RU2816371C2 - EphA4 ANTIBODY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an anti-EphA4 antibody, a method for production thereof, as well as a composition containing same. Also disclosed is an isolated nucleic acid coding said antibody, as well as a vector and a host cell containing said nucleic acid.

EFFECT: invention is effective for inhibiting phosphorylation of tau protein.

18 cl, 23 dwg, 3 tbl, 13 ex

Description

Field of technology

[0001]

The present invention relates to an antibody that binds to EphA4, a nucleic acid that encodes the antibody, a vector that contains the nucleic acid, a cell containing the vector, a method for producing the antibody, and a pharmaceutical composition containing the antibody.

State of the art

[0002]

EphA4 is a member of the receptor tyrosine kinase family. Type A and type B ephrins are known as ligands for EphA4, and when EphA4 binds to its ligand ephrin, a deadhesion signal is generated. Until now, the involvement of EphA4 in the pathology of Alzheimer's disease (hereinafter also called "AD") has been suggested (non-patent literature references 1-4) and it has been reported that inhibition of the binding between EphA4 and ephrin rescues amyloid β (Aβ) _(1-42) oligomer-mediated dysfunction of nerve impulse transmission (patent literature source 1). In AD, nerve cell death is thought to involve aggregates (neurofibrillary tangles) formed by excessively phosphorylated tau protein (Non-Patent Literature 5), and suppression of tau protein phosphorylation has also been reported to suppress neurodegeneration in the form of loss of synapses (Non-Patent Literature 6 and non-patent literature 7), and also reduces memory impairment or cognitive dysfunction (non-patent literature 8-11). There are publications that suggest that CDK5 activation is the cause of tau protein phosphorylation (Non-Patent Literature 12 and Non-Patent Literature 13). A genetically modified mouse expressing the P301L mutation, which has been found in hereditary frontotemporal dementia (rTg4510 mouse), is a mouse model of AD and similar to AD, the mouse exhibits hyperphosphorylation of tau protein and abnormal accumulation of tau protein in neurons. The rTg4510 mouse develops neurofibrillary tangles, which is a pathological feature of AD, and it causes cognitive dysfunction due to brain atrophy and neuronal loss (Non-Patent Literature 14 and Non-Patent Literature 5).

[0003]

EphA4 is abundantly expressed in the hippocampus or cortex and is cleaved in a neural activity-dependent manner by matrix metalloproteinase (MMP), ADAM (disintegrin and metalloproteinase), and γ-selectase. This EphA4 cleavage reaction is known to stabilize the spinal cord, which is a key structure in the functioning of the nervous system (Non-Patent Literature 15). Spinal cord density has been reported to be reduced in AD (Non-Patent Literature 16), and since NFT stages V and VI AD have also been shown to have a reduction in EphA4 cleavage fragments, the EphA4 cleavage reaction is thought to be involved in AD pathology (Non-Patent Literature 17) .

[0004]

Although peptide KYL and compound 1, etc. are known as existing agents that inhibit EphA4. (Patent Literature 2, Non-Patent Literature 18 and Non-Patent Literature 19), so far there have been no publications on antibodies having activity that enhances the cleavage of EphA4.

List of cited literature

[0005]

[Patent Literature 1] WO2016/019280A1.

[Patent Literature 2] WO2012/156351A1.

[0006]

[Non-Patent Literature 1] Vargas LM et al., PLoS One. 2014 Mar 21; 9(3).

[Non-Patent Literature 2] Fu AK et al., Proc Natl Acad Sci U S A. 2014 Jul 8; 111 (27): 9959-64.

[Non-Patent Literature 3] Rosenberger AF et al., Acta Neuropathol Commun. 2014 Jul 16; 2:79.

[Non-Patent Literature 4] Huang TY et al., J Exp Med. 2017 Dec 4; 214(12):3669-3685.

[Non-Patent Literature 5] Santa Cruz et al., Science. 2005 Jul 15; 309 (5733): 476-81.

[Non-Patent Literature 6] Seo J et al., J Neurosci. 2017 Oct 11; 37 (41): 9917-9924.

[Non-Patent Literature 7] Patrick GN et al., Nature. 1999 Dec 9; 402 (6762): 615-22.

[Non-Patent Literature 8] Onishi T et al., J Neurochem. 2011 Dec; 119 (6): 1330-40.

[Non-Patent Literature 9] Belfiore R et al., Aging Cell. Feb 2019; 18(1):e12873.

[Non-Patent Literature 10] Webster SJ et al., Front Genet. 2014 Apr 23; 5:88.

[Non-Patent Literature 11] Grayson B et al., Behav Brain Res. 2015 May 15; 285: 176-93.

[Non-Patent Literature 12] Cancino GI et al., Neurobiol Aging. July 2011; 32 (7): 1249-61.

[Non-Patent Literature 13] Vargas LM et al., Biochim Biophys Acta Mol Basis Dis. 2018 Apr; 1864: 1148-1159.

[Non-Patent Literature 14] Ramsden M et al., J Neurosci. 2005 Nov 16; 25 (46): 10637-47.

[Non-Patent Literature 15] Inoue E et al., J Cell Biol. 2009 May 4; 185(3):551-64.

[Non-Patent Literature 16] Boros et al., Ann Neurol. Oct 2017; 82 (4): 602-614.

[Non-Patent Literature 17] Matsui C et al., Brain Pathol. 2012 Nov; 22 (6): 776-87. doi:10.1111/j.1750-3639.

[Non-Patent Literature 18] Goldshmit et al., PLoS one. 2011; 6(9):e24636.

[Non-Patent Literature 19] Van Hoecke et al., Nature Medicine. 2012 Sep; 18(9):1418-22,2012.

Brief description of the invention

Problems solved by the present invention

[0007]

An object of the present invention is to provide an anti-EphA4 antibody that can bind to EphA4 and enhance the degradation of EphA4, and to provide a pharmaceutical composition containing the antibody as an active ingredient.

Tools for solving problems

[0008]

As a result of extensive research to find a solution to the above problems, the present inventors obtained a mouse monoclonal antibody to EphA4, which can bind to EphA4 and enhance the cleavage of EphA4, and obtained a humanized antibody to the antibody, thereby obtaining a final antibody of interest.

[0009]

The present invention covers the following features.

(1) Antibody to EphA4, where

Anti-EphA4 antibody contains heavy and light chains and also contains:

(a) a heavy chain CDR1 consisting of the amino acid sequence shown under SEQ ID NO: 44;

(b) a heavy chain CDR2 consisting of the amino acid sequence shown under SEQ ID NO: 27;

(c) a heavy chain CDR3 consisting of the amino acid sequence shown under SEQ ID NO: 28;

(d) a light chain CDR1 consisting of the amino acid sequence shown under SEQ ID NO: 29;

(e) a light chain CDR2 consisting of the amino acid sequence shown under SEQ ID NO: 30; And

(f) a light chain CDR3 consisting of the amino acid sequence shown under SEQ ID NO: 31.

[0010]

(2) An anti-EphA4 antibody according to paragraph (1), wherein the anti-EphA4 antibody is humanized.

[0011]

(3) An anti-EphA4 antibody according to items (1) or (2), wherein the anti-EphA4 antibody specifically binds to EphA4 and enhances the cleavage of EphA4.

[0012]

(4) An anti-EphA4 antibody according to any one of (1) to (3), wherein the anti-EphA4 antibody specifically binds to EphA4 and inhibits the binding between EphA4 and ephrin.

[0013]

(5) An anti-EphA4 antibody according to any of paragraphs (1) - (4), where

the heavy chain variable region consists of the amino acid sequence shown under SEQ ID NO: 45, and

the light chain variable region consists of the amino acid sequence shown under SEQ ID NO: 46.

[0014]

(6) An anti-EphA4 antibody according to any of (1) - (5), where

the heavy chain constant region and the light chain constant region contain amino acid sequences derived from a human antibody.

[0015]

(7) Antibody to EphA4 in accordance with paragraph (6), where

the heavy chain constant region is the human IgG constant region.

[0016]

(8) Antibody to EphA4 in accordance with paragraph (7), where

the human IgG constant region is the human IgG ₂ constant region.

[0017]

(9) Antibody to EphA4 in accordance with paragraph (8), where

The human IgG ₂ constant region contains the amino acid sequence shown under SEQ ID NO: 47.

[0018]

(10) Antibody to EphA4 according to any of paragraphs (6) - (9), where

the light chain constant region is the human Igκ constant region.

[0019]

(11) Antibody to EphA4 in accordance with paragraph (10), where

The human Igκ constant region contains the amino acid sequence shown under SEQ ID NO: 48.

[0020]

(12) Antibody to EphA4, where

Antibody to EphA4 contains heavy and light chains,

the heavy chain contains the amino acid sequence shown under SEQ ID NO: 59, and

the light chain contains the amino acid sequence shown under SEQ ID NO: 60.

[0021]

(13) Antibody to EphA4 in accordance with paragraph (12), where

The C-terminal lysine of the heavy chain has been removed.

[0022]

(14) Antibody to EphA4, where

Antibody to EphA4 contains heavy and light chains,

the heavy chain contains the amino acid sequence shown under SEQ ID NO: 59,

the light chain contains the amino acid sequence shown under SEQ ID NO: 60, and

The C-terminal lysine of the heavy chain has been removed.

[0023]

(15) An isolated nucleic acid encoding an antibody to EphA4 in accordance with any of paragraphs (1) to (14).

[0024]

(16) A vector containing the nucleic acid according to paragraph (15).

