubule binding domains (0N3R), as follows:

TauC3 is an extremely deleterious, nucleating (nucleating), pre-entanglement, intracellular and preferentially secreted C-terminally truncated tau fragment ending in aspartic acid 421. In contrast 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 to, for example, vaccination (seeding) and spread of pathological tau aggregation.

Pathological aggregation and diffusion of tau in the brain is 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), and the like (collectively referred to as "tauopathies").

Disclosure of Invention

It is an object of the present invention to provide a chimeric antibody which is useful for the diagnosis and treatment of neurodegenerative diseases associated with the pathological activity of TauC3 in the brain.

It is another object of the present invention to provide a humanized antibody which can be used for the diagnosis and treatment of neurodegenerative diseases associated with the pathological activity of TauC3 in the brain.

It is another object of the invention to provide human antibodies which are useful for the diagnosis and treatment of neurodegenerative diseases associated with pathological activity of TauC3 in the brain.

It is another object of the present invention to provide a chimeric antibody which is specific to the C-terminus of TauC 3.

It is another object of the present invention to provide a chimeric antibody which is specific to the C-terminus of TauC3 and has 1x10^-3 s^-1Dissociation Rate (K) of_d)。

It is another object of the invention to provide a humanized antibody which is specific for the C-terminus of TauC 3.

It is another object of the present invention to provide a humanized antibody which has specificity to the C-terminus of TauC3 and has 1x10^-3 s^-1Dissociation Rate (K) of_d)。

To facilitate the above and other objects, the present invention relates to chimeric, humanized and human antibodies specific to the C-terminus of TauC3 ("anti-TauC 3 antibodies"). The binding affinity (KD) of the anti-TauC 3antibody to TauC3 was 1X10^-10To 1x10^-12Binding affinity (KD) for full-length tau ("FLT") (SEQ ID NO: 1) is 1x10^-4To 1x10^-8And M. For example, the binding affinity (KD) of an anti-TauC 3antibody to TauC3 may be about 5x10^-12M to about 1.2x10^-10M, about 1X10^-11M to about 1x10^-10M, about 1X10^-11M to about 9x10^-11M, about 1X10^-11M to about 8x10^-11M, about 1X10^-11M to about 7x10^-11M, about 1X10^-11M to about 6x10^-11M, about 1X10^-11M to about 5x10^-11M, or about 1x10^-11M to about 4x10^-11M; and a binding affinity (KD) for FLT of 1x10-4 to 1x10-⁸And M. In a preferred embodiment, the antibody retains its binding capacity after being subjected to a temperature of about 40 ℃ to about 67 ℃ for 10 minutes and after being incubated in serum (e.g., mouse) at 37 ℃ for 21 days. The high performance of anti-TauC 3 antibodies allows the antibodies to target TauC3 without affecting the normal physiological function of FLT. In some embodiments, the specificity of the antibody allows targeting only the most harmful tau species. This may allow, for example, potentially reducing the effective therapeutic dose compared to antibodies that are not specific and do not distinguish between different classes of tau. anti-TauC 3 antibodies and antigen-binding fragments thereof are useful, for example, in diagnosis and treatment and in the brain with TauC3Including, 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 degeneration (FTLD), and the like. The anti-TauC 3antibody can have a water solubility of 50mg/ml or greater (e.g., about 50mg/ml to about 200mg/ml, about 55mg/ml to about 180mg/ml, about 55mg/ml to about 170mg/ml, about 55mg/ml to about 150mg/ml, about 55mg/ml to about 140mg/ml, about 55mg/ml to about 130mg/ml, or about 60mg/ml to about 130 mg/ml).

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

The invention also relates to chimeric, humanized and human anti-tauC 3 antibodies that have a binding affinity (KD) for TauC3 of 1x10^-10To lx10^-12Dissociation rate (Kd) of 1x10^-3s^-1And a binding affinity (KD) for FLT of 1x10^-4To 1x10^-8M。

The anti-TauC 3antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising CDR1 represented by the sequence GFTFNTYA (SEQ ID NO: 7), CDR2 represented by IRSKSNNYAT (SEQ ID NO: 8), and CDR3 represented by VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising CDR1 represented by sequence QEISVY (SEQ ID NO: 10), CDR2 represented by sequence GAF (SEQ ID NO: 11), and CDR3 represented by sequence LQYVRYPWT (SEQ ID NO: 12); and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x10^-3Dissociation Rate (K) of_d) (ii) a And has a binding affinity (KD) for FLT (SEQ ID NO: 1) of 1x10^-4To 1x10^-8M, or NO detectable binding to FLT (SEQ ID NO: 1).

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

In certain embodiments, the anti-TauC 3antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising the same CDR1 as the sequence GFTFNTYA (SEQ ID NO: 7), the same CDR2 as IRSKSNNYAT (SEQ ID NO: 8), and the same CDR3 as VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising the same CDR1 as sequence QEISVY (SEQ ID NO: 10), the same CDR2 as sequence GAF (SEQ ID NO: 11), and the same CDR3 as sequence LQYVRYPWT (SEQ ID NO: 12); and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x10^-3Dissociation ratio (Kd) and binding affinity for FLT (SEQ ID NO: 1) ((Kd))KD) is 1x10^-4To 1x10^-8M, or NO detectable binding to FLT (SEQ ID NO: 1).

In certain embodiments, the anti-TauC 3antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising CDR1 of the sequence GFTFNTYA (SEQ ID NO: 7), CDR2 of the sequence IRSKSNNYAT (SEQ ID NO: 8), and CDR3 of the sequence VGGGDF (SEQ ID NO: 9); and (b) a light chain variable region comprising CDR1 of sequence QEISVY (SEQ ID NO: 10), CDR2 of sequence GAF (SEQ ID NO: 11), and CDR3 of sequence LQYVRYPWT (SEQ ID NO: 12); and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x10^-3The following dissociation rates (Kd), and for SEQ ID NO: 1 has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or a sequence identical to SEQ ID NO: 1 has no detectable binding and is 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 degeneration (FTLD). The antibodies may also be used to diagnose 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), or frontotemporal degeneration (FTLD).

In one aspect, the invention relates to an anti-TauC 3antibody that is a humanized antibody comprising (a) a heavy chain variable region comprising a heavy chain variable region consisting of SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and the CDR2 represented by SEQ ID NO: 9, CDR 3; and (b) a light chain variable region comprising a light chain variable region consisting of SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: CDR3 denoted by 12; and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x1^0-3 s^-1Dissociation Rate (K) of_d) (e.g., 1x 10)^-4To 1x10^-3s^-1) And, for FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or no detectable binding to FLT. Thus, a humanized antibody may comprise (a) a heavy chain variable region comprising SEQ ID NO: 7, CDR1 of SEQ ID NO:8 and CDR2 of SEQ ID NO: 9 CDR 3; and (b) a light chain variable region comprising SEQ ID NO: 10, CDR1 of SEQ ID NO: 11 CDR2 and seq id NO: 12 CDR 3; and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x10^-3Dissociation Rate (Kd) of (for example, 1X 10)^-4To 1x10^-3 s^-1) And to SEQ ID NO: 1 has a binding affinity (KD) of 1x10^-4To lx10^-8M, or 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 degeneration (FTLD), and the like.

In one aspect, the invention relates to an anti-TauC 3antibody that is a humanized antibody comprising (a) a heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 7, CDR1 identical to SEQ ID NO: 8, and a CDR2 identical to SEQ ID NO: 9 an identical CDR 3; and (b) a light chain variable region comprising a sequence identical to SEQ ID NO: 7, CDR1 identical to SEQ ID NO: 11 and CDR2 identical to SEQ ID NO: 12 an identical CDR 3; and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x10^-3 s^-1Dissociation Rate (K) of_d) (e.g., 1x10^-4To 1x10^-3s^-1) And FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7, CDR1 of SEQ ID NO: 8 and CDR2 of SEQ ID NO: 9 CDR 3; and (b) a light chain variable region having the amino acid sequence of SEQ ID NO: 10, CDR1 of SEQ ID NO: 11 CDR2 and seq id NO: 12 CDR 3; and a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation rate (Kd) of 1x10^-3s^-1The following (for example, 1x 10)^-4To 1x10^-3s^-1) And to SEQ ID NO: 1 has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or no detectable binding to FLT, and are useful in the treatment of tauopathies, e.g. Alzheimer's diseaseDisease (AD), Progressive Supranuclear Palsy (PSP), frontotemporal dementia (FTD), Traumatic Brain Injury (TBI), pick's disease (PiD), corticobasal degeneration (CBD), frontotemporal degeneration (FTLD), and the like.

In one aspect, the invention relates to an anti-TauC 3antibody that is a humanized antibody comprising (a) a heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 7 CDR1 homologous to SEQ ID NO: 8 CDR2 homologous to SEQ ID NO: 9a homologous CDR 3; and (b) a light chain variable region comprising a sequence identical to SEQ ID NO: CDR1, homologous to SEQ ID NO: 11 and CDR2 homologous to SEQ ID NO: 12 homologous CDR 3; and a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation rate (Kd) of 1x10^-3s^-1The following (for example, 1x 10)^-4To 1x10^-3s^-1) And FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or no detectable binding to FLT. Thus, the humanized antibody may comprise (a) a heavy chain variable region comprising SEQ ID NO: 7, CDR1 of SEQ ID NO: 8 and CDR2 of SEQ ID NO: 9 CDR 3; and (b) a light chain variable region comprising SEQ ID NO: 10, CDR1 of SEQ ID NO: 11 CDR2 and SEQ ID NO: 12 CDR 3; and a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The following (for example, 1x 10)^-4To 1x10^-3s^-1) And to SEQ ID NO: 1 has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or 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 degeneration (FTLD), and the like.

The humanized antibody may, for example, comprise:

(a) a heavy chain variable region comprising the sequence

LVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVARIRSKSNNYATYYAASVKGRFTISRDDSKSMAYLQMDSLKTEDTAVYYCVGGGDFWGQGTLVTVSS (SEQ ID NO: 13) or a sequence similar to SEQ ID NO: 13 homologous sequence; and

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

DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWFQQKPGKAPKRLIYGAFKLQSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 14) or a sequence similar to SEQ ID NO: 14a sequence which is homologous to the sequence of the first,

DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWYQQKPGKAPKRLIYGAFTLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 15) or a sequence similar to SEQ ID NO: 15, a sequence which is homologous to the sequence,

DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWYQQKPGKAPKRLIYGAFSLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 16) or a sequence similar to SEQ ID NO: 16, a sequence which is homologous to the sequence,

DIQMTQSPSSLSASVGDRVTITCRASQEISVYLGWFQQKPGKAPKRLIYGAFKLQSGVPSRFSGSRSGTEYTLTISSLQPEDFATYYCLQYVRYPWTFGGGTKVEIK (SEQ ID NO: 17) or a sequence similar 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^-10And 9x10^-12Binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10^-4To 1x10^-8M, or no detectable binding to FLT.

In certain embodiments, the humanized antibody comprises SEQ ID NO: 13 (V) of a variable heavy chain_H) Polypeptide and SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 17 or SEQ ID NO: 18 (V) of a variable light chain_L) A polypeptide.

In certain embodiments, a humanized antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: 9, CDR3 of variable heavy chain (V)_H) Polypeptide and SEQ ID NO: 13 has at least 70% sequence identity; and (b) a variable light chain (V)_L) A polypeptide comprising SEQ ID NO: 10, CDR1, SEQ ID NO: 11 and CDR2 shown in SEQ ID NO: CDR3 of 12, the variable light chain (V)_L) Polypeptide and SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: SEQ ID NO: 18 have at least 70% sequence identity.

In certain embodiments, the humanized antibody comprises a heavy chain variable region identical to SEQ ID NO: 13V with at least 75% sequence identity_LChain polypeptide, and a polypeptide corresponding to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18V having at least 75% sequence identity_HA chain polypeptide.

In certain embodiments, the humanized antibody comprises a heavy chain variable region identical to SEQ ID NO: 13V with at least 80% sequence identity_LChain polypeptide, and a polypeptide corresponding to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18V having at least 80% sequence identity_HA chain polypeptide.

In certain embodiments, the humanized antibody comprises a heavy chain variable region identical to SEQ ID NO: 13V having at least 85% sequence identity_LChain polypeptide, and a polypeptide corresponding to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18V having at least 85% sequence identity_HA chain polypeptide.

In certain embodiments, the humanized antibody comprises a heavy chain variable region identical to SEQ ID NO: 13V having at least 90% sequence identity_LChain polypeptide, and a polypeptide corresponding to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18V having at least 90% sequence identity_HA chain polypeptide.

In certain embodiments, the humanized antibody comprises a heavy chain variable region identical to SEQ ID NO: 13V having at least 95% sequence identity_LChain polypeptide, and a polypeptide corresponding to SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18V having at least 95% sequence identity_HA chain polypeptide.

In certain embodiments, the anti-TauC 3antibody comprises a variable heavy chain (V)_H) Polypeptides and variable light chains (V)_L) Polypeptide, the variable heavy chain (V)_H) The polypeptide comprises SEQ ID NO: 13, the variable light chain (V)_L) The polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

In certain embodiments, the anti-TauC 3antibody comprises (i) a variable heavy chain (V)_H) A polypeptide comprising CDR1, CDR2 and CDR3, wherein CDRl is SEQ ID NO: 7, CDR2 is SEQ ID NO: 8, CDR3 is SEQ ID NO: 9; (ii) light chain (V)_L) A polypeptide comprising CDR1, CDR2 and CDR3, wherein CDR1 is SEQ ID NO: 10, CDR2 is SEQ ID NO: 11, CDR3 is SEQ ID NO: 12.

In certain embodiments, the anti-TauC 3antibody comprises (i) a variable heavy chain (V)_H) A polypeptide; (ii) light chain (V)_L) Polypeptide in which the variable heavy chain (V)_H) The polypeptide is SEQ ID NO: 13, variable light chain (V)_L) The polypeptide is SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17 or SEQ ID NO: 18.

The anti-TauC 3antibody may also be a chimeric antibody comprising (a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 or a sequence identical to SEQ ID NO: 7, CDR1 represented by the sequence homologous to SEQ ID NO: 8 or a variant of SEQ ID NO: 8, and CDR2 represented by the sequence homologous to SEQ ID NO: 9 or a sequence identical to SEQ ID NO: 9 CDR3 represented by the homologous sequence; and (b) a light chain variable region comprising a light chain variable region consisting of SEQ ID NO: 10 or a sequence identical to SEQ id no: 10, CDR1 represented by the sequence homologous to SEQ ID NO: 11 or a variant of SEQ ID NO: 11, and CDR2 represented by the sequence homologous to SEQ ID NO: 12 or a variant of SEQ ID NO: 12 CDR3 represented by the homologous sequence; and a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation rate (Kd) of 1x10^-3s^-1The following (for example, 1x 10)^-4To 1x10^-3s^-1) And FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or NO detectable binding to FLT (SEQ ID NO: 1).

The present invention also relates to an antigen-binding fragment of an antibody comprising (a) a heavy chain variable region comprising a heavy chain variable region consisting of SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: 9, CDR 3; and (b) a light chain variable region comprising a light chain variable region consisting of SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: CDR3 denoted by 12; and a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation rate (Kd) of 1x10^-3s^-1The following (for example, 1x 10)^-4To 1x10^-3s^-1) And FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or NO detectable binding to FLT (SEQ ID NO: 1). An 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.

The present invention also relates to an antigen-binding fragment of an antibody comprising (a) a heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 7 CDR1 homologous to SEQ ID NO: 8 CDR2 homologous to SEQ ID NO: 9a homologous CDR 3; (b) a light chain variable region comprising a heavy chain variable region substantially identical to SEQ ID NO: 10 CDR1 homologous to SEQ ID NO: 11, and CDR2 homologous to SEQ ID NO: 12 homologous CDR 3; and a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation rate (Kd) of 1x10^-3s^-1The following (for example, 1x 10)^-4To 1x10^-3s^-1) And, for FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or NO detectable binding to FLT (SEQ ID NO: 1). An 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.

The present invention also relates to an antigen-binding fragment of an antibody comprising (a) a heavy chain variable region comprising an amino acid sequence identical to SEQ ID NO: 7, CDR1 identical to SEQ ID NO: 8, and a CDR2 identical to SEQ ID NO: 9 an identical CDR 3; and (b) a light chain variable region comprising a sequence identical to SEQ ID NO: 10, CDR1 identical to SEQ ID NO: 11, and a CDR2 identical to SEQ ID NO: 12 an identical CDR 3; and 1x10 for TauC3^-10And 1x10^-12Binding affinity (KD) and 1x10^-3s^-1Dissociation Rate (K) of_d) (e.g., 1x10^-4To lx10^-3s^-1) And FLT (SEQ ID NO: 1) has a binding affinity (KD) of 1x10^-4To 1x10^-8M, or 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, or a F (ab')2 fragment or scFv fragment.

The invention also relates to methods of blocking pathological tau uptake (uptake), methods of blocking pathological tau vaccination activity, methods of inhibiting pathological tau protein aggregation, and methods of blocking the diffusion of pathological tau, tau fibrils and tau aggregates from one neuron to another or from one part of the brain to another. The method comprises administering to a subject in need thereof an effective amount of an anti-TauC 3 antibody. In some of these embodiments, anti-TauC 3 comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, CDR 3; and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

Once administered, the anti-TauC 3antibody can block or slow the spread of pathological tau from one neuron to another neuron or from one part of the brain to another part, for example, by blocking or slowing TauC3 vaccination activity (e.g., by substantially blocking or slowing intracellular uptake of TauC 3). This mechanism occurs extracellularly, and does not require anti-TauC 3 antibodies to be present inside neurons. anti-TauC 3 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 may comprise heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and/or post-translationally modified tau (truncated or hyperphosphorylated). In addition to blocking intracellular uptake of TauC3 and fibrils comprising TauC3, anti-TauC 3 antibodies may also block or slow pathological tau aggregation within cells (e.g., neurons). Since antibodies have substantially no affinity for full-length tau (e.g., 2N4R), the antibodies do not interfere with the normal nonpathological function of full-length tau. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: c is 10DR1, consisting of SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

anti-TauC 3 antibodies can also slow down the diffusion of fibrils and aggregates comprising TauC3 by binding extracellular TauC3 and aggregates comprising TauC3 released from cells, thereby preventing TauC3 and aggregates comprising TauC3 from entering neighboring cells and slowing down the diffusion of tau aggregates from one neuron to another and from one part of the brain to another. Thus, an anti-TauC 3antibody may be used as a means to prevent TauC3 or aggregates comprising TauC3 from entering cells (e.g., neurons). In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

anti-TauC 3 antibodies may also be used to reduce and/or reduce neuron-to-neuron spread of tau aggregates. For example, an anti-TauC 3antibody may promote disaggregation of protein fibrils comprising TauC3, block intracellular transformation of monomeric TauC3 into fibrils and/or aggregates comprising TauC3, and promote intracellular degradation of fibrils comprising TauC3 and/or aggregates comprising TauC 3. In addition to TauC3, fibrils and aggregates may also comprise 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-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

The anti-TauC 3antibody can reduce brain atrophy in a subject with a tauopathy. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 ofSEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

anti-TauC 3 antibodies may also inhibit the formation of insoluble aggregates comprising heterogeneous populations of full-length tau (e.g., 2N4R), tau oligomers, and/or post-translationally modified tau (truncated or hyperphosphorylated), e.g., to reduce the amount of pathological tau in the brain (e.g., TauC3, fibrils comprising TauC3, and aggregates comprising TauC 3). In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

In certain embodiments, the anti-TauC 3antibody inhibits pathological aggregation of full-length Tau (e.g., 2N 4R). In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

In certain embodiments, administration of an anti-TauC 3antibody can immunize a subject against developing a tauopathy. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

Administration of the anti-TauC 3antibody may reduce symptoms of and/or slow the progression of tauopathies in a subject. For example, in certain embodiments, administration of an anti-TauC 3antibody mayImproving cognitive function and/or motor/sensorimotor function in a subject suffering from tauopathy. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a polypeptide consisting of seq id NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

The anti-TauC 3 antibodies of the invention are useful for treating tauopathies in human subjects. Administering an anti-TauC 3antibody for, inter alia, 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-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8, and a CDR2 represented by SEQ ID NO: 9, and (b) a variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11, and CDR2 represented by SEQ ID NO: 12, CDR 3.

In certain embodiments, the invention relates to a method of reducing the spread of Tau aggregation in the brain of a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the antibody binds to TauC3, but does not bind to full-length Tau. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention further relates to a method of treating tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody sufficient to block the vaccinating activity of TauC3, wherein the anti-TauC 3antibody is a humanized antibody. In some embodiments, a humanized anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprisingConsisting of SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating a tauopathy in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody sufficient to block reuptake of TauC3 by neurons, wherein the anti-TauC 3antibody is a chimeric antibody. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating alzheimer's disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody that has a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation Rate (K)^d) Is 1x10^-3s^-1The binding affinity (KD) for FLT is 1 × 10^-4To 1x10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and a CDR2 represented by SEQ id no: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating Progressive Supranuclear Palsy (PSP) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody that has a binding affinity (KD) for TauC3 of 1x10^-10To 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10 as follows^-4To 1x10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating frontotemporal dementia (FTD) in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody that has a binding affinity (KD) for TauC3 of 1x10^-10To 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10^-4To 1x10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating Traumatic Brain Injury (TBI) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody that has a binding affinity (KD) for TauC3 of 1x10^-10To 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10^-4To 1x10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating pick's disease (PiD) in a subject comprisingAdministering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody that has a binding affinity (KD) for TauC3 of 1x10^-10To 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10^-4To 1x10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating corticobasal degeneration (CBD) in a subject comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody that has a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10^-4To 1x10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to a method of treating frontotemporal lobar degeneration (FTLD) in a subject comprising administering to the subject a therapeutically effective amount of an anti-TauC 3antibody, wherein the anti-TauC 3antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT is 1 × 10^-4To lx10^-8And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 denotesAnd CDR2 consisting of SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3.

The invention also relates to therapeutic agents and compositions for blocking intracellular uptake of pathological tau; therapeutic agents and compositions for blocking tau vaccination activity; therapeutic agents and compositions for blocking tau aggregation; and therapeutic agents and compositions for blocking pathological diffusion of tau, tau fibrils, tau aggregates, and fragments of any of the foregoing from one part of the brain to another, the pathological diffusion being induced or regulated by TauC 3. Therapeutic agents and compositions comprise anti-TauC 3 antibodies described above and below. In addition to the anti-TauC 3antibody, the composition of the invention may comprise one or more pharmaceutically acceptable excipients. The therapeutic agents or compositions may also be used for passive immunization and treatment of 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 degeneration (FTLD), and the like. In certain embodiments, the composition may further comprise an agent that prevents the production of TauC3 (e.g., a caspase inhibitor) or an agent that promotes clearance (e.g., a small molecule TauC3 aggregation inhibitor).

The invention further relates to a composition comprising an anti-TauC 3antibody and one or more pharmaceutically acceptable excipients, wherein the anti-TauC 3antibody is a humanized or chimeric antibody having a binding affinity (KD) for TauC3 of 1x10^-10And 1x10^-12Dissociation Rate (K)_d) Is 1x10^-3s^-1The binding affinity (KD) for FLT (SEQ ID NO: 1) is 1x10^-4To 1x10^{- 8}And M. In some embodiments, the anti-TauC 3antibody comprises (a) a variable heavy chain (V)_H) A polypeptide comprising a sequence defined by SEQ ID NO: 7, CDR1 represented by SEQ ID NO: 8 and CDR2 represented by SEQ ID NO: CDR3 denoted by 9, (b) variable light chain (V)_L) A polypeptide comprising a sequence defined by SEQ ID NO: 10, CDR1 represented by SEQ ID NO: 11 and CDR2 represented by SEQ ID NO: 12, CDR 3. The composition may for example be a liquid compositionA compound (I) is provided. The compositions comprise an effective amount of an anti-TauC 3antibody to treat tauopathies, 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 degeneration (FTLD), and the like. In certain preferred embodiments, the composition is stable (i.e., at least 90% of the anti-TauC 3 antibodies in the composition retain their binding ability after 21 days of storage of the composition at 37 ℃).

Drawings

FIG. 1 is the protein and DNA sequence of the Motau01 kappa light chain variable region.

FIG. 2 is the protein and DNA sequence of the Motau01 kappa heavy chain variable region.

FIG. 3 shows a pHuKLIC vector.

FIG. 4 shows the pHuG4 LIC vector.

FIG. 5 is the protein and DNA sequence of the chimeric Motau01 VK.

FIG. 6 is the protein and DNA sequence of the chimeric Motau01 VH.

FIG. 7 is a binding assay for chimeric Tau01 with tauC3 and FL Tau using a binding enzyme-linked immunosorbent assay.

FIG. 8 is a binding assay for murine and chimeric Tau01 antibodies to tauC3 and FL Tau using Octet.

FIG. 9 is the protein and DNA sequence of Tau01 HA.

FIG. 10 is the protein and DNA sequences of Tau01 HB.

FIG. 11 is the protein and DNA sequences of Tau01 HC.

FIG. 12 is the protein and DNA sequences of Tau01 KA.

FIG. 13 is the protein and DNA sequence of Tau01 KB.

FIG. 14 is the protein and DNA sequences of Tau01 KC.

FIG. 15 is a drawing of the binding of humanized and chimeric Tau01 to TauC 3: forms A and B.

FIG. 16 is a binding enzyme-linked immunosorbent assay of humanized and chimeric Tau01 with TauC 3: HA to HL variants.

Fig. 17 is an Octet screen of humanized Tau01 antibody against TauC 3: HB with KA-KC, HC, and HF.

FIG. 18 is a binding enzyme-linked immunosorbent assay of humanized Tau01 antibody with TauC 3: KA and KJ variants.

Fig. 19 is an Octet screen of humanized Tau01 antibody against TauC 3: HB with KA to KJ variants.

Figure 20 is a second round of binding enzyme-linked immunosorbent assay of humanized Tau01 antibody with TauC 3: HM, HN and HO variants.

Fig. 21 is an Octet screen of a second round of humanized Tau01 antibodies against TauC 3: HM, HN and HO variants.

FIG. 22 is a binding ELISA assay for HC and HM containing humanized antibody and TauC 3.

Fig. 23 is an Octet screen of a second round of humanized Tau01 antibodies against TauC 3: HM variants.

Fig. 24 is an Octet screen of a second round of humanized Tau01 antibodies against TauC3 and FL Tau: leader variants (Lead variants).

Figure 25 is a dissociation rate ranking of lead (lead) humanized candidates with TauC3 using Biacore.

FIG. 26 is a binding force test of Biacore against FL Tau.

FIG. 27 is a graph of the thermostability of chimeric and humanized candidate antibodies.

FIG. 28 is SEC-MALS clustering analysis of purified Mo Tau01HuG4K and Tau01HCKB HuG4K antibodies.

Fig. 29 is a DLS analysis of purified Tau01HMKN, HMKO, HMKP, HMKE and HMKM HuG4K antibodies.

FIG. 30 is a mass spectrum of purified chimeric and humanized candidate antibodies.

Figure 31 is Biacore kinetics of humanized candidate antibodies.

Figure 32 is a thermal shift analysis of humanized candidate antibodies.

FIG. 33 is a non-specific protein-protein interaction (cross-interaction chromatogram) of humanized candidate antibodies.

Figure 34 is a solubility assessment of purified humanized antibody candidates.

Figure 35 is a freeze/thaw and heat stress analysis of humanized candidate antibodies by circular dichroism.

Figure 36 is capillary isoelectric focusing to determine the isoelectric point of humanized candidates.

Figure 37 is a serum stability assessment of humanized candidate antibodies.

Definition of

As used herein, "antibody" is meant 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, e.g., pIII or pVIII or other surface proteins, or on the surface of bacteria, which are capable of binding antigen. F lacking intact antibodies_CFab, F (ab') 2 and Fv fragments of the fragment are cleared more rapidly from the circulation and are less likely to bind to non-specific tissues of 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 can also be a fully human antibody (e.g., from a transgenic mouse or phage).

As used herein, the term "humanized antibody" refers to an antibody in which Complementarity Determining Regions (CDRs) of a mouse or other non-human antibody are grafted onto a human antibody framework. Human antibody framework refers to a fully human antibody that does not include CDRs.

As used herein, the term "human antibody" refers to an antibody in which the entire sequence is derived from a human genetic library (e.g., from a transgenic mouse or phage).