[0025]

(17) A host cell containing the vector according to (16).

[0026]

(18) A method for producing an anti-EphA4 antibody, comprising the step of culturing a host cell in accordance with paragraph (17).

[0027]

(19) A pharmaceutical composition containing an anti-EphA4 antibody according to any of paragraphs (1) to (14).

[0028]

(20) The pharmaceutical composition according to paragraph (19), wherein the pharmaceutical composition contains at least one pharmaceutically acceptable carrier.

[0029]

(21) The pharmaceutical composition according to paragraphs (19) or (20) for the treatment of Alzheimer's disease.

[0030]

(22) An anti-EphA4 antibody according to any of paragraphs (1) to (14) for use in the treatment of Alzheimer's disease.

[0031]

(23) A method for treating Alzheimer's disease comprising administering to a patient in need thereof a therapeutically effective amount of an anti-EphA4 antibody according to any one of (1) to (14).

[0032]

(24) Use of an anti-EphA4 antibody according to any of paragraphs (1) to (14) for the manufacture of a pharmaceutical composition for the treatment of Alzheimer's disease.

[0033]

(25) The pharmaceutical composition according to paragraphs (19) or (20) for the treatment of tauopathy.

[0034]

(26) An anti-EphA4 antibody according to any of paragraphs (1) to (14) for use in the treatment of tauopathy.

[0035]

(27) A method of treating tauopathy comprising administering to a patient in need thereof a therapeutically effective amount of an anti-EphA4 antibody according to any of paragraphs (1) to (14).

[0036]

(28) Use of an anti-EphA4 antibody according to any of paragraphs (1) to (14) for the manufacture of a pharmaceutical composition for the treatment of tauopathy.

[0037]

(29) A pharmaceutical composition, an anti-EphA4 antibody, a method of treatment, or a use according to any one of (25) to (28), wherein the tauopathy is Alzheimer's disease or frontotemporal lobar degeneration with tau protein pathology.

[0038]

(30) Pharmaceutical composition, anti-EphA4 antibody, method of treatment or use according to (29), wherein the tauopathy is Alzheimer's disease.

[0039]

(31) A pharmaceutical composition, an anti-EphA4 antibody, a method of treatment or use according to paragraph (29), wherein the tauopathy is frontotemporal lobar degeneration with tau protein pathology.

[0040]

(32) A pharmaceutical composition, an anti-EphA4 antibody, a method of treatment or use according to paragraph (31), wherein frontotemporal lobar degeneration with tau protein pathology is progressive supranuclear palsy, corticobasal degeneration, dementia with argyrophilic granules, senile dementia by type of neurofibrillary tangle or Pick's disease.

[0041]

(33) The pharmaceutical composition, anti-EphA4 antibody, method of treatment or use according to paragraph (32), wherein frontotemporal lobar degeneration with tau pathology is progressive supranuclear palsy.

[0042]

(34) The pharmaceutical composition, anti-EphA4 antibody, method of treatment or use according to paragraph (32), wherein the frontotemporal lobar degeneration with tau protein pathology is corticobasal degeneration.

[0043]

(35) A pharmaceutical composition, an anti-EphA4 antibody, a method of treatment or use according to paragraph (32), wherein frontotemporal lobar degeneration with tau protein pathology is dementia with argyrophilic granules.

[0044]

(36) A pharmaceutical composition, an anti-EphA4 antibody, a method of treatment or use according to paragraph (32), wherein frontotemporal lobar degeneration with tau protein pathology is senile dementia of the neurofibrillary tangle type.

[0045]

(37) A pharmaceutical composition, an anti-EphA4 antibody, a method of treatment or use according to paragraph (32), wherein the frontotemporal lobar degeneration with tau protein pathology is Pick's disease.

Effects of the invention

[0046]

The present invention relates to an anti-EphA4 antibody that can bind to EphA4 and enhance the cleavage of EphA4, a nucleic acid that encodes the antibody, a vector that contains the nucleic acid, a cell containing the vector, a method for producing the antibody, and a pharmaceutical composition containing the antibody as an active agent. ingredient.

Brief description of graphic materials

[0047]

Figure 1 shows the binding affinity of the anti-EphA4 monoclonal antibody (Antibody A) to mouse and human EphA4.

Figure 2 shows the activity of enhancing EphA4 cleavage by an anti-EphA4 monoclonal antibody (Antibody A) using hippocampal neurons.

Figure 3 shows the inhibitory activity of an anti-EphA4 monoclonal antibody (Antibody A) against murine ligand-binding mouse EphA4.

Figure 4 shows the inhibitory activity of an anti-EphA4 monoclonal antibody (Antibody A) against human ligand-binding human EphA4.

Figure 5 shows the selectivity of the anti-EphA4 monoclonal antibody (Antibody A) for each human Eph receptor.

Figure 6 shows the selectivity of the anti-EphA4 monoclonal antibody (Antibody A) for each mouse Eph receptor.

Figure 7 shows the reactivity of an anti-EphA4 monoclonal antibody (Antibody A) to mouse, rat, monkey and human EphA4.

Figure 8 shows the reactivity of an anti-EphA4 monoclonal antibody (Antibody A) to the human EphA4 extracellular region (ECD), ligand binding domain (LBD), fibronectin type III domain 1 (FN1) and fibronectin type III domain 2 (FN2).

Figure 9 shows the effect of an anti-EphA4 monoclonal antibody (Antibody A) on increasing the number of spines in a hippocampal neuron.

Figure 10 shows the effect on tau phosphorylation suppression for the anti-EphA4 monoclonal antibody in vivo .

In Figure 11A, the horizontal axis shows the amino acids of the EphA4 ligand binding domain (EphA4-LBD) and the vertical axis shows the structural region of the A-Fab antibody. Black fragments show the intersection points of combinations in which interaction is present.

Figure 11B shows the surface structure of the EphA4 ligand binding domain (EphA4-LBD). In Figure 11B, the amino acid names and residue numbers contained in the binding region are shown at the corresponding positions, and the CDRs of the H chain and L chain of the A-Fab binding antibody are shown as a ribbon model.

Figure 12 shows the affinity of a humanized monoclonal antibody against EphA4 (antibody B) for human EphA4.

Figure 13 shows activity to enhance EphA4 cleavage in a hippocampal neuron for a humanized monoclonal antibody to EphA4 (Antibody B).

Figure 14 shows inhibitory activity against the human ligand binding domain of human EphA4 for a humanized anti-EphA4 monoclonal antibody (Antibody B).

Figure 15 shows the inhibitory activity against murine ligand-binding murine EphA4 for a humanized monoclonal antibody against EphA4 (Antibody B).

Figure 16 shows the selectivity of the humanized monoclonal antibody to EphA4 (Antibody B) for the human Eph receptor.

Figure 17 shows the selectivity of the humanized monoclonal antibody to EphA4 (Antibody B) for the mouse Eph receptor.

Figure 18 shows the reactivity of a humanized monoclonal anti-EphA4 antibody (antibody B) to mouse, rat, monkey and human EphA4.

Figure 19 shows the reactivity of a humanized monoclonal antibody to EphA4 (antibody B) towards the human EphA4 extracellular region (ECD), ligand binding domain (LBD), fibronectin type III domain 1 (FN1) and fibronectin type III domain 2 (FN2). .

Figure 20 shows the effect of a humanized monoclonal antibody against EphA4 (Antibody A) on increasing the number of spines in a hippocampal neuron.

Figure 21 shows activity for enhancing the cleavage of human EphA4 in a hippocampal neuron for a humanized monoclonal antibody to EphA4 (Antibody B).

Figure 22 shows the effect of a humanized monoclonal antibody against EphA4 (Antibody A) via MMP and ADAM to increase the number of spines in a hippocampal neuron.

Figure 23 shows the effect of suppressing tau phosphorylation on a humanized anti-EphA4 monoclonal antibody (antibody B) in vivo .

Description of Embodiments

[0048]

The regions indicated or encoded by SEQ ID NO: are used in this document as follows.