As used herein, the term "homologous" means that the sequence has at least 80% identity to the sequence to which it is homologous, and that a polymeric peptide (e.g., an antibody) comprising the homologous sequence has substantially the same biological activity as a polymeric peptide comprising the sequence to which it is homologous. For example, a humanized antibody comprises (a) a heavy chain variable region comprising CDR1 represented by the sequence GFTFNTYA (SEQ ID NO: 7), CDR2 represented by IRSKSNNYAT (SEQ ID NO: 8), and CDR3 represented by VGGGDF (SEQ ID NO: 9); (b) a light chain variable region comprising CDR1 represented by sequence QEISVY (SEQ ID NO: 10), CDR2 represented by sequence GAF (SEQ ID NO: 11), and CDR3 represented by sequence LQYVRYPWT (SEQ ID NO: 12); and wherein one or more CDR sequences are replaced by homologous sequencesThe binding affinity (KD) of the body to TauC3 was 1X10^-10And 1x10^-12And a binding affinity (KD) for FLT of 1x10^-4To 1x10^-8M, or no detectable binding to FLT. By definition, homologous antibodies have a substantially similar three-dimensional shape.

As used herein, "CDR" refers to "complementarity determining region". CDRs may also be referred to as hypervariable regions. Unless otherwise indicated, CDR sequences disclosed herein are defined by the IMGT numbering system.

As used herein, "consisting of SEQ ID NO: means "that the sequence of the CDR is identical or homologous to the SEQ ID NO.

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 human constant regions.

The term "murine anti-TauC 3 antibody" as used herein refers to antibodies described in nichols, s.b., s.l.deos, c.commns, c.nobuhara, r.e.bennett, d.l.corju, e.maury et al 2017, "chromatography of TauC3 antibodies and purification of bits potential to block tau amplification," PLoS ONE 12 (5): e0177914. the "TauC 3 antibody" in (1).

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.

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

As used herein, the term "affinity" refers to the strength of binding of an antibody molecule to its epitope. Affinity was determined by Surface Plasmon Resonance (SPR) using Biacore kinetics.

The term "KD" as used herein refers to the equilibrium dissociation constant (KD ═ KD/Ka, where KD is the dissociation rate constant and Ka is the association rate constant).

As used herein, the term "immunodepletion" refers to the removal of a protein by the use of an antibody. 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.

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

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

The term "TauC 3" refers to a C-terminally truncated tau fragment ending at aspartic acid 421 of htau40(SEQ ID NO: 1).

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

The term "treating" or "treatment" includes the reduction, reversal or amelioration of at least one symptom or symptom sign associated with tauopathy.

The term "seeding" refers to extracellular activity prior to intracellular aggregation of tau 3, fibrils comprising tau 3, and/or aggregates comprising tau 3 as part of a heterogeneous population of full-length tau (e.g., 2N4R), tau oligomers, and/or post-translationally modified tau (truncated or hyperphosphorylated).

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

"expihho" is an abbreviation for chinese hamster ovary (CHO high density/serum free) cells.

"A" is an abbreviation for adenine.

"bp" is an abbreviation for base pair.

"° c" is an abbreviation for degrees celsius.

"C" is an abbreviation for cytosine.

"MEM" is an abbreviation for minimum essential medium.

"DNA" is an abbreviation for deoxyribonucleic acid.

"ELISA" is an abbreviation for enzyme-linked immunosorbent assay.

"EC 50" is an abbreviation for antibody concentration that elicits 50% of maximal response.

"EC 80" is an abbreviation for antibody concentration that causes 80% of the maximal response.

"ECD" is an abbreviation for extracellular domain.

"g" is an abbreviation for grams.

"G" is an abbreviation for guanine.

"HRP" is an abbreviation for horseradish peroxidase.

"IgG" is an abbreviation for immunoglobulin-G.

"K" is an abbreviation for G or T (IUPAC protocol).

"LIC" is an abbreviation for ligase independent cloning.

"min" is an abbreviation for minutes.

"M" is an abbreviation for A or C (IUPAC protocol).

"nm" is an abbreviation for nanometers.

"OD" is an abbreviation for optical density.

"PBS" is an abbreviation for phosphate buffered saline.

"PCR" is an abbreviation for polymerase chain reaction.

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

"RT" is an abbreviation for room temperature.

"s" is an abbreviation for seconds.

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

"T" is an abbreviation for thymine.

"TBS" is an abbreviation for Tris buffered saline.

"UV" is an abbreviation for ultraviolet light.

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

"VCI" is an abbreviation for vernier (vernier) residues, canonical (canonical) residues, and interface (interface) residues.

"VH" is an abbreviation for immunoglobulin heavy chain variable region.

"VK" is an abbreviation for immunoglobulin kappa light chain variable region

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

"Y" is an abbreviation for C or T (IUPAC convention).

Detailed Description

TauC3 is one of the many high molecular weight species that exist and is responsible for tau aggregation and vaccination activities. 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). Other neurodegenerative diseases besides sporadic AD have been shown to have increased levels of TauC 3.

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

anti-TauC 3 antibodies

The anti-TauC 3 antibodies of the invention recognize an aggregated, extracellular form of pathological TauC 3. The anti-TauC 3antibody of the invention may be, for example, a chimeric, humanized or human anti-TauC 3 antibody.

When tested against recombinant TauC3 protein, the anti-TauC 3antibody showed very tight binding specificity for the target caspase-cleaved Tau protein. In certain embodiments, the anti-TauC 3antibody blocks vaccination in the biosensor assay and effectively blocks entry into neurons of the species responsible for inducing intracellular tau aggregation (i.e., effectively blocks entry of TauC3 and TauC3 fibrils into cells).

anti-TauC 3 antibodies typically have sub-nanomolar specificity for TauC3, and specificity for TauC3 is at least 100-fold greater than for full-length Tau (2N4R) (e.g., specificity for TauC3 is 100-fold or greater than for full-length Tau). For example, the specificity of an anti-TauC 3antibody for TauC3 may be 150 to 5000 times greater than the specificity for full-length Tau (2N 4R). In certain embodiments, the anti-TauC 3antibody is 500 to 2500 times more specific for TauC3 than for full-length Tau (2N 4R). In certain embodiments, the anti-TauC 3antibody is 750 to 2000 times more specific for TauC3 than for full-length Tau (2N 4R). In certain embodiments, the anti-TauC 3antibody is 1000 to 1500 times more specific for TauC3 than for full-length Tau (2N 4R). In all of these embodiments, the anti-TauC 3antibody may not detectably bind to full-length Tau (2N 4R).

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

In certain embodiments, the antibody is at 1x10^-10M to 1x10^-11The equilibrium constant KD of M binds to TauC 3; and has an equilibrium constant KD of 1x10 with 2N4R^-4M to 1x10^-8M or showed no detectable binding to full-length Tau (e.g. 2N 4R). In a preferred embodiment, the anti-TauC 3antibody is at 1x10^-9M to 1x10^-12M has an equilibrium constant KD of binding to TauC3 and at 1X10^-8M to 9x10^-8The equilibrium constant KD of M binds to full length (e.g. 2N4R) or shows no detectable binding to 2N 4R. In some of these embodiments, the antibody has a very slow off-rate for TauC3 (i.e., an off-rate (Kd) of 1x 10)^-4To 1x10^-3s^-1) And has substantially no affinity for 2N4R (i.e., ka is less than 100,0001/MS).

In certain embodiments, the anti-TauC 3antibody is a chimeric or humanized antibody having a KD value to TauC3 of about 5pM to about 90pM, about 10pM to about 80pM, about 10pM to about 70pM, about 10pM to about 60pM, about 10pM to about 50pM, about 10 to about 40pM, or about 10pM to about 35 pM.

In certain embodiments, the anti-TauC 3antibody is a chimeric or humanized antibody having a KD value of about 10 to about 90pM for TauC3 and a very slow off-rate, e.g., less than 2x10^-3s^-1Kd values of (A) are shown. In other words, these antibodies are highly specific for TauC3(TauC3 is the target protein produced in diseased states) and have a slow dissociation rate, both of which are ideal for antibodies used in immunization strategies.

anti-TauC 3 antibodies include, but are not limited to, monoclonal, chimeric, humanized, single chain, Fab fragments, and Fab expression libraries. The anti-TauC 3antibody may be native or recombinant, immobilized, free in solution, or displayed on the surface of various molecules or bacteria, viruses, or other surfaces.

In certain embodiments, the anti-TauC 3antibody recognizes sequence SSTGSIDMVD (SEQ id No.23) at the C-terminus of TauC3, but does not recognize this sequence when the same sequence is present inside the FLT.

anti-TauC 3 antibodies (e.g., humanized antibodies) useful according to the invention may be administered to a subject who may be susceptible to or suffering from a tauopathy to block vaccination and/or aggregation of TauC3 and thereby treat one or more symptoms of the tauopathy.

In yet another embodiment of the invention, an anti-TauC 3antibody of the invention can be conjugated to a cytoprotective agent or an agent that will facilitate and/or improve the ability of the antibody to cross the blood-brain barrier ("BBB"). The cytoprotective agent may be an antioxidant (e.g., melatonin); an agent that promotes or improves the ability of an antibody to cross the blood-brain barrier is a hydrophobic substance that is capable of crossing the blood-brain barrier and is generally recognized by the U.S. food and drug administration ("FDA") as sage (GRAS). The cytoprotective agent or agent that promotes or improves the ability of an antibody to cross the BBB may be conjugated to the antibody directly or through a linker. The linker may be selected from: hydrazine linkers, disulfite linkers, thioether linkers, peptide linkers. In certain embodiments, the antibody has an equilibrium constant KD for TauC3 that is 2-3 orders of magnitude higher than the equilibrium constant KD for antibody to 2N4R, and the cytoprotective agent is melatonin.

Application method

In one aspect, the invention provides an anti-TauC 3antibody for use in a live human suffering from or at risk of developing a tauopathy. Tauopathies include, 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 degeneration (FTLD), and the like.

Method for blocking pathological Tau aggregate proliferation

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

In one aspect, the invention relates to a method of blocking the activity of TauC3 vaccination in the brain of a subject.

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

The invention further relates to a method of reducing the diffusion of an aggregate comprising TauC3 in the brain of a subject.

The invention further relates to a method of reducing the diffusion of fibrils comprising TauC3 in the brain of a subject.

In another aspect of the invention, the invention relates to a method of reducing intracellular aggregation of tau induced by intracellular uptake of tau 3 and tau 3 fibrils.

In each aspect, the method comprises administering to the human a therapeutically effective amount of an anti-TauC 3 antibody. anti-TauC 3 antibodies are uniquely able to recognize aggregated extracellular forms of pathological tau without binding to physiological tau. In a preferred embodiment, the critical part of the epitope for the anti-TauC 3antibody is the carboxyl group that forms a neo-epitope at the C-terminal residue of the peptide corresponding to the last ten C-terminal residues of TauC3 (e.g., TauC3 or SEQ ID NO: 23). The anti-TauC 3antibody has an equilibrium constant KD for TauC3 that is 2-3 orders of magnitude higher than the equilibrium constant KD of the antibody for 2N4R and one or more pharmaceutically acceptable excipients. anti-TauC 3antibody at 1X10^-10M to 1x10^-11M has an equilibrium constant KD for binding to TauC3, but an equilibrium constant KD for full-length tau (e.g., 2N4R) of 1x10^-4M to 1x10^-8M, or no detectable binding to full-length tau (e.g., 2N 4R). In a preferred embodiment, the anti-TauC 3antibody is at 1x10^-11M to 9x10^-11M has an equilibrium constant KD of 1X10 in combination with TauC3^-8M to 9x10^-8The equilibrium constant KD of M binds to 2N4R, or shows no detectable binding to 2N 4R. The anti-TauC 3antibody preferably has a very slow off-rate from TauC3 and essentially no affinity for 2N4R (i.e., ka is less than 100,0001/MS). The antibody may, for example, be selected from a humanized antibody, a chimeric antibody, or an immunological fragment of any of the foregoing antibodies. In a preferred embodiment, the antibody is an antibody selected from the group consisting of humanized anti-TauC 3 antibodies described herein.

Prior to administration of a therapeutically effective amount of an anti-TauC 3antibody, a human may or may not have symptoms associated with tau aggregation. In other words, the human may or may not experience symptoms associated with tau vaccination and/or aggregation. One of ordinary skill in the art will appreciate that pathological tau inoculation and aggregation may begin prior to the onset of symptoms diagnosed or associated with tau aggregation. In some embodiments, the human has symptoms associated with tau vaccination and/or aggregation. In other embodiments, the human does not have symptoms associated with tau vaccination and/or aggregation. In other embodiments, the human has detectable tau pathology, but no 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 may reduce the development and/or progression of symptoms associated with pathological inoculation and/or aggregation of tau.

Thus, preventing, inhibiting, or slowing the diffusion of pathological tau aggregates can be used to treat pathologies associated with the production and diffusion of tau aggregates. One definition of symptoms associated with tau vaccination and/or aggregation refers to any symptoms caused by tau aggregate formation consisting in part of tau fibrils. Exemplary disorders with symptoms associated with tau aggregation include, but are not limited to, progressive supranuclear palsy, dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia and parkinson's disease associated with chromosome 17, Lytico-Bodig disease (parkinson dementia of guam), tangle-predominant dementia, ganglioglioma and gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, plumbum encephalopathy, tuberous sclerosis, globus nigrosis (hallervvorden-Spatz disease), lipofuscinosis, pick's disease, corticobasal degeneration, silvery granulosis (AGD), frontotemporal degeneration, alzheimer's disease and frontotemporal dementia. Methods for diagnosing these diseases are known in the art.

Exemplary symptoms associated with tau vaccination or aggregation may include, for example, impaired cognitive function, altered behavior, mood disorders, seizures, and impaired nervous system structure or function. Impaired cognitive function includes, but is not limited to, difficulties in memory, attention, language, abstract thinking, creativity, executive function, planning and organization. Altered behaviors include, but are not limited to, physical or verbal attacks, impulses, decreased stamina, apathy, decreased aggressiveness, character changes, alcohol abuse, tobacco or drugs, and other addiction related behaviors. Mood disorders include, but are not limited to, depression, anxiety, mania, irritability, and loss of mood control. 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 paresis of one limb, hemiplegia, quadriplegia, ataxia, twitching and tremor. This also includes sensory loss or dysfunction, including smell, touch, taste, vision, and hearing. In addition, this includes autonomic nervous system injuries such as bowel and bladder dysfunction, sexual dysfunction, blood pressure and temperature disorders. Finally, this includes hormonal damage attributable to hypothalamic and pituitary dysfunction, such as deficiencies 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 assessing symptoms associated with tau aggregation are known in the art.

In some embodiments, the symptom associated with tau aggregation is dementia. Dementia is not a specific disease per se, but rather a general term that describes a wide range of symptoms associated with a decline in memory or other mental capacity, the severity of the decline being sufficient to reduce one's ability to perform daily activities. Dementia is also a common clinical feature of many diseases associated with tau protein aggregation. Skilled 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 a simple mental state detection table (MMSE), a simple mental state detection table (AMTS), a modified simple mental state detection table (3MS), a cognitive dysfunction screening table (CASI), a trace-makingtest, a clock plot test, an questionnaire for aged cognitive decline, a general practitioner cognitive assessment, a Clinical Dementia Rating (CDR), an 8-entry dementia screening table (AD 8).

In some embodiments, the severity of dementia symptoms is quantified using a clinical dementia score. Using the clinical dementia score, a score of 0 indicates no symptoms, a score of 0.5 indicates very mild symptoms, a score of 1 indicates mild symptoms, a score of 2 indicates moderate symptoms, and a score of 3 indicates severe symptoms. Thus, any increase in a human's clinical dementia score indicates a cognitive deterioration and an increase in dementia. In addition, a change in clinical dementia score from 0 to greater than 0 indicates the development or onset of dementia.

In some embodiments, the symptom associated with tau vaccination or aggregation is referred to as a tauopathy or tauopathy. The term "tauopathy" or "tauopathy" refers to pathological inoculation or aggregation of tau. In some embodiments, the 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 fold higher than levels detected in individuals without disease.

Administration of

Administration of an anti-TauC 3antibody described herein may be used as a treatment or therapy for immune tauopathies.

Preferably a pharmaceutical grade, therapeutically effective amount of the antibody (including immunoreactive fragments) may be administered to a human. Administration is carried out using standard effective techniques, including peripheral administration (i.e., no 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, buccal, sublingual, or suppository administration. Local administration, including direct access to the Central Nervous System (CNS), includes, but is not limited to, controlled release formulations via lumbar, intracerebroventricular, or intraparenchymal catheters or using surgical implants.

Suitable for treatment of people including risk of disease but not symptoms of the individual, as well as the present symptoms of the subjects. In the case of alzheimer's disease, almost anyone is at risk for alzheimer's disease. Thus, the present method can be administered prophylactically to the general population without any assessment of the subject's risk. Such prophylactic administration may be initiated, for example, at an age of 50 years or older. The method is particularly useful for individuals who do have a known genetic risk of tauopathies (e.g., Alzheimer's disease). Such individuals include those with relatives who have suffered from the disease, as well as those at risk as determined by genetic or biochemical marker analysis. For example, genetic markers of risk of alzheimer's disease include mutations in the APP gene, particularly the mutation at position 717 and the mutations at positions 670 and 671, referred to as Hardy mutation and Swedish mutation, respectively. Other risk markers are the presenilin genes (PS1 and PS2) and mutations in ApoE4, family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from alzheimer's disease can be identified from the characteristic dementias by the presence of the risk factors mentioned above. In addition, many diagnostic tests are available to identify individuals with AD. These include imaging and/or measuring CSF tau and Α β 42 levels. Elevated tau and reduced a β 42 levels indicate the presence of AD. Individuals with alzheimer's disease can also be diagnosed by standards of the association of alzheimer's disease and related diseases.

In asymptomatic subjects, treatment may begin at any age (e.g., 10, 20, 30, 40, 50, or 60). However, typically, treatment need not be initiated until the subject reaches the age of 40, 50, 60, 70, 75, or 80 years. Treatment typically requires multiple administrations over a period of time. Treatment can be monitored by assaying the response of the antibody, or activated T cells or B cells, to the therapeutic agent over time. If the response is decreasing, a booster dose is indicated. In the case of a potential down syndrome subject, treatment may begin prenatally by administering a therapeutic agent to the mother or shortly after birth.

In prophylactic applications, the pharmaceutical composition or medicament is administered to a subject susceptible to or at risk of developing a tauopathy, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during the course of disease progression, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease. In therapeutic applications, the compositions or medicaments are administered to a subject suspected of having or having had such a disease, including complications and intermediate pathological phenotypes in the course of disease progression, in an amount sufficient to cure or at least partially arrest the biochemical, histological and/or behavioral symptoms of the disease. In some methods, administration of the agent reduces or eliminates mild cognitive impairment. An amount sufficient to effect a therapeutic or prophylactic treatment is defined as a therapeutically or prophylactically effective dose or amount. In prophylactic and therapeutic regimens, the agent is typically administered in several doses until a sufficient immune response is obtained. Typically, the immune response is monitored and repeated doses are given as the immune response begins to diminish.

The effective dosage of the compositions of the invention for treating the above conditions will depend on a number of different factors, including the mode of administration, the target site, the physiological state of the subject, the other drug being administered, and whether the treatment is prophylactic or therapeutic. Therapeutic doses need to be tested case by case in clinical trials and often titrated to optimize safety and efficacy. Another advantage of the anti-TauC 3 antibodies of the invention in certain embodiments may be that, for an equivalent mass dose, the anti-TauC 3antibody dose of the invention comprises a higher molar dose of effective clearing and/or "inactivating" antibodies compared to an antibody composition comprising anti-TauC 3 antibodies according to the invention that are less specific for TauC 3. Typically, the anti-TauC 3 antibodies of the invention will be administered by intravenous infusion or subcutaneous injection. The amount of anti-TauC 3antibody administered by intravenous infusion may vary from 0.5 to 10mg per subject. Subcutaneous injections generally require higher doses to reach the brain in sufficient quantities. Antibodies (e.g., intact IgG molecules) can be administered once a month.

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 indicated range. In this case, both or more antibodies may be directed against, for example, truncated tau. Alternatively, one or more antibodies may be directed to, for example, truncated tau, and one or more additional antibodies may be directed to amyloid- β (a β) peptide associated with alzheimer's disease. Antibodies are typically administered in multiple doses. The interval between the individual doses may be hourly, daily, weekly, monthly or yearly. In some methods, the dose is adjusted to achieve a plasma antibody concentration of 1-1000. mu.g/ml, and in some methods 25-300. mu.g/ml. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequency of administration is required.

The dose and frequency will vary depending on the half-life of the antibody in the subject. In general, human antibodies have the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic 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 remainder of their lives. In therapeutic applications, it is sometimes desirable to use relatively high doses over relatively short intervals until the progression of the disease is slowed or terminated, preferably until the subject exhibits partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be treated prophylactically.

The dose of anti-TauC 3antibody blocking the vaccination with TauC3 is not necessarily the same as the dose of anti-TauC 3antibody inhibiting TauC3 aggregation. Based on the information provided in the specification, specific dosages can be determined by routine experimentation.

The efficacy of administration/treatment can be assessed by measuring the levels of pathogenic or phosphorylated tau in plasma and/or CSF. Based on this evaluation, the dose and/or frequency of administration may be adjusted accordingly.

In certain embodiments, cognitive effects may also be evaluated.

The effect can also be assessed by the extent of brain atrophy determined by MRI

Safety of dosing/treatment can be assessed by the number of participants experiencing Adverse Events (AEs), severe AEs and clinical laboratory examination abnormalities, vital signs, ECG, MRI, and physical and neurological examinations, as well as cognitive deterioration. Based on this evaluation, the dose and/or frequency of administration may be adjusted accordingly.

The anti-TauC 3antibody and immunogen may be administered intranasally, subcutaneously, intramuscularly, IV infusion, transdermally, orally, and the like, or as described in more detail below.

Pharmaceutical composition

The pharmaceutical composition according to the invention comprises an anti-TauC 3antibody or fragment thereof as described herein, and one or more pharmaceutically acceptable excipients. anti-TauC 3antibody at 1X10^-10M to 1x10^-11M has an equilibrium constant KD for binding to TauC3, while the equilibrium constant KD for full-length tau (e.g., 2N4R) is 1x10^-4M to 1x10^-8M, or no detectable binding to full-length tau (e.g., 2N 4R). In a preferred embodiment, the anti-TauC 3antibody is at 1x10^-11M to 9x10^-11M has an equilibrium constant KD of 1X10 in combination with TauC3^-8M to 9x10^-8The equilibrium constant KD of M binds to 2N4R or shows no detectable binding to 4 RTau. Preferably, the anti-TauC 3antibody dissociates at a very slow rate from TauC3 (i.e., a dissociation rate of 1x 10)^-4To 1x10^-3s^-1) And has substantially no affinity for 4RTau (i.e., ka is less than 100,0001/MS). The antibody may be, for example, a humanized, chimeric or human (e.g., from a tg mouse) antibody.

The pharmaceutical compositions are designed to be suitable for the selected mode of administration, and pharmaceutically acceptable excipients, such as compatible dispersants, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers and the like, are suitably used.

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 understood.

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% to as high as about 95 or about 99.9% by weight and will be selected primarily based on fluid volume, viscosity, etc., the particular mode of administration being selected as desired. In certain embodiments, the antibody may comprise about 15% or about 20% by weight of the composition.

The composition for injection into a subject may be prepared as any one or combination of a sterile buffered water containing 1-250ml of phosphate buffered saline and about 1-5000mg of an anti-TauC 3antibody of the invention. The preparation may be sterile filtered after preparation, or made in other microbiologically acceptable ways. Typical compositions for intravenous infusion may have a volume of 1-250ml of fluid (e.g., sterile ringer's solution) and the anti-tau antibody concentration may be 1-100mg/ml or higher. The therapeutic agents of this discovery may be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. Lyophilization and reconstitution can result in varying degrees of loss of antibody activity (e.g., IgM antibodies tend to have greater loss of activity than IgG antibodies when using conventional immunoglobulins).

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

In one aspect, a typical dose comprises from about 0.1mg to about 10mg of an anti-TauC 3antibody described herein. In certain embodiments, a typical dose comprises from about 0.5mg to about 10mg of the anti-TauC 3 antibody. The dosage may range from about 0.55mg/kg to about 10 mg/kg. The frequency of administration of intact IgG antibodies is usually monthly, while antibody fragments need to be administered more frequently due to their shorter half-life to effectively treat the symptoms.

The time of treatment administration relative to the disease itself and the duration of treatment will depend on the circumstances surrounding the case. After diagnosis of a disease associated with tau protein aggregation, treatment may be initiated. Alternatively, treatment may be initiated after clinical confirmation of symptoms associated with tau aggregation. In addition, treatment may begin after tau pathology is detected. Treatment may begin immediately at the hospital or clinic, or may begin after discharge or after an outpatient visit. The duration of treatment can range from a single dose, one-time administration, to treatment over the course of a lifetime.

While the above methods appear to be the most convenient, suitable and effective for the administration of proteins such as humanized antibodies, other effective administration techniques, such as intracerebroventricular, transdermal and oral administration, may be employed with appropriate adaptation, provided that appropriate formulations are employed herein.

Based on the information provided herein and knowledge available in the art, typical effective amounts or dosages can be determined and optimized using standard clinical techniques and will depend on the mode of administration.

Example 1: (determination of mouse Motau01 antibody sequence)

Sequencing of the Motau01 antibody

RNA was prepared from hybridoma cells.

Cryoprecipitates of mouse hybridoma cells (MoTau01) stored at-80 ℃ were provided by Genscript for Tau-Biologic and processed using Qiagen RNeasy kit to isolate RNA according to the manufacturer's protocol.

First Strand cDNA Synthesis

Motau01RNA (. about.21. mu.g) was reverse transcribed using the GE Life Sciences first Strand cDNA Synthesis kit to generate cDNA and purified as described in example 5, according to the manufacturer's protocol. This was repeated twice to generate 3 independent cDNA products (rounds 1, 2 and 3) to detect and avoid reverse transcriptase-induced cDNA mutations.

cDNA sequence determination

The Motau01 cDNA was amplified by PCR as described in example 5. Immunoglobulin cDNA was PCR amplified using Phusion Flash high fidelity PCR premix 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 produce any product.

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

TABLE 1 PCR primers for amplifying mouse VK

Fuzzy base code: w is A or T; y ═ C or T; k ═ G or T

MKV represents a primer that hybridizes to the leader sequence of the mouse kappa light chain variable region gene; MKC represents a primer that hybridizes to the mouse kappa constant region gene. The bold underlined section indicates either the M13 forward or M13 reverse sequencing primer. Wobble base pairs (Wobble base) are defined in the definitions section.

TABLE 2 PCR primers for amplification of mouse VH

Fuzzy base code: r ═ a or G; k ═ G or T; and M is A or C.

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 a mouse constant region gene. The bold underlined section indicates either 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 MHCG 3. "wobble" base pairs are defined in the definitions section.

The result of each PCR reaction was a single amplification product that was purified using the QIAquick PCR purification kit and sequenced in both directions (by Eurofins/GATC Genomics) using M13-forward and M13-reverse primers (table 3) to obtain three sets of independent sequence information for each immunoglobulin chain.

TABLE 3 Universal PCR and sequencing primers

Name (R)	Sequence (5 '→ 3')
		HCMVi promoter	TGTTCCTTTCCATGGGTCTT(SEQ ID NO：59)
HuG4_ LIC _ Reversal	CTCTCGGAGGTGCTCCTGGAG(SEQ ID NO：60)
		Huk LIC reverse	GCAGTTCCAGATTTCAACTG(SEQ ID NO：61)
M13-Forward	TGTAAAACGACGGCCAGT(SEQ ID NO：62)
		M13-reverse	CAGGAAACAGCTATGACC(SEQ ID NO：63)

VK and VH MoTau01DNA sequences

The consensus DNA sequences of the MoTau01 VK PCR product and the MoTau01 VH PCR product are shown in fig. 1 and 2, respectively. The obtained variable region DNA sequence 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 x 01 and the heavy chain was murine VH1 IGHV10-1 x 02.

Example 2: (Generation of chimeric Motau01 antibody)

Construction of chimeric Motau01 expression vector

The genes for Motau01 VH and VK were synthesized by GenScript. Sequences of MoTau01 VH and VK were optimized by silent mutagenesis using a software algorithm proprietary to GenScript to use codons preferentially utilized and synthesized by human cells.