SEQ No. Sequence area SEQ No. Sequence area 1 Mouse EphA4 (amino acid sequence) 32 Monkey EphA4 (amino acid sequence) 2 Extracellular region of mouse EphA4 (amino acid sequence) 33 Extracellular region of monkey EphA4 (amino acid sequence) 3 Mouse extracellular region protein EphA4-SEAP-His (amino acid sequence) 34 Human EphA4 signal sequence (amino acid sequence) 4 Mouse EphA4 signal sequence (amino acid sequence) 35 Preprotrypsin signal sequence (amino acid sequence) 5 Human EphA4 (amino acid sequence) 36 Extracellular region of human EphA4 (amino acid sequence) 6 Extracellular region of human EphA4 (amino acid sequence) 37 Human EphA4 ligand binding domain (amino acid sequence) 7 ad29S DNA oligonucleotide (nucleic acid sequence) 38 Human EphA4 fibronectin type III domain 1 (amino acid sequence) 8 DNA oligonucleotide ad29AS (nucleic acid sequence) 39 Human EphA4 fibronectin type III domain 2 (amino acid sequence) 9 5' forward primer (nucleic acid sequence) 40 FR IGKV1-17*01 human antibody light chain (amino acid sequence) 10 3' Reverse Primer for Mouse IgG Heavy Chain (Nucleic Acid Sequence) 41 FR JK4 human antibody light chain (amino acid sequence) eleven 3' Reverse Primer for Mouse Ig κ Light Chain (Nucleic Acid Sequence) 42 FR IGHV3-33*03 human antibody heavy chain (amino acid sequence) 12 Antibody A heavy chain signal sequence (amino acid sequence) 43 FR JH6 human antibody heavy chain (amino acid sequence) 13 Antibody A heavy chain variable region (amino acid sequence) 44 Antibody B heavy chain CDR1 (amino acid sequence) 14 Antibody A light chain signal sequence (amino acid sequence) 45 Antibody B heavy chain HK2-42 variable region (amino acid sequence) 15 Antibody A light chain variable region (amino acid sequence) 46 Antibody B light chain variable region L1-8 (amino acid sequence) 16 Antibody A heavy chain signal sequence (nucleic acid sequence) 47 Human IgG ₂ Antibody B heavy chain constant region (amino acid sequence) 17 Antibody A heavy chain variable region (nucleic acid sequence) 48 Human IgG antibody B κ light chain constant region (amino acid sequence) 18 Antibody A light chain signal sequence (nucleic acid sequence) 49 Antibody B heavy chain CDR1 (nucleic acid sequence) 19 Antibody A light chain variable region (nucleic acid sequence) 50 Antibody B heavy chain CDR2 (nucleic acid sequence) 20 Antibody A heavy chain 5' forward primer (nucleic acid sequence) 51 Antibody B heavy chain CDR3 (nucleic acid sequence) 21 Antibody A light chain 5' forward primer (nucleic acid sequence) 52 Antibody B light chain CDR1 (nucleic acid sequence) 22 3' Reverse Primer for Antibody A Heavy Chain (Nucleic Acid Sequence) 53 Antibody B light chain CDR2 (nucleic acid sequence) 23 Antibody A light chain 3' reverse primer (nucleic acid sequence) 54 Antibody B light chain CDR3 (nucleic acid sequence) 24 Antibody A heavy chain constant region (amino acid sequence) 55 Antibody B heavy chain variable region HK2-42 (nucleic acid sequence) 25 Antibody A light chain constant region (amino acid sequence) 56 Antibody B light chain variable region L1-8 (nucleic acid sequence) 26 Antibody A heavy chain CDR1 (amino acid sequence) 57 Human IgG ₂ Antibody B heavy chain constant region (nucleic acid sequence) 27 Antibody A heavy chain CDR2 (amino acid sequence) 58 Human IgG Antibody B κ light chain constant region (nucleic acid sequence) 28 Antibody A heavy chain CDR3 (amino acid sequence) 59 Antibody B full-length heavy chain sequence (amino acid sequence) 29 Antibody A light chain CDR1 (amino acid sequence) 60 Antibody B light chain full length sequence (amino acid sequence) thirty Antibody A light chain CDR2 (amino acid sequence) 61 Antibody B full-length heavy chain sequence (nucleic acid sequence) 31 Antibody A light chain CDR3 (amino acid sequence) 62 Full-length Antibody B light chain sequence (nucleic acid sequence)

[0049]

The present invention relates to an anti-EphA4 antibody that binds to EphA4.

An anti-EphA4 antibody according to the present invention is an antibody that can recognize and bind to EphA4, and, as described below, the antibody may be an intact antibody or may be a synthetic antibody (such as a recombinant antibody, a chimeric antibody, a humanized antibody, and etc.), provided that it has binding affinity for EphA4. By EphA4 herein can be understood EphA4 derived from human, mouse, rat and monkey. EphA4 derived from human, mouse, rat and monkey can be obtained from a publicly available database in which sequence information is registered, such as Genbank, provided by the US National Center for Biotechnology Information, or EphA4 gene sequence information can be obtained by designing primers on based on information about the nucleotide sequence of EphA4 from a closely related animal species, and then cloning from RNA isolated from the desired animal species. For example, nucleotide sequence information for human, mouse, rat, and monkey EphA4 is registered in the database under Genbank accession numbers NM_004438.5, NM_007936.3, NM_001162411.1, and NM_001260870.1, respectively.

[0050]

In one aspect, an anti-EphA4 antibody is an antibody that specifically binds to EphA4. The term "specifically binding" is a term well known to those skilled in the art, and methods for determining specific binding between an antibody or antigen binding fragment thereof and an antigen or epitope are well known. In one embodiment, "specific binding" is understood to mean that an anti-EphA4 antibody can bind to EphA4 via an immunological reaction with higher binding affinity and binding activity, faster and/or for a longer period of time compared to binding to other target molecules . This does not mean that an antibody that specifically binds to EphA4 does not bind to other target molecules. In another embodiment, "specific binding" may be demonstrated by an antibody having a KD for EphA4 of at least about 10 ^-7 M, or at least about 10 ^-8 M, or at least about 10 ^-9 M, or less. Moreover, in another further embodiment, "specific binding" is understood as binding to EphA4 through an immunological reaction, but essentially without binding to molecules of another Eph receptor family.

[0051]

In one aspect, an anti-EphA4 antibody is an antibody that binds to the extracellular region of EphA4. In one embodiment, an anti-EphA4 antibody is an antibody that binds to the ligand binding domain (LBD) in the extracellular regions of EphA4.

[0052]

In one embodiment, an anti-EphA4 antibody may specifically bind to EphA4 and enhance the degradation of EphA4. In a specific embodiment, an anti-EphA4 antibody may specifically bind to EphA4 and enhance cleavage of the extracellular domain of EphA4 by matrix metalloproteinase (MMP) or ADAM (disintegrin and metalloproteinase).

[0053]

In one embodiment, an anti-EphA4 antibody may specifically bind to EphA4 and inhibit the binding between EphA4 and its ligand ephrin.

[0054]

In another embodiment, an anti-EphA4 antibody may specifically bind to EphA4 and increase the number of spines in a hippocampal neuron or stabilize spines in a hippocampal neuron.

[0055]

In one embodiment, the present invention provides an anti-EphA4 antibody that can specifically bind to at least one of human EphA4, mouse EphA4, rat EphA4 and monkey EphA4 and inhibit binding to their ligand. In another embodiment, the present invention provides an anti-EphA4 antibody that can specifically bind to two or more of human EphA4, mouse EphA4, rat EphA4 and monkey EphA4 and inhibit binding to their ligand. In a further embodiment, the present invention provides an anti-EphA4 antibody that can specifically bind to all of human EphA4, mouse EphA4, rat EphA4 and monkey EphA4 and inhibit binding to their ligand.

[0056]

For a method of measuring the antigen-binding property (such as binding affinity and cross-species reactivity) of an anti-EphA4 antibody, methods well known to those skilled in the art can be used. For example, binding affinity can be measured using, but not limited to, a Biacore™ biosensor, a KinExA biosensor, proximity scintillation assay, ELISA, ORIGEN immunoassay (IGEN), flow cytometry, fluorescence quenching, metastatic fluorescence, yeast display, and/or immunostaining. The neutralizing activity of an anti-EphA4 antibody against binding between EphA4 and its ligand can be measured using, without limitation, the Biacore™ biosensor, ELISA and/or flow cytometry.

[0057]

The anti-EphA4 antibody of the present invention may be a monoclonal antibody as long as it binds to EphA4.

[0058]

The anti-EphA4 antibody of the present invention may be of any class such as IgG, IgA or IgM (or subclasses thereof) and is not limited to a particular class. Immunoglobulins are divided into different classes depending on the amino acid sequence of the heavy chain constant region (may be called the H chain) of the antibody. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), for example: IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Moreover, the light chain (may be called L chain) of an antibody is divided into the following types: λ chain and κ chain. The anti-EphA4 antibody according to the present invention may be an IgG antibody, for example, may be an IgG ₁ antibody or an IgG ₂ antibody and the like. Moreover, the anti-EphA4 antibody of the present invention may in some cases be in the form of a monomer, dimer or multimer.

[0059]

The variable region of an antibody according to the present invention may mean an antibody light chain variable region and/or an antibody heavy chain variable region, and an antibody constant region may mean an antibody light chain constant region and/or an antibody heavy chain constant region. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three CDRs, also known as complementarity determining regions. The CDRs on each chain are held nearby by the FR and, together with the CDRs on the other chain, participate in the formation of the antigen-binding site in the antibody. Technologies for determining CDRs include, but are not limited to, (1) an approach based on interspecies sequence variability (eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5 ^th ed., 1991, National Institutes of Health, Bethesda MD); and (2) an approach based on the study of the crystal structure of antigen-antibody complexes (Alazikani et al., 1997 J. Molec. Biol. 273: 927-948). These and other approaches can be used in combination.

[0060]

Monoclonal antibody as used herein may mean an antibody derived from a population of substantially homogeneous antibodies. In other words, the individual antibodies contained in the population are identical, with the exception of naturally occurring mutants, which may possibly be present in small quantities. Monoclonal antibodies target specific antigenic sites and are highly specific. Additionally, unlike a typical polyclonal antibody, which targets different antigens or different epitopes, each monoclonal antibody targets a single epitope of the antigen. The definition of "monoclonal" refers to the property of an antibody obtained from a population of essentially homogeneous antibodies, and should not be construed as limited to the need to obtain antibodies using a particular method.