Construction of the chimeric expression vector requires cloning of the synthetic variable regions into an IgG/kappa vector using Ligase Independent Cloning (LIC) (pHuK and pHuG 4-FIGS. 3 and 4, respectively). The vector (pCMV modified) was digested with BfuA1(BspM1) and then a compatible overhang was generated with T4 DNA polymerase 3 '-5' exonuclease activity (+ dATP).

The antibody sequences (fig. 5 and 6) were generated as follows: the synthesized variable region was first amplified by PCR using primers containing the 3' end of the leader sequence (most of the sequence was present in the vector) -the forward primer-or the beginning of the constant region (IgG4 or κ) -the reverse primer, followed by the beginning of the variable region (in each direction), table 4.

TABLE 4 cloning and mutagenesis primers

Complementary overhangs are generated in the PCR product by T4 DNA polymerase + dTTP treatment (protocol provided in example 5). The vector and insert were incubated at room temperature, transformed into chemically active TOP10 bacteria and inoculated onto kanamycin plates. Several clones were isolated and screened by PCR using the forward primer HCMVi promoter and HuG4 LIC reverse primer (for VH) or HuK LIC reverse primer (for VK) (table 3).

Clones producing the correct size PCR product were selected, mini-prepared using QIAGEN kit and sequenced using the same primers.

Generation of chimeric antibodies

Expifcho suspension cells grown in expifcho transfection medium and antibiotics were co-transfected with MoTau01_ vh. phug4 and MoTau 01. vk. phuk (1 μ g of DNA each) using expifctamine CHO reagent. Cells were grown in 1mL growth medium for 7 days. Motau01HuG4k antibody was measured up to 160. mu.g/mL (Table 14A) in conditioned medium by Octet quantification.

TauC3 binding Activity of chimeric antibodies

The TauC3 and FL Tau antigens were produced and purified by Genscript and were provided at concentrations of 2.54mg/ml or 0.24mg/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 0.7nM (fig. 7A), but no binding to FL Tau was observed (fig. 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). Mice and chimeric antibodies were assayed against a concentration series of 20nM to 0.31nM TauC3 (FIG. 8A and FIG. 8B). Both mouse and chimeric antibodies were able to bind TauC3 in a concentration-dependent manner. Binding of mouse and chimeric antibodies to FL Tau was also tested, but low or no binding was observed (fig. 8C and 8D), confirming that the binding of the antibodies was specific for TauC 3.

Example 3: design of Tau01 humanized antibody variants

Human VH and VK cDNA database

The protein sequences of human and mouse immunoglobulins in the fifth edition of the immunological protein sequences derived from the international immunogenetics database 2009(Lefranc, 2015) and the Kabat database (last update 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.

Molecular model of Motau01

The MoTau01 VH and VK sequences were used to design humanized versions of the MoTau01 antibody. A homology model for the variable region of the MoTau01 antibody has been used to predict panel production using the antibody in Maestro 11.5. The human framework selected was used to generate 10 cycle models, which were prepared using a one-step protein preparation wizard. Protein reliability reports were generated for all 10 models, and no significant differences were found in model quality. All 10 models were used to determine CDR loopsThe identity of the internal residues to capture the different orientations of the CDRs.

Selection of human frame

Humanization requires the identification of appropriate human v regions. Use of sequencesThe analysis program Gibbs, used various selection criteria, queries human VH and VK databases with MoTau01 VH and VK protein sequences. Using Maestro 11.5Software, CDR residues (defined by IMGT) in the structure of mouse Tau01 antibody were identifiedInner FW residue and its name "Adjacent residues ". In thatAn alignment of the human VH sequences with the highest identity to the MoTau01 VH among the adjacent residues is shown in table 5. Table 6 lists these envelope (envelope) residues and VCI, as well as FW, VCI orThe number of residues in the adjacent (proximity) residues that are identical to the mouse equivalent positions of the sequences of table 5.

Humanized and incomplete sequences were removed from the analysis. The sequence DQ840895.1 was selected as a human heavy chain donor candidate. This sequence scored very high in sequence identity and similarity, and there were only 2 somatic mutations from its IGHV3-73 x 01 VH germline. It has eightThe adjacent residues and one VCI residue were changed, but this was the least change available (table 8).

Likewise, sequence L33034 was chosen as a human kappa light chain donor candidate. The sequence has a high sequence identity and similarity score with Tau01 VK, and there are only 1 somatic mutation from IGKV1-17 x 01 germline. It has five potentialAdjacent residues and one VCI residue.

The sequences of the kappa light chain humanization strategy are shown in table 12.

Design of humanized heavy chain variants of Tau01

Once the appropriate human framework has been determined, synthetic protein and DNA sequences can be designed. The initial design of the humanized form of Tau01 was to graft CDRs 1, 2 and 3 of the MoTau01 VH to the receptor FW of DQ840895.1, resulting in a variant Tau01 HA. In the humanized form Tau01 HB, 8 were then addedThe adjacent residues and 1 VCI residue (at positions 1, 4, 35, 49, 58, 61 and 76-78) were back-mutated to mouse equivalent residues, one at a time in the following variants: the sequences were assembled in a computer and designated Tau01 HD through Tau01 HL. Table 8 compares the murine and humanized versions of the Tau01 VH protein sequences.

Design of a humanized light chain variant of Tau01

The framework from L33034 was used to design DNA and proteins for the humanized constructs. CDRs 1, 2 and 3 from Tau01 VK are shown grafted into the receptor FW of L33034 to generate the original form of humanized Tau 01. There are five unmatched at positions 34, 36, 44, 53, 69 and 71 of Tau01 KAAdjacent residues and one VCI residue, which were back mutated to the mouse equivalent residue in variant Tau01 KB (table 12).

In the following variants, these residues are mutated one at a time: the sequences were assembled in silico and named Tau01 KD through Tau01 KI. In the Tau01 KG form, residue K, was back-mutated to mouse residue T and also to human germline residue S. This additional variant was named Tau01 KJ.

Design of heavy and light chain C forms

After the design of the original humanized variants, a homology model of Tau01 HAKA was established and evaluated. The latter model overlaps with the mouse antibody model. Each position determined for the back-mutation and those around it are highlighted and examined in the modelAnd (c) a residue. Based on this data, it was predicted which residues were most important for back mutation, 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).

Example 4: (Generation and characterization of humanized antibody)

Generation of humanized antibody to Tau01

The sequences of Tau01 HA/B/C and KA/B/C were codon optimized to use codons preferentially utilized 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 form was then modified by PCR mutagenesis using the primers in table 4 to obtain the other humanized variants annotated in table 8 or 12 respectively.

Clones were sequenced using QIAGEN Plasmid Miniprep kit or Qiagen Plasmid Maxiprep kit and Plasmid DNA was prepared. The expression construct sequences (HA, HB, HC, KA, KB and KC) are shown in FIGS. 9 to 14.

Antibody expression

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 harvested in conditioned medium containing secreted antibody. The concentration of IgG4 kappa antibody in ExpicHO cell conditioned media was measured by octet and is shown in tables 14A-C. Most antibodies are produced at good expression levels.

TABLE 14 IgG levels in conditioned media of transfected ExpicHO cells

Expression of the transfection control Hu1210 HuG1K was also reduced (expected level-100 μ g/ml).

Antigen binding of humanized antibody original form

Binding of the humanized variants to the provided TauC3 antigen was tested by binding ELISA as described in example 5. The data shown in FIG. 15 show that TauC3 binds to an ELISA of a humanized antibody consisting of HA/HB heavy chain and KA/KB light chain. The HAKA and HAKB humanized antibodies do not bind to TauC 3. The MoTau01HuG4K chimera bound to TauC3 with an EC50 value of 0.65nM, HBKA and HBKB variants bound with similar EC50 values, with the HBKB form being the closest (0.78 nM).

In view of this data, other forms of humanized heavy and light chains were expressed, each with a single back mutation (tables 8 and 12). FIG. 16 shows the results of heavy chain single mutants and HC versions in combination with KA-KC, tested for binding to TauC3 by ELISA. The data show that the heavy chain single mutant is not able to bind TauC3 as does the chimeric antibody. The HB and HC versions combined with KA-KC bound to TauC3 with the highest EC50 values, and HBKB and HBKC showed the closest values to the chimeric antibody (0.81 nM and 0.84nM, respectively). These results were confirmed by screening experiments using one concentration of TauC3 on an Octet instrument as described in section 8.12 (fig. 17). The Octet data was not optimal as biphasic association and dissociation events could be observed, probably due to the nature of the TauC3 protein. However, Octet data was sufficient to screen and rank humanized candidates.

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

Second round humanized antibody design and Generation

Based on suboptimal binding results for the initial humanized variants, second round variants were designed by incorporating additional back mutations into both HC and KC forms (HM-HO and KL and KM, respectively) or the two back of 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 table 14D and table 14E. Most antibodies are produced at good expression levels.

TABLE 14 IgG levels in conditioned media of transfected ExpicHO cells

Antigen binding of the second round of humanized Tau01 antibody

To evaluate the heavy chains, antibody variants comprising HM, HN and HO in combination with light chains KA, KB, KG, KI, KJ were tested for binding activity against TauC3 by ELISA (fig. 20) and Octet (fig. 21). The HM and HO variants ranked the highest of the two assays. The only difference between the HO and HM variants is the additional back-mutation of L-V in position 4, which is also present in the HN variant. Since the HN form does not bind as well as the HM form, it is believed that the L-V back-mutation is not required, and therefore the HM was selected from the second round of design as the head heavy chain.

Expressing a leader heavy chain HM and a plurality of light chains; KA-KC, previously highlighted single mutants (KE, KG, KI, KJ) and KL, KN, KO, KP for the second round of design. 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 (figure 22). Overall, the HM variant bound with a higher EC50 than the HC variant, and the former variant was therefore evaluated in the Octet screening experiment (fig. 23). Antibody variants HMKE, HMKN, HMKO and HMKP ranked highest in both assays. KM light chains were also screened in combination with HC/HM and compared to the lead antibody variants by Octet (fig. 24A). HMKM format ranked very high in Octet screening experiments, while HCKM ranked the lowest. Humanized pilots were also tested for binding to FL Tau using Octet to determine how much selectivity was retained (fig. 24B). The signal obtained with FL Tau at 500nM is very low (0.03-0.07nM), in the range of non-specific binding.

To further evaluate the binding data observed on octets, dissociation rate ranking experiments were performed on a Biacore200 instrument. Biacore offers higher selectivity than Octet and facilitates the immobilization of TauC3 onto CM5 chips by amine coupling of the stabilized antigen. This will yield higher quality data with a stable baseline and a good reproducible response. Once TauC3 was immobilized, a concentration (5nM) of antibody was added, followed by the dissociation and regeneration steps (section 8.20). The dissociation rate was fitted using a two-phase decay model (fig. 25B) or only the second dissociation rate was fitted using a single-phase decay model (fig. 25A). Candidates are then ranked according to the dissociation rate obtained using fitting 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 the humanized variants with the slowest dissociation rate. Biacore also demonstrated the lack of binding of FL Tau to chimeric and humanized candidates by loading the antibody onto Protein G chips and adding 250nM of FL Tau. FIG. 26 shows that no binding of Motau01HuG4k or Tau01HCKB was observed.

Thermostability of humanized Tau01 candidate antibodies

The objective of this experiment was to test the thermostability of chimeric and some humanized antibodies (Tau01 HCKG, HCKN, HMKE, HMKN, HMKO, HMKP) when subjected to higher temperatures, from 35 ℃ to 95 ℃ for 10 minutes, cooled to 4 ℃, and used for binding ELISA at the EC80 concentration of each candidate (section 8.15). All humanized candidate antibodies were more stable than the chimeric antibody, maintaining the ability to bind to TauC3 until 67-68 ℃, and then reduced binding to TauC3 (fig. 27). The humanized variants exhibited greater thermostability with the binding remaining only up to about 55 ℃ compared to the chimeric antibody MoTau01HuG4 k. The variants containing HM heavy chain may remain bound at slightly higher temperatures than the HC heavy chain variants tested.

Selection of lead humanized Tau01 candidate antibodies

All these results were put together and lead humanized antibody variants Tau01HMKE, HMKM, HMKN, HMKO and HMKP were selected for amplification and purified 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 weakly to TauC3 were also amplified for expression and purification to be used as comparative antibodies for ranking in vaccination trials. This would enable us to further test the correlation between antibody affinity in vitro and potency in cell assays.

Aggregation of humanized Tau01 candidate antibodies

As part of the post-purification QC process, the antibody samples were subjected to SEC-MALS/DLS followed by mass spectrometry. To determine absolute molar mass and check for aggregation, purified antibody samples were injected into size exclusion columns in an HPLC system and analyzed by multi-angle light scattering. The graphs of MoTau01HuG4k and Tau01 HMKM show no evidence of aggregation, with average molecular weights of approximately 147-8kDa, which is the expected range of IgG monomers in this analytical setup (fig. 28A). The Tau01HCKB antibody spectrum had a broad peak that biased the data, resulting in a molecular weight of 179.6kDa (fig. 28B). All three antibodies were monodisperse (Mw/Mn < 1.05) and showed no evidence of aggregation.

Dynamic light scattering is a complementary technique to static light scattering (SEC-MALS) for the detection of soluble aggregates and for the QC humanized variants Tau01HMKN, HMKO, HMKP and HMKE. The Z-Ave or hydrodynamic diameter of the antibody is expected to be about 10, and if the sample is monodisperse, the polydispersity index (PdI) should be < 0.1. As shown in fig. 29, all antibody samples contained a major population that was monodisperse and consistent with the size of the monoclonal antibodies.

To confirm the exact molecular weight of the antibody, mass spectrometry analysis was performed on the intact and reduced antibody samples, as shown in fig. 30A to 30G. The molecular weights matched the predicted molecular weights of all antibodies tested and the amino acid sequences were confirmed. No other redundancies were marked. In general, purified chimeric and humanized antibodies passed QC.

Kinetic study of humanized Tau01 candidate antibody against tauC3

To determine the affinity of the binding interaction, a biacore kinetic analysis was developed which involved immobilizing TauC3 on a CM5 chip by amine coupling and injecting a range of concentrations of the corresponding antibody thereon. The chimeric antibody bound to TauC3 with a KD of 57pM (fig. 31A).

Fig. 31B shows that Tau01HCKB is the weakest Tau c3 binding agent (1.2nM), followed by HMKM, which binds Tau c3 with an affinity of 110pM (fig. 31G). Tau01 HMKO, HMKN, HMKP and HMKE shown in fig. 31C-F have KD values comparable or tighter than chimeric antibodies. However, the dissociation rate was slightly faster for all humanized candidates compared to the chimeras (0.001-0.004 for humanization and 0.0007 for chimeras). It should be noted that ka is at the limit of the instrument and therefore it should be prudent to compare absolute values. It can be concluded that the antibodies HMKN, HMKP and HMKE bind in the picomolar range and have the slowest off-rate among all the tested humanized variants.

Determination of melting temperature (Tm) of humanized Tau01 candidate antibody

In order to determine the melting temperature of the lead antibodies Tau01HMKE, HMKM, HMKN, HMKO, HMKP, thermal displacement measurements were performed. Samples were incubated with fluorescent dye (Sypro Orange) for 71 cycles, each cycle increasing by 1 ℃, in a qPCR thermocycler (section 8.21). The Tm of the humanized antibody was calculated to be 68-69 ℃. (FIG. 32).

Non-specific protein-protein interaction (CIC) of humanized Tau01 candidate antibody

Cross-interaction chromatography using large amounts of purified human polyclonal IgG is a technique for monitoring nonspecific protein-protein interactions and can provide an indication of any solubility issues that may cause downstream manufacturing problems, as explained in example 5. An increase in retention index (k') indicates a tendency to self-interact and low solubility. Humanized Tau01HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies showed retention indices below 0.038, indicating a low propensity for non-specific interactions and good solubility (fig. 33).

Solubility of humanized Tau01 candidate antibody

The humanized Tau01HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies were concentrated using a solvent absorption concentrator (MWCO 7500kDa) and the concentrations were measured at regular intervals. Tau01 HMKP was concentrated to 123mg/mL and Tau01HMKN, HMKM and Motau01HuG4K antibodies were concentrated to 87-88mg/mL (FIG. 34). Tau01 HMKO HuG4K was concentrated to 59mg/ml with no significant precipitation, Tau01HMKE HuG4K was concentrated to 57 mg/ml. The data show that the antibody does not precipitate easily at concentrations as high as 57 mg/mL.

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

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

Freeze-thaw (FT) stress experiments involve subjecting purified candidate antibody samples to 10 cycles of 15 minutes at-80 ℃ followed by 15 minutes of thawing at room temperature. For heat stress experiments, purified candidate antibody samples were exposed to temperatures of a)4 ℃, b) Room Temperature (RT), c)37 ℃ and d)50 ℃ for 25 days.

The samples were then analyzed by circular dichroism to check whether secondary structure remained (fig. 35). All humanized variants tested passed our internal threshold. Overall, the data indicate that heat stress and freeze-thaw cycling do not affect the secondary structure of the humanized Tau01HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies.

Isoelectric Point analysis of humanized Tau01 candidate antibody

pI analysis of humanized candidate antibodies was performed using capillary isoelectric focusing (cIEF). This technique allows the separation of antibodies according to their isoelectric point (pI) using a pH gradient across the capillary. Figure 36 shows chromatograms, and table 15 shows the major pI isoforms (defined as peak areas greater than 10%) for each antibody and the pI range for each antibody. The main isoelectric points of the humanized Tau01HMKE, HMKM, HMKN, HMKO and HMKP candidates are 8.86-8.81.

Evaluation of serum stability of humanized Tau01 candidate antibody

Purified samples of chimeric and humanized antibodies were incubated in mouse, human and cynomolgus monkey sera for 21 days. The binding of Tau01HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies incubated in 3 different sera were compared to antibodies incubated in PBS and a 4 ℃ positive control sample by binding ELISA to TauC3 (figure 37). Tau01HMKE, HMKM, HMKN, HMKO, HMKP candidate antibodies retained their binding capacity after incubation in mouse, human and cynomolgus serum.

Summary of data for lead Tau01 HMKEHMKN, HMKO, HMKP, HMKM humanized candidates compared to MoTau01HuG4k

Table 16 shows a summary of the binding, kinetic affinities and biophysical properties of the lead humanized Tau01HMKE, HMKN, HMKO, HMKP, HMKM antibody candidates compared to the chimeric antibody MoTau01HuG4 k. All humanized candidates bound in the picomolar range, but of these, Tau01HMKN, HMKO, and HMKP had the highest affinity and the slowest off-rate, and also passed all of our biophysical assays. Considering all the data, Tau01 HMKP was selected as the primary humanized candidate because it has the highest affinity, slow dissociation rate and shows no potential redundancy in biophysical analysis. Tau01HMKN and Tau01 HMKO and KMKE are good backup humanization lead candidates because they also have superior properties.

TABLE 16

Final humanized candidate antibody summary

Conclusion

The objective of this study was to humanize the MoTau01 antibody and ensure that the resulting antibody binds TauC3 with comparable affinity as the chimeric antibody. The MoTau01 antibody has been designed and expressed as a humanized antibody without significant loss of binding affinity. Tau01HMKE, HMKM, HMKN, HMKO, HMKP humanized antibodies showed high affinity in binding ELISA, Octet ordering and kinetic studies using Biacore, in the picomolar range (fig. 31), and also passed all our biophysical assays.

The Tau01 HMKP antibody showed the best drug-like profile and excellent binding kinetics and was therefore selected as the primary candidate (table 16). To our opinion, the combination of excellent binding, expression, thermostability, affinity and biophysical properties makes Tau01 HMKP a candidate antibody suitable for further development. Tau01HMKN, HMKO and HMKE also exhibit excellent properties and are very good backup humanization lead candidates.

Example 5 (protocol)

The following protocol/procedure was used in examples 1-4.

RNeasy Mini protocol for isolation of Total RNA (Qiagen)

1. Cells were destroyed by addition of buffer RLT. For the pelleted cells, the cell pellet was loosened thoroughly by flicking the tube. Buffer RLT (600. mu.l) was added and then step 2 was continued. Note that: incomplete loosening of the cell pellet may result in inefficient lysis and reduced yield.

2. Cells passed through the lysate were homogenized at least 5 times by means of an 18-20 gauge needle mounted on an RNase-free syringe.

3. To the homogenized lysate was added 1 volume of 70% ethanol and mixed well by pipette. Without centrifugation. The volume of lysate may be less than 600 μ l due to losses during homogenization.

4. Up to 700. mu.l of sample (including any precipitate that may have formed) was transferred to an RNeasy spin column placed in a 2mL collection tube. The lid was lightly closed and centrifuged at 8000Xg or more for 15 seconds. The flow-through was discarded (flow-through). The centrifuge tube in step 5 was reused.

5. To the RNeasy column 700. mu.l of buffer RW1 was added. The lid was gently closed and centrifuged at 8000Xg or more for 15 seconds to wash the column membrane. The flow-through was discarded. The centrifuge tube in step 6 was reused.

6. To the RNeasy column was added 500. mu.l of buffer RPE. The lid was gently closed and centrifuged at 8000Xg or more for 15 seconds to wash the column membrane. The flow-through was discarded. The centrifuge tube is reused in step 7.

6. Then 500. mu.l of buffer RPE was added to the RNeasy column. The lid was gently closed and centrifuged at > 8000Xg for 2 minutes to dry the RNeasy spin column membrane.

7. RNeasy spin columns were placed in new 2mL collection tubes and old collection tubes with flow-through were discarded. The lid was gently closed and centrifuged at full speed for 1 min.

8. For elution, the RNeasy column was transferred to a new 1.5mL collection tube. Mu.l RNase free water was added directly to the RNeasy spin column membrane. The tube was gently closed. Standing for 1 minute, and then centrifuging at > 8000Xg for 1 minute.

First Strand cDNA Synthesis protocol (GE Life Sciences)

1. The RNA samples were placed in a microcentrifuge tube and RNase-free water was added to bring the RNA to the appropriate volume (20. mu.L-12 fold dilution, see Table A).

2. The RNA solution was heated to 65 ℃ for 10 minutes and then cooled on ice. A large amount of the first strand cDNA reaction mixture was gently pipetted to obtain a uniform suspension. (BSA may precipitate in the mixture during storage; this precipitate will dissolve during incubation).

3. A large volume of first strand cDNA reaction mixture (11. mu.L) was added to a sterile 1.5 or 0.5mL microcentrifuge tube. mu.L of DTT solution, 1. mu.L (0.2. mu.g, 1: 25 dilution) of NotI-d (T)18 primer and heat-denatured RNA were added to the tube. Mix several times up and down the pipettor.

4. Incubate at 37 ℃ for 1 hour, and heat at 95 ℃ to inactivate the transcriptase for 5 minutes.

TABLE A volume of Components in the first chain reaction

A large amount of first chain reaction mixture	Primer and method for producing the same	DTT	RNA	First chain reaction Final volume
					11μL	1μL	1μL	20μL	33μL

cDNA purification

1. A simple protocol is aimed at removing contaminating first strand cDNA primers that may interfere with subsequent PCR reactions.

2. 99. mu.l of buffer QG (from Qiagen gel extraction kit, cat # 28704) and 33. mu.l of IPA were added. Mix and add to QiaQuick gel extraction column. The flow-through was spun down and discarded.

3. The column was washed once with 500. mu.l of buffer QG. The flow-through was discarded.

4. The column was washed once with 750. mu.l of buffer PE. The flow-through was discarded.

5. The column was rotated to remove any residual alcohol and the column was dried.

6. The cDNA was eluted with 50. mu.l of distilled water preheated to 65 ℃.

PCR cloning of mouse variable regions

1. PCR reactions were performed on the purified cDNAs using the primers in tables 1 and 2. Different forward primers (MHV1-12 and 14 and MKV1-11) were used in each reaction as follows:

2. and (3) circulation:

3. mu.l of the sample from each PCR reaction was electrophoresed on a 2% (w/v) agarose gel to determine which lead primer produced the PCR product. The size of the positive PCR clone was about 420-500 bp.

4. For positive clones, the remaining samples were PCR purified using QIAGEN PCR purification kit and eluted into 40 μ L nuclease-free water. Sent to an outside contractor (e.g., GATC) and PCR fragment sequencing was performed using the M13 forward primer and the M13 reverse primer.

QIAquick PCR purified microcentrifuge and vacuum protocol (QIAGEN)

1. In a conventional benchtop microcentrifuge, all centrifugation steps are performed at a speed of 17,900Xg (13,000 rpm).

2. Buffer PB was added to 1 volume of PCR reaction in 5 volumes and mixed. If the mixture is orange or purple in color, 10. mu.l of 3M sodium acetate, pH5.0, are added and mixed. The color of the mixture will change to yellow.

3. The QIAquick column was placed into a provided 2mL collection tube or vacuum manifold.

4. To bind the DNA, the sample was applied to a QIAquick column and centrifuged for 30-60 seconds or vacuum was applied to the manifold until all the sample passed through the column. The flow-through was discarded and the QIAquick column was returned to the same tube.

5. For washing, 0.75mL of buffer PE was added to the QIAquick column, and centrifuged for 30-60 seconds or evacuated. The flow-through was discarded and the QIAquick column was returned to the same tube.

6. The column was centrifuged for 1 min in a 2mL collection tube (provided).

7. Each QIAquick column was placed into a clean 1.5mL microcentrifuge tube.

8. To elute the DNA, 40-50. mu.l of buffer EB (10mM Tris. Cl, pH 8.5) was added to the center of the QIAquick membrane, and the column was then centrifuged for 1 minute.

Production of mAb expression vectors by LIC

Preparation for insertion

1. Sequences were used to generate LIC primers.

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

3. Setting PCR reaction:

note that: polymerases that produce blunt-ended PCR products must be used in this step. Other polymerases that generate T overhangs are not suitable.

4. And (3) circulation:

5. run 5. mu.L of PCR product on gel to ensure correct product size-should be around 370 bp.

PCR purification products were nucleotide and primer removed using Qiagen PCR purification kit. Elute to 40 μ L nuclease free water.

T4 DNA polymerase treatment insert:

8. incubate at room temperature for 30 minutes, then inactivate the enzyme at 70 ℃ for 20 minutes.

Preparation of the support

9. The LlC vector (vector maps shown in FIGS. 3 and 4) was digested with BfuAI by incubation at 50 ℃ for 3 hours or overnight:

adding after BfuAI digestion: mu.L of Bam HI and incubated at 37 ℃ for 2 hours.

11. The digested vector was run on a 1% (w/v) agarose/1 × TAE gel containing 1 × SYBR Safe DNA stain. Two bands could be seen-the higher MW band was cut out and extracted using the gel extraction kit, eluting in 50 μ Ι _ of EB.

T4 DNA polymerase treatment of the vector as follows:

13. incubate at room temperature for 30 minutes, then inactivate the enzyme at 70 ℃ for 20 minutes.

Cloning

14. mu.L of insert was mixed with 0.5. mu.L of vector in a total of 10. mu.L nuclease-free water for 20 minutes at room temperature. Vector-only transformation was always performed.

15. 25-50 μ L of chemically active Invitrogen TOP10 bacteria were transformed with the ligation mixture and plated on 90mm diameter LB agar plates containing kanamycin (50 μ g/mL) according to the manufacturer's instructions. Incubate overnight at 37 ℃.

Selection of clones from transformants

16. PCR confirmation was performed using Phusion PCR premix:

reagent	Volume (for 20. mu.l final reaction volume)
		2x Phusion PCR premix	25μL
HCMVi primer	1μL
		HuG4/HuK LIC primer	1μL
dH20	To 23 μ L
		DNA	Bacterial colony dipping (daily culture of the same bacterial colony)

17. Each PCR reaction was run on a 2% agarose E-Gel cartridge and run for 15 minutes to determine the size of any PCR product bands on the Gel.

18. The initial culture was cultured overnight using LB supplemented with kanamycin, the construct was miniprepped, and the DNA of at least two independent positive clones of the variable gene was sequenced (using the same primers) to identify any possible errors due to the PCR reaction itself.

^TMTransformation of TOP10 E.coli (Invitrogen protocol)

1. Vials containing the ligation reaction were briefly centrifuged and placed on ice.

2. A50. mu.L vial of One Shot cells was thawed on ice for One or two ligations/transformations.

3. 1 to 2. mu.L of each ligation reaction was directly added to a vial of competent cells by pipette and mixed with gentle tapping. Do not mix up and down by pipetting. The remaining ligation mixture can be stored at-20 ℃.