[0061]

The anti-EphA4 antibody of the present invention may be a murine antibody, a chimeric antibody, or a humanized antibody. A chimeric antibody is, for example, an antibody in which the variable region of a non-human antibody (such as a mouse or rat) is fused to a constant region of a human antibody, and may refer to, for example, an antibody in which the variable region is derived from an antibody non-human, and the constant region is derived from a human antibody. A humanized antibody is, for example, an antibody in which a complementarity determining region (CDR (may also be called a hypervariable region)) of a non-human antibody is introduced into a human antibody, and, for example, may refer to an antibody in which the CDR is derived from a non-human antibody, and the remaining regions of the antibody are derived from a human antibody. It should be noted that the boundary between a chimeric antibody and a humanized antibody does not need to be clear-cut, and the antibody may be in a state that may be referred to as a chimeric antibody or a humanized antibody. Moreover, in a chimeric antibody or humanized antibody, the region of the antibody derived from a human antibody (FR, constant region) need not consist entirely of amino acids derived from a human antibody, but may contain one or more amino acids derived from an antibody other than human, provided that it can be commonly used in a human subject. One embodiment of a humanized antibody is an antibody in which the CDR is derived from a rodent antibody and the remaining regions of the antibody are derived from a human antibody. A specific embodiment of a humanized antibody is an antibody in which the CDRs are derived from a murine antibody and the remaining regions of the antibody are derived from a human antibody. In such embodiments, the CDR may contain one or more amino acids derived from a non-rodent antibody, or one or more amino acids derived from a non-mouse antibody, and regions of the antibody other than the CDRs may contain one or more amino acids derived from a non-human antibody. As used herein, "several" means, without limitation, 2-20 or 2-15, such as 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2, or within 10 %, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2% or within 1% of the number of amino acids in amino acid sequence. Humanization can be carried out using the CDR grafting method (Kontermann and Dubel, Antibody Engineering, Springer Lab Manual (2001) and Tsurushita et al., Methods 36: 69-83 (2005)) and additionally can also be carried out by replacing the CDR sequence with the corresponding sequence in a human antibody using methods well known in the art (see, for example, Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988) and Verhoeyen et al., Science 239: 1534-1536 (1988)).

[0062]

To reduce antigenicity, when producing a humanized antibody, it may be important to choose to use human variable regions in both the light and heavy chains. In a "best match" method, the variable region sequence of a rodent antibody is screened against the entire library of known human FR sequences. The human sequence that is closest to the rodent sequence is then taken as the human FR of the humanized antibody. See, for example, Sims et al., J. Immunol. 151: 2296-2308 (1993) and Chothia et al., J. Mol. Biol. 196:901-917 (1987). Another method uses a specific framework region derived from a sequence common to all human antibodies in a specific light or heavy chain subset. The same framework region can be used for several different humanized antibodies. See, for example, Carter et al., Proc. Natl. Acad. Set USA 89: 4285-4289 (1992) and Presta et al., J. Immunol. 151: 2623-2632 (1993).

[0063]

In addition, it is generally desirable that a humanized antibody retain high antigen binding affinity and other advantageous biological properties. To this end, in one method, humanized antibodies are generated through a parent sequence analysis step and various conceptual humanized products using three-dimensional models of the parent and humanized sequences. Generally, three-dimensional immunoglobulin models are available and are known to those skilled in the art. Computer programs are available that illustrate and present promising three-dimensional conformations of selected candidate immunoglobulin sequences. Investigation of such representations will provide an opportunity to analyze the possible roles of residues in the functions of candidate immunoglobulin sequences, i.e., to analyze residues that affect the ability of candidate immunoglobulins to bind their antigens. Using this method, FR residues can be selected from recipient and imported sequences and used in combination to achieve desired antibody properties, such as increasing binding affinity to one or more target antigens (such as EphA4 or a fragment thereof).

[0064]

It is clear that the antibody to EphA4 in accordance with the present invention also covers an antibody with a corresponding change (such as a modification of the antibody or a partial substitution, addition and/or deletion in the amino acid sequence of the antibody) in the chimeric or humanized antibody illustrated above while maintaining the function of the antibody (or with the addition or improvement of antibody function). More specifically, the present invention also includes an antibody having a change in the amino acid sequence of the constant region for the purpose of modifying the effector function of the antibody. For example, also included within the scope of the present invention is an antibody containing valine (Val) at position 234 (Eu numbering) of a human IgG ₂ antibody substituted for alanine (Ala), and containing glycine (Gly) at position 237 substituted for alanine (Ala) , in order to reduce activity like antibody-dependent cell-mediated cytotoxicity (ADCC) and/or activity like antibody-dependent cellular phagocytosis (ADCP), etc. Also included within the scope of the present invention is a bispecific antibody that has an antigen binding region with the CDR sequence of an anti-EphA4 antibody according to the present invention, together with an antigen binding region that binds another antigen (Kontermann (2012), mAbs 4, 182-97 ).

[0065]

If necessary, the antibody to EphA4 in accordance with the present invention can be modified. The modification of the anti-EphA4 antibody may be a modification that changes (a) the three-dimensional structure of the amino acid sequence in the region to be modified, such as sheet or helix conformation, etc.; (b) the charge or hydrophobic state of the molecule at the target site; or (c) the effect of modification in maintaining side chain volume, or may be a modification in which these changes are not clearly defined.

[0066]

Modification of the anti-EphA4 antibody according to the present invention can be accomplished, for example, by substitution, deletion, addition, or the like. constituent amino acid residues.

[0067]

Amino acid is used herein in its broadest sense and includes not only naturally occurring amino acids such as serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr), cysteine (Cys) , methionine (Met), phenylalanine (Phe), tryptophan (Trp) and proline (Pro), but also unnatural amino acids such as amino acid variants and derivatives. Those skilled in the art will naturally appreciate, given this broad definition, that examples of amino acids as used herein include, for example, L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and amino acid derivatives; amino acids that will not be protein components in the body, such as norleucine, β-alanine and ornithine; and chemically synthesized compounds having amino acid properties well known to those skilled in the art, and the like. Examples of a non-naturally occurring amino acid may include, for example, α-methylamino acids (such as α-methylalanine), D-amino acids (such as D-aspartic acid and D-glutamic acid), histidine-like amino acids (such as 2-aminohistidine, β- hydroxyhistidine, homohistidine, α-fluoromethylhistidine and α-methylhistidine), amino acids with an excess of methylene groups on the side chain ("homo"-amino acids), and amino acids in which the carboxylate functional group on the amino acid side chain is replaced by a sulfonate group (such as cysteic acid).

[0068]

Naturally occurring amino acid residues can be divided into the following groups based on general side chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Asn, Gln, Cys, Ser, Thr;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro; And

(6) aromatic: Trp, Tyr, Phe.

[0069]

A non-conservative replacement of an amino acid sequence constituting an antibody can be accomplished by replacing an amino acid belonging to one of these groups with an amino acid belonging to another group. A more conservative substitution can be made by replacing an amino acid belonging to one of these groups with another amino acid in the same group. Likewise, a deletion or substitution in an amino acid sequence can be made accordingly.

[0070]

Modification of the amino acids that give shape to the antibody may be, for example, carbohydrate glycosylation or post-translational modifications such as acetylation or phosphorylation. The antibody may be glycosylated at a conserved position in its constant region. Antibody glycosylation is typically either N-linked or O-linked. N-linked means the binding of the carbohydrate moiety to the side chain of the aspartic residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine (where X is any amino acid other than proline) are recognition sequences for the enzymatic addition of a carbohydrate moiety to the asparagine side chain. If one of these tripeptide sequences is present in an antibody, then a potential glycosylation site is present. O-linked glycosylation may be the binding of N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid (such as serine or threonine), and in some cases may be the binding of 5-hydroxyproline or 5-hydroxylysine. Those skilled in the art will be able to appropriately select the glycosylation conditions (such as the type of host cell or cellular environment and pH, etc., if glycosylation is carried out by biological means) according to the desired purpose.

[0071]

The anti-EphA4 antibody of the present invention may be further modified by other modification methods, alone or in combination, based on general technical knowledge well known to those skilled in the art.

[0072]

Antibody to EphA4 in accordance with the present invention can be obtained using a method well known to specialists in this field. For example, an antibody can be produced by inserting a nucleic acid encoding an anti-EphA4 antibody according to the present invention into an expression vector, introducing the expression vector into a host cell, and culturing the host cell. Accordingly, the present invention covers a nucleic acid encoding an anti-EphA4 antibody, a vector that contains the nucleic acid, a host cell containing the vector, and a method for producing an anti-EphA4 antibody, comprising the step of culturing the host cell.

[0073]

The nucleic acid encoding an anti-EphA4 antibody according to the present invention may contain DNA encoding a signal sequence, or may contain DNA encoding a signal sequence at the 5' end of the DNA encoding a heavy chain variable region and the DNA encoding a light chain variable region . A signal sequence is an amino acid residue present at the N-terminus of a protein that is required for a secretory protein or integral membrane protein to pass through a lipid bilayer after synthesis on a ribosome, and is not particularly limited in the present invention provided that it is a sequence that has this function. Signal sequences that may be contained in the anti-EphA4 antibody of the present invention include those derived from human, mouse, rat, rabbit, donkey, goat, horse, bird, dog, cat, yeast, etc. Specifically, in the present invention, a peptide containing the amino acid sequence represented by SEQ ID NO: 12 or 16 may be included as a heavy chain signal sequence, and a peptide containing the amino acid sequence represented by SEQ ID NO: 14 or 18 may be included as a signal sequence related to the light chain. Moreover, provided that it is functionally equivalent, the signal sequence may be characterized by the substitution, addition and/or deletion of one or more (such as 2, 3, 4 or 5) amino acids in the amino acid sequence represented by SEQ ID NO: 12 or 16, and the amino acid sequence shown under SEQ ID NO: 14 or 18.

[0074]

Antibody to EphA4 in accordance with the present invention can be isolated or purified in accordance with a method well known to specialists in this field.