4. The vials were incubated on ice for 15-30 minutes.

5. Incubate in a 42 ℃ water bath for exactly 30 seconds and then place on ice for 2 minutes.

6. To each vial was added 250 μ L of pre-heated s.o.c. medium.

7. The vial was shaken at 37 ℃ for exactly 1 hour in a shaking incubator at 225 rpm.

8. 200 μ L of each transformation flask was smeared onto a separate, labeled LB agar plate containing 500 μ g/mL kanamycin.

9. The plates were inverted and incubated overnight at 37 ℃.

Use of Miniprep isolation of plasmid DNA (Oiagen protocol)

1. The pelleted bacterial cells were resuspended in 250. mu.L of buffer P1 and transferred to a microcentrifuge tube. Ensure that RNase A has been added to buffer P1.

2. Add 250. mu.L of buffer P2 and gently invert the tube 4-6 times to mix well.

3. 350 μ L of buffer N3 was added and the tube was immediately inverted gently 4-6 times. The solution should become cloudy.

4. Centrifuge at 13,000rpm (. about.17,900 Xg) for 10 minutes in a benchtop microcentrifuge. Compact white particles will be formed.

5. The supernatant from step 4 was added to a QIAprep spin column by pipetting.

6. And centrifuging for 30-60 seconds. The flow-through was discarded.

7. The column was washed by adding 0.5mL buffer PB and centrifuging for 30-60 s.

8. The column was washed by adding 0.75mL buffer PE and centrifuging for 30-60 s.

9. The flow-through was discarded and centrifuged for 1 min.

10. The QIAprep column was placed into a clean 1.5mL microcentrifuge tube. The DNA was eluted, 50. mu.L of nuclease-free water was added to the center of the QIAprep spin column, allowed to stand for 1 minute, and then centrifuged for 1 minute.

^TMExpicHO transfection in 24-well plates 1ml transfection (ExpicHO expression System kit-Invitrogen)

1. Passaging and expanding expiCHO cells until cell density reaches about 4-6x10⁶Viable cells/mL.

2. One day before transfection (day-1), the expiCHO cultures were divided into 3-4X10⁶Final density of individual viable cells/mL, cells were allowed to grow overnight.

3. Cells were diluted to 6x10⁶Viable cells/mL.

4. 0.9mL of cells were dispensed into each well of a 24-well plate for transfection.

5. Expifeacmine/DNA complexes were prepared.

6. Plasmid DNA (1. mu.g plasmid DNA per ml culture volume to be transfected) was diluted by adding 1. mu.L of DNA to each well to be transfected in a final volume of 50. mu.L OptiPro.

7. For each well to be transfected, 4 μ L of expifctamine CHO reagent was diluted in 46 μ L of OptiPro medium (no incubation time required).

8. Diluted expifctamine CHO was added to the diluted DNA and mixed by gentle pipetting 3-4 times (incubation for 1 to 5 minutes).

9. To each well containing the culture in a 24-well plate, 100 μ L of the complex mixture was added.

10. The plate is covered with a gas permeable cover.

11. The 24-well plate was placed on an orbital shaker in a 37 ℃ incubator with 8% CO2 (for a shaker with an orbital path of 19 mm, a shaker speed of 225rpm was recommended).

12. Expifactamine enhancer (6ul ExpicHO enhancer) and ExpicHO feed (190ul ExpicHO feed) were added 18-22 hours post-transfection

13. Protein expression is usually complete 7-8 days after transfection and the supernatant is ready for collection.

Quantification of IgG by Octet

1. A100. mu.l standard curve and ExpicHO supernatant at each concentration was prepared as follows:

HuG4K isoform standards at concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.81, 3.9. mu.g/ml using ExpicHO expression medium as diluent;

b. the (unknown) sample was tested.

2. Protein G-coated biosensors (Pall ForteBio) were pre-soaked (. gtoreq.10 min) in 200. mu.l ExpicHO expression medium.

3. 45 μ l of standards and test samples were dispensed into 384-well inclined plates in duplicate, including the medium-only control. Plates were sealed and spun in a bench top centrifuge (1000rpm, 1 minute).

4. The plate seal was removed and the plate and prepreg sensor were inserted into the Octet.

5. Quantification was performed as follows:

a. the protein G coated sensor was regenerated in 10mM glycine (pH 1.5) for 5 seconds and neutralized in ExpicHO expression medium for 5 seconds. Repeating for three times.

b. The standard or sample is measured for 120 seconds.

c. The above regeneration and neutralization steps are repeated.

6. Data were imported into analytical software and fitted to a 5PL weighted fit of dose response to give IgG concentration (in μ g/ml).

TauC3 binding ELISA

1. In each well of the 94/384 well MaxiSorp plate, 50/30. mu.L of 1. mu.g/mL TauC3 aliquots of PBS were plated in each well of the 96/384 well plate, respectively. Incubate overnight at 4 ℃.

2. Washed 3 times with PBS-T (0.1% Tween 20).

3. In 96/384 well plates, each well was blocked with 150/80 μ L PBS + 5% BSA + 0.1% Tween 20.

4. Incubate at 37 ℃ for 1 hour. Washed 3 times with PBS-T (0.1% Tween 20).

5. 50/30 μ l of primary antibody serially diluted in PBS + 0.2% BSA + 0.1% Tween 20 was added to the test plates (96/384 well plates, respectively). A3-fold dilution series from 4. mu.g/mL was used. The incubation and washing steps are repeated (step 4).

6. 3ul of anti-human kappa chain HRP (Sigma A7164-1mL) was diluted per 10mL in PBS + 0.2% BSA + 0.1% Tween 20 and 50/30. mu.L was added to each well of 96/384 well plates. The incubation and washing steps are repeated (step 4).

7. 75/20 μ L of K-Blue substrate (Neogen) was added to each well and incubated for 5-10 minutes at room temperature.

8. The reaction was stopped by adding 50/10 μ l of RED STOP solution (Neogen) to each well of an 96/384 well plate.

9. The optical density at 650nm was read using the Pheastar Plus.

Tau01 variant screening and affinity determination by Octet

1. Just before use, the protein G-coated sensor (Pall ForteBio) was placed on HBS-P⁺Incubate in buffer for 10 min.

2. HBS-P⁺1. mu.g/mL of the antibody in (1) was loaded on the Protein G sensor for 600 seconds (fixed level 0.8-1 nm).

3. Having sensors on HBS-P⁺Middle equilibrium for 180 seconds.

4. For the screening experiments, the binding step was performed for 600 seconds using 10nM TauC 3. For kinetic analysis, in HBS-P⁺Loading 0.5. mu.g/mL antibody for 10 minutes and performing the binding step using TauC3 at a concentration ranging from 20nM to 0.31nM for 10 minutes.

5. In HBS-P⁺The dissociation step was performed for 600 seconds.

6. The sensor was regenerated with 10mM glycine (pH1.5-2.0) for 5-30 seconds and then passed through HBS-P⁺The buffer was incubated for 30-60 seconds to neutralize. Repeating for three times.

QuikChange Lightning site-directed mutagenesis kit (Stratagene)

1. The reaction was prepared as follows:

a.5. mu.L of 10 × reaction buffer

0.12 μ L (25ng) of RHA or RKA template

c.1.3. mu.L (125ng) of oligonucleotide mutation Forward primer

d.1.3. mu.L (125ng) of oligonucleotide mutation reverse primer

e.1. mu.L of dNTP mix

f.1.5. mu.L of Quiksolution reagent

ddH2O to a final volume of 50. mu.L

h.1 μ L of QuikChange Lightning enzyme.

2. Each reaction was cycled using the cycling parameters listed in the table below:

3. 2. mu.L of Dpn I restriction enzyme was added.

4. Each reaction was gently mixed thoroughly, briefly microcentrifuged, and immediately incubated at 37 ℃ for 5 minutes to digest the parental dsDNA.

5. mu.L of Dpn I treated DNA from each reaction was converted to a single 45. mu.L (+ 2. mu.L. beta. -ME) aliquot of XL10-Gold super competent cells (see TOP 10)^TMTransformation of E.coli).

6. Colonies were screened using Phusion method, mini prep and sequence to check for correct mutations.

Qiagen high speed Maxiprep system scheme

1. Selection from freshly streaked selective plates or glycerol stocks of target clones, and inoculation with 2-5ml starting cultures of LB medium supplemented with kanamycin. Incubate at 37 ℃ for about 8 hours with shaking at 250-300 rpm.

2. The starting culture was diluted 1/1000 and inoculated with 150-250mL LB medium supplemented with kanamycin from the starting culture and incubated overnight (12-16 hours) at 37 ℃ with shaking at 250-300 rpm.

3. Cells were harvested at 6,000 Xg for 15 minutes. The supernatant was discarded.

4. The cell pellet was completely resuspended in 10mL of buffer P1 by vortexing or pipetting.

5. 10mL of buffer P2 was added. Mix by vigorously inverting 4-6 times. Incubate at room temperature for 5 minutes.

6. 10mL of cooled buffer P3 was added. Mix by vigorously inverting 4-6 times.

7. The lysate was poured into the barrel of a QIAfilter Cartridge. Incubate at room temperature for 10 minutes.

8. 10ml of buffer QBT was added to equilibrate the HiSpeed Maxi Tip and emptied by gravity flow.

9. The lysates were filtered into equilibrated HiSpeed Maxi Tip using QIAfilter. The lysate was allowed to flow by gravity into the resin.

10. HiSpeed Maxi Tip was washed with 60ml buffer QC.

11. The DNA was eluted with 15ml of buffer QF.

12. 10.5ml of isopropanol was added to the eluted DNA to precipitate the DNA. Mix and incubate for 5 minutes at room temperature.

13. The eluate/isopropanol mixture was transferred to a 30ml syringe and filtered through a qiaaprecipitator module.

14. The DNA in QIAprecipitator was washed with 2ml of 70% ethanol. The film was dried by passing air through the qiaaprecipitator by multiple squeezes.

15. The DNA was eluted in 1ml nuclease-free water using a 5ml syringe. The eluate was transferred to a syringe and subjected to a second elution.

Comparison of thermal stability

1. Fully humanized antibody and chimeric control were diluted to 1 μ g/mL in PBS/0.2% tween and aliquoted into PCR tubes at the appropriate volume of EC80 concentration. The volume was adjusted to 100. mu.l using the same buffer.

2. Each tube was heated at a temperature between 30 ℃ and 85 ℃ for 10 minutes at 5 ℃ intervals, respectively, and then cooled to 4 ℃.

3. The stock solution at 1. mu.g/mL was frozen for 1 hour and then diluted to EC80 concentration.

4. Binding assays against TauC3 were performed in 96-well plates using 100 μ l of each antibody per well (duplicate for each temperature assay) (section 8.11).

Biacore dissociation rate ranking and kinetic study of Tau01 humanized antibodies

Off rate ordering

1. Amine coupling to 0.5. mu.g/mL human TauC3 was performed in acetate buffer pH5 on 1 flow channel in a CM5 chip (GE Healthcare). Fixed wizards (immobilization wizards) were used to guide (aim) -15 RUs with HBS-EP + as run buffers.

2. Antibody supernatant loaded with 2.5nM HBS-EP + buffer at 30. mu.L/min for 300 seconds and then dissociated for 600 seconds using 3M MgCl₂Regeneration was carried out for 30 seconds. Raw data was derived, buffer baseline was subtracted and data was fitted using monophasic or biphasic decay in GraphPad Prism.

Dynamics of

1. Amine coupling to 0.5. mu.g/mL human TauC3 was performed on 1 flow channel of a CM5 chip in acetate buffer pH5. Fixed wizards were used to guide-15 RU, with HBS-EP + as running buffer.

2. Each antibody was diluted to 5nM and produced a 2-fold dilution series to 0.08nM in HBS-EP + buffer. Injection at 30. mu.L/min for 300 seconds and then dissociation for 600 seconds for each concentration, using 3M MgCl₂Regeneration was 30 seconds with a stabilization period of 600 seconds between cycles. Data were fitted using a 1: 1 global fit.

Binding assay FL Tau

1. HBS-EP + buffer solution of 0.25. mu.g/mL antibody was loaded on a Protein G chip (GE Healthcare) at a rate of 10. mu.L/min for 30 seconds. The flow rate was increased to 30. mu.L/min and 250nM FL Tau was added to HBS-EP + buffer for 180 sec. Regeneration with 10mM glycine (pH 1.5) for 30 seconds

Purification of antibody candidates

The instrument comprises the following steps: GE HealthcarePurification system

Software: UNICON

A chromatographic column: HiTrap Mabselect SuRe, 1 mL; HiLoad 16/600Superdex200pg

Mobile phase: IgG elution buffer; dulbecco's 1 XPBS

Sample preparation: filtration through 0.22 μm

Injection amount: 200mL Expi293 conditioned Medium (1: 1) in DPBS

Flow rate: the sample loading amount is 0.5 mL/min; performing gel filtration at 1.5 mL/min; eluting at 1mL/min

SEC-MALS

1. Mu.l of each sample (1mg/mL) was injected onto a SEC chromatography column (Advance Bio SEC)4.6 × 150mm, 2.7 μm, LC column, agilent), then detected by three tandem detectors:

UV (Agilent 1260Infinity HPLC system with constant temperature column oven)

b. Light scattering (Wyatt Technology DAWN HELEOS)

c. Differential refractometer (Wyatt Technology Optilab TRex)

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

3. Data were analyzed using Wyatt technology astra software (version 6.1.2.83) and the refractive index delta (dn/dc) was set to 0.185 (i.e., for protein analysis).

4. All samples were stored at 4 ℃ prior to SEC-MALS analysis.

Dynamic Light Scattering (DLS)

1. 50 μ l of a 1.3mg/ml sample (in Dulbecco PBS; Sigma D8537) was prepared and aliquoted into 384 well polypropylene plates (Greiner bio-one).

2. Data are recorded on a Zetasizer APS (Malvern). All values were recorded in triplicate and processed using the relevant Zetasizer software (version 7.11).

3. Cumulant analysis was performed to obtain the mean particle size (z-average) and polydispersity index (PDI).

Mass spectrometry

Mass spectrometry analysis of purified chimeric candidate antibodies and humanized candidate antibodies is depicted in figure 30.

Thermal displacement comparison

1. Samples were prepared directly into 96-well white PCR plates in a final volume of 25. mu.L (final concentrations of purified antibody of 1. mu.M and 2. mu.M).