[0075]

As used herein, “isolated” or “purified” means artificially isolated or purified from its natural state. If a molecule or composition occurs naturally, it is "isolated" or "purified" if it has been altered or removed from its original environment, or both. Examples of the isolation or purification method include, but are not limited to, methods by electrophoresis, molecular biology, immunology or chromatography, etc., in particular, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase HPLC chromatography, isoelectric focusing or alkaline extraction method, etc. .

[0076]

In one embodiment, the anti-EphA4 antibody contains the following CDRs:

(a) a heavy chain CDR1 consisting of the amino acid sequence shown under SEQ ID NO: 44;

(b) a heavy chain CDR2 consisting of the amino acid sequence shown under SEQ ID NO: 27;

(c) a heavy chain CDR3 consisting of the amino acid sequence shown under SEQ ID NO: 28;

(d) a light chain CDR1 consisting of the amino acid sequence shown under SEQ ID NO: 29;

(e) a light chain CDR2 consisting of the amino acid sequence shown under SEQ ID NO: 30; And

(f) a light chain CDR3 consisting of the amino acid sequence shown under SEQ ID NO: 31.

[0077]

In one embodiment, the anti-EphA4 antibody is a humanized antibody or a chimeric antibody, and in a particular embodiment, a humanized antibody.

[0078]

In another embodiment, an anti-EphA4 antibody comprises a heavy and a light chain, wherein the heavy chain variable region comprises the amino acid sequence shown under SEQ ID NO: 45 and the light chain variable region contains the amino acid sequence shown under SEQ ID NO: 46. Note Attention is drawn to the fact that in an embodiment, the heavy chain variable region and/or the light chain variable region may comprise an amino acid sequence in which one or more amino acids are substituted, added and/or deleted in the amino acid sequence shown under SEQ ID NO: 45, and/or the amino acid sequence shown under SEQ ID NO: 46. As used herein, “several” is not limited so long as binding affinity for EphA4 is maintained and cleavage of EphA4 is enhanced, and means 2-15 or 2-10, such as, 9, 8, 7, 6, 5, 4, 3 or 2, or within 10%, such as within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2% or within 1% of the number of amino acids in the amino acid sequence.

[0079]

In one embodiment, the anti-EphA4 antibody heavy chain comprises a human IgG ₂ constant region.

[0080]

In a specific embodiment, the human IgG ₂ constant region comprises the amino acid sequence of SEQ ID NO: 47.

[0081]

In one embodiment, the anti-EphA4 antibody light chain comprises a human Igκ constant region.

[0082]

In a specific embodiment, the human Ig κ chain constant region comprises the amino acid sequence of SEQ ID NO: 48.

[0083]

In one embodiment, an anti-EphA4 antibody comprises a heavy chain comprising the amino acid sequence shown under SEQ ID NO: 59 and a light chain containing the amino acid sequence shown under SEQ ID NO: 60.

[0084]

In another embodiment, for example, for reasons such as decreased uniformity of antibodies produced by antibody-producing cells (US Patent Application Publication No. 2010/0297697 or Liu H et al., MAbs. 2014 Sep-Oct; 6(5): 1145-1154 ), the anti-EphA4 antibody has the lysine located at the C-terminus (carboxy-terminus) of the heavy chain removed. In the present invention, an anti-EphA4 antibody with the C-terminal heavy chain lysine deleted also includes an anti-EphA4 antibody with the C-terminal heavy chain lysine deleted, removed by genetic modification, or an anti-EphA4 antibody with the C-terminal heavy chain lysine deleted, post-translationally cleaved by carboxypeptidase etc. Moreover, in the present invention, the anti-EphA4 antibody with the C-terminal lysine of the heavy chain deleted includes not only the anti-EphA4 antibody with the C-terminal lysine deleted in both heavy chains, but also the anti-EphA4 antibody with the C-terminal lysine deleted in only one heavy chain.

[0085]

In one aspect, the present invention provides an isolated nucleic acid encoding an anti-EphA4 antibody. An isolated anti-EphA4 antibody-encoding nucleic acid refers to one or more nucleic acid molecules encoding the heavy chain and/or light chain of an anti-EphA4 antibody. In one embodiment, the nucleic acid in accordance with the present invention encodes the heavy chain of an anti-EphA4 antibody. In another embodiment, the nucleic acid of the present invention encodes an anti-EphA4 antibody light chain. In another further embodiment, the nucleic acid of the present invention encodes the heavy and light chains of an anti-EphA4 antibody. The nucleic acid of the present invention also includes a first nucleic acid molecule encoding an anti-EphA4 antibody heavy chain and a second nucleic acid molecule encoding an anti-EphA4 antibody light chain.

[0086]

In another aspect, the present invention provides a vector containing an isolated nucleic acid encoding an anti-EphA4 antibody. A vector in accordance with the present invention refers to one or more vectors containing an isolated nucleic acid encoding an antibody to EphA4. In one embodiment, a vector in accordance with the present invention is a vector comprising a nucleic acid encoding an anti-EphA4 antibody heavy chain and a nucleic acid encoding an anti-EphA4 antibody light chain. In another embodiment, the vector in accordance with the present invention is a vector containing a nucleic acid encoding the heavy and light chains of an anti-EphA4 antibody. In another further embodiment, a vector in accordance with the present invention comprises a first vector comprising a nucleic acid encoding an anti-EphA4 antibody heavy chain and a second vector containing a nucleic acid encoding an anti-EphA4 antibody light chain. The vector of the present invention may be, without limitation, a plasmid, cosmid, virus, phage, or the like. For example, the vector of the present invention also includes a viral vector, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a herpes simplex virus vector and the like.

[0087]

In yet another aspect, the present invention also includes a host cell containing a vector in accordance with the present invention, and a method for producing an anti-EphA4 antibody, comprising the step of culturing the host cell. The host cell of the present invention may be, but is not limited to, E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, NS0 cells, and the like. In one embodiment, a method for producing an anti-EphA4 antibody comprises the step of culturing a host cell and the step of isolating an anti-EphA4 antibody secreted from the host cell (or the culture medium of the host cell).

[0088]

In one aspect, the present invention provides a pharmaceutical composition comprising an anti-EphA4 antibody. The pharmaceutical composition according to the present invention can be manufactured in accordance with known methods, such as those described in the Japanese Pharmacopoeia (JP), the United States Pharmacopoeia (USP), or the European Pharmacopoeia (EP), and the like.

[0089]

The anti-EphA4 antibody of the present invention may be useful for the treatment of Alzheimer's disease. In other words, in other aspects, the present invention provides a method of treating Alzheimer's disease, comprising the step of administering a therapeutically effective amount of an anti-EphA4 antibody to a subject having Alzheimer's disease. Moreover, in other aspects, the present invention covers the use of an anti-EphA4 antibody for the manufacture of a therapeutic drug for Alzheimer's disease. In other aspects, the present invention provides an anti-EphA4 antibody for use in the treatment of Alzheimer's disease.

[0090]

The anti-EphA4 antibody of the present invention may be useful for the treatment of tauopathy. In other words, in other aspects, the present invention provides a method of treating tauopathy, comprising the step of administering a therapeutically effective amount of an anti-EphA4 antibody to a subject suffering from the tauopathy. Moreover, in other aspects, the present invention covers the use of an anti-EphA4 antibody for the manufacture of a therapeutic drug for tauopathy. In other aspects, the present invention provides an anti-EphA4 antibody for use in the treatment of tauopathy. The tauopathy of the present invention includes Alzheimer's disease or frontotemporal lobar degeneration with tau pathology (FTLD-tau). Moreover, frontotemporal lobar degeneration with tau pathology includes progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic dementia (AGD), senile dementia of the neurofibrillary tangle type (SD-NFT), Pick's disease (PiD), etc.

[0091]

The anti-EphA4 antibody of the present invention can be used alone in a therapeutic method or in combination with other agents or compositions. For example, the anti-EphA4 antibody of the present invention may be administered at the same time or at different time points as another agent. Such combination therapy involves combined administration (two or more agents included in the same or separate formulation) and separate administration (such as simultaneous or sequential). When two or more agents are administered separately, administration of the anti-EphA4 antibody of the present invention may be administered before or after a concomitant therapeutic modality.

[0092]

The subject matter for administering the pharmaceutical composition according to the present invention is not limited, and can be used, for example, in relation to a human or a non-human mammal (such as a monkey, mouse, rat, rabbit, cow, horse and goat).

[0093]

The method of administering the pharmaceutical composition according to the present invention to a subject (such as route of administration, dosage, number of administrations per day and schedule of administration) is not limited and can be appropriately determined by those skilled in the art (such as a physician) depending on the condition the health of the subject, the extent of the disease, the type of agents used in combination, etc.

[0094]

Those skilled in the art will understand that, so long as it is not technically inconsistent, the present invention may be practiced in suitable combination with any one or more of all possible aspects described herein. In addition, those skilled in the art will understand that, so long as it is not technically inconsistent, it is preferred that the present invention be practiced in an appropriate combination of all possible preferred or advantageous aspects described herein.

[0095]

All literature disclosures cited herein are to be considered as expressly cited herein by reference, and those skilled in the art will be able to understand the relevant content of the disclosures in those literatures by reference to them as part of this specification in accordance with the context of this document without deviations from the essence and scope of the present invention.

[0096]

The literature cited herein is presented solely for the purpose of disclosing the state of the relevant technology prior to the filing date of this application, and should not be construed as an admission by the inventors of the present invention that the present invention is not entitled to be considered prior to said disclosures as an earlier invention or for any other reason. All descriptions of all these literature sources are based on information available to the authors of this application and are in no way an admission that the contents of these descriptions are accurate.