Sypro Orange-stock solution 1: 100 in PBS buffer and then added to the final sample 1: 10 (e.g., 2.5. mu.L in 25. mu.L)

3. Loaded into qPCR machine and using MxPro software, SYBR Green method, (filter ═ FRROX, no reference dye). Thermal profile set-71 cycles with each 1 degree increase

4. The results are plotted and Tm is determined.

Cross-interaction chromatography (CIC)

1. Samples were analyzed by two separate 20 μ l injections (0.5 mg/mL); first on 1mL of NHS activated resin (GE Healthcare) conjugated with 30mg of human polyclonal IgG (Sigma 14506) and then on a blank conjugated with 1mL of NHS activated resin as a control column.

2. The mobile phase consisted of Dulbecco PBS (Sigma D8537) containing 0.01% sodium azide (0.1mL/min) and all experiments were performed at 25 ℃.

3. Eluted samples were detected by uv absorbance (Agilent 1260Infinity HPLC system with thermostatted column oven) and the data was analyzed using Wyatt technology astra software (version 6.1.2.83) to determine sample peak retention times. These are then used to calculate the retention factor k':

wherein T is_rIs the retention time of the sample on a poly-IgG column, T_mIs the retention time on the simulated (control) column.

Solubility in water

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

1. Antibody concentration was monitored every 10 minutes and small samples were taken on a Nanodrop 2000 (e ═ 1.4) for measurement and then continued until the concentration volume reached a dead volume of-30-50 μ Ι.

2. Concentration values (mg/ml) were plotted against corresponding time points to generate concentration curves.

Circular dichroism

1. 30 μ L of sample was prepared at a concentration of 1mg/ml (in Dulbecco's PBS; Sigma D8537).

2.1 mg/mL of the sample was diluted to 0.15 fold with 10mM phosphate buffer.

3. Readings were taken from 1mM spectropure quartz cuvettes. Readings were taken in 1nm steps at a DIT of 4 seconds and a scan speed of 20 nm/min.

4. The average blank spectrum is subtracted from the sample spectrum and the spectrum is then converted to Δ ε. The spectra were then zeroed relative to their 256-260nm values. Smoothness is performed by the savitsky-golay filter through a custom excel function sgFilter () using a quadratic polynomial with a window size of 7(-2, 3,6,7, 6, 3, -2). The spectra show error bars, which are the mean of the standard deviation of +/-2nm wavelength.

PI analysis Using cIEF

1. Samples were concentrated to > 5mg/ml and desalted to < 50mM NaCl levels, then 10. mu.l was added to 240. mu.l of Pharmalyte/urea gel stock containing 4.5/5.1/9.5 and 10 isoelectric point labels.

2. The samples were mixed for at least 5 minutes and then 200. mu.l was added to the PCR sample vial.

3. The samples were loaded into the PA800 sample block along with the cIEF gel, catholyte, anolyte, and chemical flow agent wash buffer. The PA800 was equipped with a neutral capillary and the default "condition" method was run to prepare the capillary for sample analysis.

4. Each sample was run using the correct "split" method, which was dependent on the level of urea present in the sample.

5. Data were analyzed using 32Karat software and pI markers provided a standard curve to quantify the peak pI values of the samples.

Antibody serum stability assessment

1. 600 μ l of 0.4mg/mL polishing antibody was prepared in PBS.

2. Mouse serum from Seralab (SCD-808), human serum (S-123) and Cyno serum (S-118) were used. 150ul of serum and PBS control were dispensed into round bottom 96 well plates and 50ul of 0.4mg/mL antibody solution (100 ug/mL final concentration in PBS) was added to each serum type in triplicate in tissue culture cabinet (BSL-2). Some were stored at 4 ℃ to be used as controls.

Serum culture plate layout

PBS	Mouse serum	Human serum	Cyno serum
				PBS	Mouse serum	Human serum	Cyno serum
PBS	Mouse serum	Human serum	Cyno serum

3. Seal plate, incubate at 37 ℃.

4. Under sterile conditions (BSL-2), 20ul samples were taken at specific time intervals (e.g., day 10, day 20) to avoid contamination. Frozen at-20 ℃ until analysis.

5. The longest incubation time was analyzed first. Samples were diluted appropriately and binding of antigen to TauC3 was determined by generating ELISA binding curves for each sample (3 x dilution) (section 8.11). Using the non-incubated antibody as a control (NI), PBS/all sera of each mAb sample on the same plate were compared.

All references, publications, and patent documents cited herein, and the text presented in the figures and in the sequence listing, are incorporated by reference in their entirety for all purposes to the same extent as if each were individually indicated.

In the foregoing specification, the invention has been described with reference to specific exemplary embodiments and examples thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention as set forth in the appended claims. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

The citation of any document herein is not an admission that such document is pertinent prior art, or material regarded as patentable with any claims of the present application. Any statement as to the content or date of any document is based on the information available to the applicant at the time of filing and does not constitute an admission as to the correctness of such statement.

Sequence listing

<110> TAUC3 Biochemical Co., Ltd (TAUC3 BIOLOGICS LIMITED)

<120> anti-TAUC 3antibody and application thereof

<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 (Artificial Sequence)

<220>

<223> htau 40

<400> 1

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

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Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

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Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu

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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

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Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro

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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

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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

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Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln

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Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser

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Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn

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Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala

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Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser

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Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser

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Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val

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Ser Ala Ser Leu Ala Lys Gln Gly Leu

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> Tau 2N3R

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Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

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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

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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

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Val Ser Ala Ser Leu Ala Lys Gln Gly Leu

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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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> VGGGDF

<400> 9

Val Gly Gly Gly Asp Phe

1 5

<210> 10

<211> 6

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<220>

<223> light chain CDR2

<400> 11

Gly Ala Phe

1

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> LQYVRYPWT

<400> 12

Leu Gln Tyr Val Arg Tyr Pro Trp Thr

1 5

<210> 13

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H 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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> primer

<400> 20

tgtaaaacga cggccagtat gaagttgcct gttaggctgt tggtgctg 48

<210> 21

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 21

tgtaaaacga cggccagtat ggagwcagac acactcctgy tatgggtg 48

<210> 22

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 22

tgtaaaacga cggccagtat gagtgtgctc actcaggtcc tggsgttg 48

<210> 23

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 23

tgtaaaacga cggccagtat gaggrcccct gctcagwtty ttggmwtctt g 51

<210> 24

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 24

tgtaaaacga cggccagtat ggatttwagg tgcagattwt cagcttc 47

<210> 25

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 25

tgtaaaacga cggccagtat gaggtkckkt gktsagstsc tgrgg 45

<210> 26

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 26

tgtaaaacga cggccagtat gggcwtcaag atggagtcac akwyycwgg 49

<210> 27

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 27

tgtaaaacga cggccagtat gtggggayct ktttycmmtt tttcaattg 49

<210> 28

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 28

tgtaaaacga cggccagtat ggtrtccwca sctcagttcc ttg 43

<210> 29

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 29

tgtaaaacga cggccagtat gtatatatgt ttgttgtcta tttct 45

<210> 30

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 30

tgtaaaacga cggccagtat ggaagcccca gctcagcttc tcttcc 46

<210> 31

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 31

tgtaaaacga cggccagtat gragtywcag acccaggtct tyrt 44

<210> 32

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 32

tgtaaaacga cggccagtat ggagacacat tctcaggtct ttgt 44

<210> 33

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 33

tgtaaaacga cggccagtat ggattcacag gcccaggttc ttat 44

<210> 34

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 34

tgtaaaacga cggccagtat gatgagtcct gcccagttcc tgtt 44

<210> 35

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 35

tgtaaaacga cggccagtat gaatttgcct gttcatctct tggtgct 47

<210> 36

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 36

tgtaaaacga cggccagtat ggattttcaa ttggtcctca tctcctt 47

<210> 37

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 37

tgtaaaacga cggccagtat gaggtgccta rctsagttcc tgrg 44

<210> 38

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 38

tgtaaaacga cggccagtat gaagtactct gctcagtttc tagg 44

<210> 39

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 39

tgtaaaacga cggccagtat gaggcattct cttcaattct tggg 44

<210> 40

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 40

caggaaacag ctatgaccac tggatggtgg gaagatgg 38

<210> 41

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 41

tgtaaaacga cggccagtat gaaatgcagc tggggcatst tcttc 45

<210> 42

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 42

tgtaaaacga cggccagtat gggatggagc trtatcatsy tctt 44

<210> 43

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 43

tgtaaaacga cggccagtat gaagwtgtgg ttaaactggg ttttt 45

<210> 44

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 44

tgtaaaacga cggccagtat gractttggg ytcagcttgr ttt 43

<210> 45

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 45

tgtaaaacga cggccagtat ggactccagg ctcaatttag ttttcctt 48

<210> 46

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 46

tgtaaaacga cggccagtat ggctgtcytr gsgctrctct tctgc 45

<210> 47

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 47

tgtaaaacga cggccagtat ggratggagc kggrtctttm tctt 44

<210> 48

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 48

tgtaaaacga cggccagtat gagagtgctg attcttttgt g 41

<210> 49

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 49

tgtaaaacga cggccagtat ggmttgggtg tggamcttgc tattcctg 48

<210> 50

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 50

tgtaaaacga cggccagtat gggcagactt acattctcat tcctg 45

<210> 51

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 51

tgtaaaacga cggccagtat ggattttggg ctgatttttt ttattg 46

<210> 52

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 52

tgtaaaacga cggccagtat gatggtgtta agtcttctgt acctg 45

<210> 53

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 53

tgtaaaacga cggccagtat gaacaggctt acttcctcat tgctgctgc 49

<210> 54

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 54

caggaaacag ctatgaccca gtggatagac agatggggg 39

<210> 55

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 55

caggaaacag ctatgaccca gtggatagac cgatggggc 39

<210> 56

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 56

caggaaacag ctatgaccca gtggatagac tgatggggg 39

<210> 57

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 57

caggaaacag ctatgaccca agggatagac agatggggc 39

<210> 58

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 58

tgtaaaacga cggccagtga ggtgcaggtt gttgagtctg g 41

<210> 59

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 59

tgttcctttc catgggtctt 20

<210> 60

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 60

ctctcggagg tgctcctgga g 21

<210> 61

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 61

gcagttccag atttcaactg 20

<210> 62

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 62

tgtaaaacga cggccagt 18

<210> 63

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 63

caggaaacag ctatgacc 18

<210> 64

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 64

ctctggctcc ctgataccac cggagaggtg caggtggtgg agagc 45

<210> 65

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 65

ctctggctcc ctgataccac cggagatatc cagatgacac agtct 45

<210> 66

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 66

gggcccttgg tggaggcgga gctcactgtc agggcggt 38

<210> 67

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 67

cgcttggtgc tgccacagtt ctcttgatct ccagctttgt gccg 44

<210> 68

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 68

ctctggctcc ctgataccac cggactggtg cagctggtgg aaagcg 46

<210> 69

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 69

ctctggctcc ctgataccac cggagaggtg caggtggtgg aaagcg 46

<210> 70

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 70

gggcccttgg tggaggcgga gctcactgtc accagggtg 39

<210> 71

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 71

cctgatacca ccggagaggt gcagctggtg 30

<210> 72

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 72

caccagctgc acctctccgg tggtatcagg 30

<210> 73

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 73

cggactggtg caggtggtgg aaagcgg 27

<210> 74

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 74

ccgctttcca ccacctgcac cagtccg 27

<210> 75

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 75

tttaacacat atgcaatgaa ctgggtgcgg cagg 34

<210> 76

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 76

cctgccgcac ccagttcatt gcatatgtgt taaa 34

<210> 77

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 77

ctggagtggg tggcccggat cagatct 27

<210> 78

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 78

agatctgatc cgggccaccc actccag 27

<210> 79

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 79

ctaagagcaa caattatgca acatattatg cagcatctgt gaagggcag 49

<210> 80

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 80

ctgcccttca cagatgctgc ataatatgtt gcataattgt tgctcttag 49

<210> 81

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 81

tatgcaacag catatgcaga ttctgtgaag ggcaggttca 40

<210> 82

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 82

tgaacctgcc cttcacagaa tctgcatatg ctgttgcata 40

<210> 83

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 83

ccgcgacgat tctaagagta cagcctatct gcaga 35

<210> 84

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 84

tctgcagata ggctgtactc ttagaatcgt cgcgg 35

<210> 85

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 85

tctcccgcga cgattctaag aatatggcct atctgcagat 40

<210> 86

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 86

atctgcagat aggccatatt cttagaatcg tcgcgggaga 40

<210> 87

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 87

gacgattcta agaatacagt ctatctgcag atggactcc 39

<210> 88

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 88

ggagtccatc tgcagataga ctgtattctt agaatcgtc 39

<210> 89

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 89

ctctggctcc ctgataccac cggagacatc cagatgaccc agtctc 46

<210> 90

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 90

ctctggctcc ctgataccac cggagacatc cagatgacac agtctc 46

<210> 91

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 91

cgcttggtgc tgccacagtt ctcttgatct ccacctttgt gccg 44

<210> 92

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 92

ttctgctgat accagctcag gtacacggag atc 33

<210> 93

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 93

tgtacctggg ctggtttcag cagaagccc 29

<210> 94

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 94

gggcttctgc tgaaaccagc ccaggtaca 29

<210> 95

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 95

gaagcccggc aaggccatta agcggctgat ctac 34

<210> 96

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 96

gtagatcagc cgcttaatgg ccttgccggg cttc 34

<210> 97

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 97

atctacggcg ccttcacgct gcagtccg 28

<210> 98

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 98

cggactgcag cgtgaaggcg ccgtagat 28

<210> 99

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 99

ggatccagat ctggcagcga gtttaccctg a 31

<210> 100

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 100

tcagggtaaa ctcgctgcca gatctggatc c 31

<210> 101

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 101

cagatctggc accgagtata ccctgacaat ctcta 35

<210> 102

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 102

tagagattgt cagggtatac tcggtgccag atctg 35

<210> 103

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 103

ctacggcgcc ttcagcctgc agtccggagt 30

<210> 104

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 104

actccggact gcaggctgaa ggcgccgtag 30

<210> 105

<211> 115

<212> PRT

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<220>

<223> primer

<400> 139

tgtaaaacga cggccagtga ggtgcaggtt gttgagtctg g 41

<210> 140

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 140

tgtaaaacga cggccagtat gacattgaac atgctgttgg ggc 43

<210> 141

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer

<400> 141

gatctccgtg tacctgagct ggtatcagca gaa 33