[0097]

The terms used herein are used to describe specific embodiments and are not intended to limit the present invention.

[0098]

The term "comprise" as used herein, unless the content is expressly implied otherwise, means the presence of the described objects (such as components, stages, elements or numbers) and does not exclude the presence of other objects (such as components, stages, elements and numbers). The term "consist of" covers aspects described by the terms "consist of" and/or "actually consist of".

[0099]

As used herein, the term “neutralizing activity” means the activity of inhibiting the binding between EphA4 and its ligand and/or the activity of inhibiting signal transduction or molecular expression response or changing the functional properties of cells that are induced by EphA4 due to binding to its ligand in the human body.

[0100]

Unless otherwise specified, all terms used herein (including technical and scientific terms) have the same meanings as are commonly known to those skilled in the art to which this invention relates. Terms used herein, unless expressly stated otherwise, should be understood to have the meanings consistent with their meanings herein and in the relevant fields of art, and should not be construed as having simplistic or overly formal meanings.

[0101]

Terms such as first and second are used to refer to various elements, and it should be understood that these elements should not be limited to these terms. These terms are used solely for the purpose of distinguishing between one element and another, and, for example, one may describe a first element as a second element and likewise describe a second element as a first element without departing from the scope of the present invention.

[0102]

Numerical values used herein to indicate the content of a component or a range of numerical values, etc., unless expressly indicated, are to be understood as modified by the term “about.” For example, unless expressly stated otherwise, "4°C" should be understood to mean "approximately 4°C", and it will be understood by those skilled in the art to reasonably understand its scope in accordance with technical common sense and the meaning of the parts presented herein text.

[0103]

It should be understood that, unless the context clearly indicates otherwise, when used herein in the specification and claims, each aspect presented in the singular form may also appear in the plural form unless technically inconsistent and vice versa. .

[0104]

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention can be practiced in various aspects and should not be construed as limited to the examples described herein. Those skilled in the art will be able to implement the present invention with various modifications, additions, deletions, substitutions, and the like. without changing the spirit or scope of the present invention.

Examples

[0105]

Comparative Example 1: Preparation of Anti-EphA4 Monoclonal Antibody

(A) Generation of mouse monoclonal antibody to EphA4

To generate a monoclonal antibody that binds to mouse EphA4 (Genbank accession no. NP_031962.2, SEQ ID NO: 1), a secretory alkaline phosphatase (SEAP) and histidine tag-containing protein fused to the extracellular region of mouse EphA4 (positions 20-547) (SEQ ID NO: 2) (hereinafter referred to as “mouse extracellular region protein-EphA4-SEAP-His”, SEQ ID NO: 3) was prepared using the following steps.

[0106]

First, DNA sequences encoding the signal sequence (SEQ ID NO: 4) and extracellular region (SEQ ID NO: 2) of mouse EphA4 were amplified by RT-PCR from total RNA obtained from mouse brain and cloned into the Sal I/ site. Not I vector pENTR1A (Invitrogen/LifeTechnologies) having a DNA sequence encoding SEAP and a histidine tag. The DNA sequence encoding the signal sequence and extracellular region of mouse EphA4, SEAP and histidine tag was then transferred into the vector pcDNA 3.1_rfcB by LR reaction using the Gateway system (Invitrogen/LifeTechnologies) to construct the expression vector pcDNA 3.1-extracellular region-EphA4- SEAP-His mice. The constructed mouse pcDNA 3.1-extracellular region-EphA4-SEAP-His expression vector was transfected into HEK293 EBNA cells (Invitrogen/LifeTechnologies) using TransIT-LT1 (TAKARA). After 6 days of incubation (5% CO ₂ , 37°C), the culture supernatant was isolated. From the isolated culture supernatant, mouse extracellular region protein-EphA4-SEAP-His (SEQ ID NO: 3) was purified using a Protino column (MACHEREY-NAGEL).

[0107]

Twenty micrograms of mouse extracellular region protein-EphA4-SEAP-His was mixed with the same amount of TiterMax Gold adjuvant (TiterMax, USA) or GERBU adjuvant (GERBU Biotechnik GmbH) and injected subcutaneously into the footpad of Balb/c mice. Then, mouse extracellular region protein-EphA4-SEAP-His was administered in a similar manner on days 3, 7 and 10. In this case, TiterMax Gold adjuvant (TiterMax, USA) was used only on day 10, and GERBU adjuvant (GERBU Biotechnik GmbH) was used on days 3, 7 and 10. Mice were sacrificed on day 13 and peripheral lymph nodes were isolated to obtain lymph node cells. The resulting lymph node cells and P3U1 myeloma cells (provided by Kyoto University) were fused at a ratio of 5:1 in the presence of GenomeONE-CF (Ishihara Sangyo Kaisha, Ltd.). Confluent cells were cultured in a 96-well plastic plate. After 7 days of incubation (5% CO ₂ , 37°C), the culture supernatant was isolated.

[0108]

Using the resulting culture supernatant, wells that were reactive to mouse, rat, and human EphA4 were selected.

[0109]

Reactivity to mouse, rat and human EphA4 was assessed by ELISA using proteins having the Fc region of human IgG ₁ and a histidine tag fused to the extracellular region of mouse EphA4, the extracellular region (positions 20-547) of rat EphA4 (GenBank, no. NP_001155883.1) or the extracellular region (positions 20-547) (SEQ ID NO: 6) of human EphA4 (Genbank accession no. NP_004429.1, SEQ ID NO: 5) (hereinafter referred to as “extracellular region protein-EphA4 -Fc-His mouse", "extracellular region protein-EphA4-Fc-His" or "extracellular region protein-EphA4-Fc-His" respectively).

[0110]

Mouse, rat or human extracellular region-EphA4-Fc-His proteins were prepared using the following steps. Mouse, rat or human extracellular region-EphA4-Fc-His pcDNA 3.1 expression vectors were initially constructed. First, DNA sequences encoding the signal sequence and extracellular region of mouse, rat, or human EphA4 were amplified by RT-PCR from total RNA obtained from mouse, rat, or human brain and cloned into the Sal I/Not I site of the pENTR1A vector (Invitrogen/ LifeTechnologies), containing a DNA sequence encoding an Fc and a histidine tag. DNA sequences encoding the signal sequence and extracellular region of mouse, rat or human EphA4, Fc and histidine tag were then transferred into the pcDNA 3.1_rfcB vector by LR reaction using the Gateway system (Invitrogen/LifeTechnologies) to construct pcDNA 3.1-extracellular expression vectors region-EphA4-Fc-His of mouse, rat or human. These constructed expression vectors were transfected into HEK293 EBNA cells (Invitrogen/LifeTechnologies) using TransIT-LT1 (TAKARA). After 6 days of incubation (5% CO ₂ , 37°C), the culture supernatant was isolated.

[0111]

ELISA using mouse, rat or human extracellular region proteins-EphA4-Fc-His was performed according to the steps below. Anti-human IgG antibody (Jackson ImmunoResearch Laboratories) was coated into the wells of a 96-well plate (Nunc). After incubation at 4°C overnight, wells were blocked at room temperature for one hour using 1 x Block ACE (Dainippon Seiyaku). After washing three times with 0.02% Tween 20/PBS (Nacalai Tesque), culture supernatant containing mouse, rat, or human extracellular region protein-EphA4-Fc-His (final concentration 1 nM) was added to each well and incubated at room temperature for one hour. After washing three times, the supernatant of the confluent cell culture was added to each well. After incubation at room temperature for one hour and washing three times, horseradish peroxidase-labeled anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was added and the mixture was incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to each well and incubated at room temperature for 5-20 minutes. An equal amount of stop solution (2N H ₂ SO ₄ , Wako Pure Chemical) was added to each well and the absorbance was read at 450 nm using a microplate reader (PerkinElmer).

[0112]

Hybridomas collected at the above steps were cloned from the wells using a limited dilution method, and hybridoma clones were ultimately obtained expressing mouse anti-EphA4 antibodies having binding activity to mouse, rat or human EphA4.

[0113]

The resulting hybridoma clones were cultured and mouse monoclonal antibody to EphA4 was purified from the culture supernatant using protein A (GE Healthcare).

[0114]

(B) Assessment of EphA4 cleavage enhancing activity

The preparation of rat hippocampal neurons was performed according to the steps below. Fetuses were removed from rats on day 18 of pregnancy (Charles River Laboratories, Japan) and their heads were cut to remove the brain. An area of the hippocampus was dissected under a stereomicroscope and placed in a digestion solution (137 mM NaCl (Wako Pure Chemical), 5 mM KCl (Wako Pure Chemical), 7 mM _{Na2HPO4 (} Wako Pure Chemical), 25 mM Hepes (DOJINDO), 0 .5 mg/ml DNase (Sigma) and 0.25% trypsin (Life technologies) and shaken at 37°C for 10 minutes. The solution was removed and 20% fetal bovine serum/Hank's buffer (Sigma) was added. After removing the solution and washing twice in Hanks buffer, the hippocampal tissue was pipetted in Hanks buffer to obtain a cell suspension. Cells were seeded in a 96-well plate (Falcon) coated with a culture fluid containing poly-L-lysine (neurobasal medium (Life technologies), 1 x B-27 supplement (Life technologies) and 0.5 mM L-glutamine (Life technologies )).

[0115]

EphA4 cleavage enhancing activity was assessed using hippocampal neurons according to the steps below. Rat hippocampal neurons seeded in a 96-well plate (Falcon) were treated with anti-EphA4 monoclonal antibody (67 nM) and the γ-selectase inhibitory drug Compound E (50 nM, Enzo Life Sciences). After sixteen hours, it was washed with PBS (Wako Pure Chemical), added with SDS sample buffer (Laemmli sample buffer (Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) to extract cells and boiled for 5 minutes. SDS-PAGE was performed on this sample, Western blotting was performed with an anti-EphA4 monoclonal antibody (Abnova), the band concentration was quantified, and the EphA4 C-terminal fragment/full-length EphA4 value was calculated.

[0116]

A mouse monoclonal antibody to EphA4 was generated that has activity that enhances the cleavage of EphA4 (antibody A). Antibody A isotype was determined using a monoclonal antibody isotyping kit (Serotec) and was IgG ₁ for the heavy chain and κ for the light chain.

[0117]

(C) Sequence analysis of antibody A

The DNA sequence encoding the signal sequence and variable regions of the heavy and light chains of Antibody A was amplified using the 5'-RACE (rapid amplification of 5' cDNA ends) method. Total RNA was prepared from the hybridoma using the RNeasy kit (QIAGEN) and treated with DNase (QIAGEN, RNase-Free DNase Kit). Double-stranded cDNA was prepared from total RNA using a cDNA synthesis kit (TAKARA). A 5' adapter obtained by hybridizing an ad29S DNA oligonucleotide (ACATCACTCCGT) (SEQ ID NO: 7) and an ad29AS DNA oligonucleotide (ACGGAGTGATGTCCGTCGACGTATCTCTGCGTTGATACTTCAGCGTAGCT) (SEQ ID NO: 8) was added to the cDNA. The resulting cDNA was amplified with a forward 5' primer (primer 5'-PCR4, AGCTACGCTGAAGTATCAACGCAGAG) (SEQ ID NO: 9), and a reverse 3' primer (GCCAGTGGATAGACTGATGG (SEQ ID NO: 10) was used to amplify the mouse IgG heavy chain gene and GATGGATACAGTTGGTGCAGC (SEQ ID NO: 11) was used to amplify the murine Ig κ light chain). The amplified cDNA was inserted into the pCR2.1 vector (Invitrogen/LifeTechnologies). The antibody A gene sequence was analyzed using an ABI 3130XL. With respect to the amino acid sequences encoded by the antibody A gene sequence identified by the present analysis, the heavy chain signal sequence was the sequence shown under SEQ ID NO: 12, the heavy chain variable region was the sequence shown under SEQ ID NO: 13, the light chain signal sequence was the sequence shown under SEQ ID NO: 14, and the light chain variable region was the sequence shown under SEQ ID NO: 15. As for the nucleotide sequences encoding the antibody A gene sequence, the heavy chain signal sequence was is the sequence shown at SEQ ID NO: 16, the heavy chain variable region is the sequence shown at SEQ ID NO: 17, the light chain signal sequence is the sequence shown at SEQ ID NO: 18, and the light chain variable region is the sequence shown under SEQ ID NO: 19.

[0118]

The full-length sequences of the heavy and light chains of Antibody A were obtained using the following steps. Total RNA was prepared from the hybridoma using the RNeasy kit (QIAGEN) and treated with DNase (QIAGEN, RNase-Free DNase Kit). Reverse transcription products were obtained from total RNA using an RNA PCR kit (TAKARA). Using the reverse transcription products obtained from the templates, the gene sequence encoding the heavy and light chains of Antibody A was amplified by PCR with a 5' forward primer (GCGAAGCTTGCCGCCACCATGGCTGTCCTGGTGCTGCTCC (primer ID 7455) (SEQ ID NO: 20), which was used to amplify the heavy chain, and GCGAAGCTTGCCGCCACCATGGACATGAGGGTTCCTGCTCACG (primer ID 7453) (SEQ ID NO: 21), which was used for light chain amplification) and a 3' reverse primer (GCGGAATTCATCATTTACCAGGAGAGTGGGAGAGGC (primer ID 7257) (SEQ ID NO: 22), which was used for heavy chain amplification chain, and CGCGAATTCACTAACACTCATTCCTGTTGAAGCTCTTGAC (primer ID 7249) (SEQ ID NO: 23), which was used for light chain amplification), and were respectively cloned into vectors pEE6.4 and pEE12.4 (Lonza). The gene sequence was analyzed using ABI3130XL. With respect to the amino acid sequences encoded by the antibody A gene sequence identified by the present analysis, the heavy chain constant region was the sequence shown under SEQ ID NO: 24, and the light chain constant region was the sequence shown under SEQ ID NO: 25.

[0119]

The CDR of Antibody A was determined by the following method. The amino acid sequence of Antibody A was numbered according to the Kabat numbering system using Abysis software (UCL). Based on this numbering, a decision was made in accordance with the Kabat definition for identifying CDRs. The amino acid sequences of the Antibody A CDR are shown in Table 1.

[Table 1] Amino acid sequences of the CDR of antibody A Name Subsequence Heavy chain CDR1 RYGVH (SEQ ID NO: 26) Heavy chain CDR2 VIWRGGSTDYNAAFMS (SEQ ID NO: 27) Heavy chain CDR3 ESLFGVYYDYGYYSMDY (SEQ ID NO: 28) Light chain CDR1 RASQEISGYLS (SEQ ID NO: 29) Light chain CDR2 AASTLDS (SEQ ID NO: 30) Light chain CDR3 LQYASYPLT (SEQ ID NO: 31)

[0120]

Comparative Example 2: Binding Affinity of Anti-EphA4 Monoclonal Antibody to Mouse and Human EphA4

The binding affinity of Antibody A for mouse and human EphA4 was determined by surface plasmon resonance (SPR method) using a Biacore T200 (GE Healthcare). First, anti-His antibody (GE Healthcare, 28-9950-56) was fixed to the CM5 sensor chip. Fixation was accomplished by the amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and ethanolamine was used for blocking (sensor chip and all fixation reagents were purchased from GE Healthcare ). It was diluted to 3.5 μg/ml with fixation buffer (10 mM sodium acetate, pH 4.5) and fixed to the sensor chip according to the protocol included with the Biacore T200. Mouse or human extracellular region protein-EphA4-SEAP-His10 was diluted in HBS-EP running buffer (GE Healthcare, BR-1001-88) and the solution was introduced into the flow cell for 120 seconds for capture (capture amount was approximately 10 RU). Antibody A, serially diluted to a range of 100, 50, 25, 12.5, 6.3, 3.2, 1.6 and 0 nM with HBS-EP, was then added to the sensor chip for 120 seconds and the curve was subsequently monitored binding reactions from the moment of addition (binding phase, 120 seconds) until the moment after completion of addition (dissociation phase, 600 seconds). After completion of each tracking, 4 M MgCl ₂ (60 seconds, Wako Pure Chemical) was added to regenerate the sensor chip. The resulting binding reaction curve was fit to a 1:1 binding model using the BIA evaluation software supplied with the system, and the binding affinity (KD=kd/ka) for mouse and human EphA4 was calculated.

[0121]

The binding affinities of Antibody A for mouse and human EphA4 (KD value) were 1.32 x 10 ^-9 M and 1.19 x 10 ^-9 M, respectively (Figure 1). Other binding parameters with respect to mouse and human EphA4 were almost to the same extent. Therefore, it was hypothesized that Antibody A had the same degree of binding affinity for mouse and human EphA4.

[0122]

Comparative Example 3 EphA4 Cleavage Enhancing Activity of Anti-EphA4 Monoclonal Antibody in Hippocampal Neurons

For Antibody A, EphA4 cleavage enhancing activity was assessed using hippocampal neurons according to the steps below. Rat hippocampal neurons seeded in a 96-well plate (Falcon) were treated with Antibody A (2.0, 6.7 and 20 nM) and the γ-selectase inhibitory drug Compound E (50 nM, Enzo Life Sciences) . After twenty-four hours, it was washed with PBS (Wako Pure Chemical), sample buffer with SDS (Laemmli sample buffer (Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) was added to extract cells, and boiled for 5 minutes. SDS-PAGE was performed on this sample, Western blotting was performed with an anti-EphA4 monoclonal antibody (Abnova), the band concentration was quantified, and the EphA4 C-terminal fragment/full-length EphA4 value was calculated.

[0123]

Antibody A enhanced the EphA4 cleavage response in hippocampal neurons in a concentration-dependent manner (Figure 2).

[0124]

Comparative Example 4: Inhibitory Activity of Anti-EphA4 Monoclonal Antibody on Mouse Ligand-Binding Mouse EphA4

For Antibody A, evaluation of binding inhibitory activity between mouse EphA4 and mouse ligand was performed according to the steps below. Alkaline phosphatase antibody (Thermo SCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.02% Tween 20/PBS (Thermo SCIENTIFIC), mouse extracellular region protein-SEAP-His EphA4 (final concentration 10 nM) was added to the wells and incubated as indicated at room temperature for one hour. After washing three times, ligand and antibody A (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 nM) were added to each well. It should be noted that biotinylated mouse Ephrin A1-Fc chimera (R&D Systems, final concentration 6 nM) and biotinylated mouse Ephrin B2-Fc chimera (R&D Systems, final concentration 2.5 nM) were used as ligands. After incubation at room temperature for one hour and washing three times, horseradish peroxidase-labeled streptavidin (GE Healthcare) was added and the mixture was incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells and incubated at room temperature for 2 minutes. An equal amount of stop solution (1N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells and the absorbance was read at 450 nm using a microplate reader (PerkinElmer).

[0125]

Antibody A inhibited binding between mouse EphA4 and mouse ligand in a concentration-dependent manner, and the IC ₅₀ values for binding of mouse Ephrin A1 and Ephrin B2 were approximately 5.9 nM and 3.1 nM, respectively (Figure 3). Accordingly, Antibody A was shown to potently inhibit the binding between mouse EphA4 and the mouse ligand.

[0126]

Comparative Example 5: Inhibitory Activity of Anti-EphA4 Monoclonal Antibody on Human EphA4 Ligand-Binding

For Antibody A, evaluation of binding inhibitory activity between human EphA4 and human ligand was performed according to the steps below. Alkaline phosphatase antibody (Thermo SCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), human EphA4 extracellular region protein-SEAP-His (final concentration 10 nM) was added to the wells and incubated at room temperature for one hour. After washing three times, ligand and serially diluted Antibody A (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 and 3000 nM) were added to each well ). It should be noted that biotinylated human Ephrin A5-Fc chimera (R&D Systems, final concentration 0.7 nM) and biotinylated human Ephrin B3-Fc chimera (R&D Systems, final concentration 2.3 nM) were used as ligands. After incubation at room temperature for one hour and washing three times, horseradish peroxidase-labeled streptavidin (GE Healthcare) was added and the mixture was incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells and incubated at room temperature for 2-5 minutes. An equal amount of stop solution (1N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells and the absorbance was read at 450 nm using a microplate reader (Molecular Devices or PerkinElmer).

[0127]

Antibody A inhibited binding between human EphA4 and human ligand in a concentration-dependent manner, and the IC ₅₀ values for human Ephrin A5 and Ephrin B3 binding were approximately 2.8 nM and 1.4 nM, respectively (Figure 4). Accordingly, Antibody A was shown to potently inhibit the binding between human EphA4 and the human ligand.

[0128]

Comparative Example 6: Selectivity of EphA4 Monoclonal Antibody for Human Eph Receptor

According to the method for producing the extracellular region-SEAP-His protein of mouse EphA4 described in Comparative Example 1, the DNA sequences encoding the signal sequence and the extracellular region of each human Eph receptor (EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10 , EphB1, EphB2, EphB3, EphB4, and EphB6) were amplified by RT-PCR from tissue-derived total RNA and cloned into the pENTR1A vector (Invitrogen/LifeTechnologies) having a DNA sequence encoding SEAP and a histidine tag. DNA sequences encoding the signal sequence and extracellular region of each human Eph receptor, SEAP, and histidine tag were then transferred into the pcDNA 3.1_rfcB vector by an LR reaction using the Gateway System (Invitrogen/Life Technologies) to construct vectors (referred to as “protein expression vector” Eph receptor extracellular region-SEAP-His") expressing a protein having a SEAP and a His tag fused to the extracellular region of each human Eph receptor (referred to as "Eph receptor extracellular region-SEAP-His protein").

[0129]

Each human Eph receptor extracellular region-SEAP-His protein expression vector was then introduced into Expi293F cells (Gibco/ThermoFisher) with the Expi293 expression system (Gibco/ThermoFisher). After 5 days of culture (5% CO ₂ , 37°C, 120 rpm), the culture supernatant was collected and centrifuged at room temperature at 1500 rpm for 5 minutes. The centrifuged supernatant was filtered through a 0.45 μm pore size filter (Millipore).

[0130]

For Antibody A, human Eph receptor binding activity was assessed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with rabbit anti-6-His antibody (Bethyl Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), each human Eph receptor extracellular region-SEAP-His protein (at a final concentration of 1 nM) was seeded into each well and incubated at room temperature for one hour. After washing three times, a solution of human IgG (100 μg/ml, Mitsubishi Pharma Corporation) and antibody A (10 μg/ml) was added to the wells and incubated at room temperature for one hour. Horseradish peroxidase-labeled donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was added to this and incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells, and after moderate staining was confirmed, an equal amount of stop solution (1 N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells ) and absorbance at 450 nm were read using a microplate reader (PerkinElmer).

[0131]

Among the human Eph receptor family, Antibody A had specific binding activity only to human EphA4 (Figure 5).

[0132]

Comparative Example 7: Selectivity of EphA4 Monoclonal Antibody for Mouse Eph Receptor

According to the method for producing the extracellular region protein-Fc-His EphA4 in accordance with Comparative Example 1, the DNA sequences encoding the signal sequence and the extracellular region of each mouse Eph receptor (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2 , EphB3, EphB4, and EphB6) were amplified by RT-PCR from tissue-derived total RNA and cloned into the pENTR1A vector (Invitrogen/LifeTechnologies) having a DNA sequence encoding the human IgG ₁ Fc region and a histidine tag. DNA sequences encoding the signal sequence and extracellular region of each mouse Eph receptor, Fc, and histidine tag (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB6, and EphB4) were then transferred into the vector pcDNA 3.1_rfcB by LR reaction using the Gateway System (Invitrogen/LifeTechnologies) to construct an expression vector for the extracellular region of each mouse Fc-His protein Eph receptor. In constructing the mouse EphA2-Fc-His extracellular region protein expression vector, DNA sequences encoding the signal sequence and extracellular region of mouse EphA2 were amplified by RT-PCR from total tissue-derived RNA and cloned into the pcDNA 3.1 vector having the DNA sequence , encoding Fc and a histidine tag, to construct an expression vector for the mouse extracellular region protein-Fc-His EphA2.

[0133]

Each mouse Eph receptor Fc-His extracellular region protein expression vector was then introduced into Expi293F cells (Gibco/ThermoFisher) with the Expi293 expression system (Gibco/ThermoFisher). After 5 days of culture (5% CO ₂ , 37°C, 120 rpm), the culture supernatant was collected and centrifuged at room temperature at 1500 rpm for 5 minutes. The centrifuged supernatant was filtered through a 0.45 μm pore size filter (Millipore).

[0134]

For Antibody A, mouse Eph receptor binding activity was assessed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with rabbit anti-6-His antibody (Bethyl Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), each mouse Eph receptor extracellular region-Fc-His protein (at a final concentration of 1 nM) was seeded into each well and incubated at room temperature for one hour. After washing three times, a solution of human IgG (100 μg/ml, Sigma) and antibody A (10 μg/ml) was added to the wells and incubated at room temperature for one hour. Horseradish peroxidase-labeled donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was added to this and incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells, and after moderate staining was confirmed, an equal amount of stop solution (1 N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells ) and absorbance at 450 nm were read using a microplate reader (PerkinElmer).

[0135]

Among the mouse Eph receptor family, antibody A had specific binding activity only to mouse EphA4 (Figure 6).

[0136]

Comparative Example 8: Reactivity of EphA4 Monoclonal Antibody to Mouse, Rat, Monkey and Human EphA4

The preparation of mouse, rat, monkey and human extracellular region proteins-Fc-His EphA4 was carried out according to the steps below. First, according to the production method of EphA4 extracellular region-Fc-His protein in Comparative Example 1, a monkey EphA4 extracellular region-Fc-His protein expression vector was constructed. The amino acid sequence of monkey EphA4 used in the vector construction is shown at SEQ ID NO: 32, and its extracellular region is shown at SEQ ID NO: 33. Various extracellular region-Fc-His proteins of EphA4 were produced using the extracellular region-Fc-His protein expression vector. Monkey His EphA4, as well as the extracellular protein expression vectors-Fc-His EphA4 of mouse, rat and human, which are described in Comparative Example 1.

[0137]

For Antibody A, assessment of binding activity to various extracellular regions of EphA4 was performed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with donkey anti-human IgG antibody (Jackson ImmunoResearch Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), mouse, rat, monkey and human extracellular region-Fc-His EphA4 proteins (1 nM final concentration) were seeded into the wells and incubated at room temperature for one hour . After washing three times, a solution of human IgG (100 μg/ml, Mitsubishi Pharma Corporation) and antibodies A (0, 0.00013, 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, and 10) were added to the wells. µg/ml) and incubated at room temperature for one hour. Horseradish peroxidase-labeled donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) was added to this and incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells, and after moderate staining was confirmed, an equal amount of stop solution (1 N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells ) and absorbance at 450 nm were read using a microplate reader (PerkinElmer).

[0138]

Antibody A had equivalent binding activity to all of mouse, rat, monkey and human EphA4 (Figure 7).

[0139]

Comparative Example 9. Reactivity of EphA4 Monoclonal Antibody to the Extracellular Region, Ligand Binding Domain, Type III Fibronectin Domain 1, Type III Fibronectin Domain 2 of Human EphA4

Preparation of proteins containing the extracellular region (ECD), ligand binding domain (LBD), fibronectin type III domain 1 (FN1) or fibronectin type III domain 2 (FN2) of human EphA4 fused to maltose binding protein (MBP) and a histidine tag (hereinafter referred to herein as “human EphA4 extracellular region protein-MBP-His”, “human EphA4 ligand binding domain protein-MBP-His”, “human EphA4 fibronectin type III domain 1-MBP-His protein” and “human human EphA4 fibronectin type III domain 2-MBP-His") was carried out according to the steps below. First, pcDNA 3.4 expression vectors were constructed for the extracellular region, ligand binding domain, fibronectin type III domain 1, or fibronectin type III domain 2-MBP-His of human EphA4. First, the human EphA4 signal sequence (SEQ ID NO: 34) or preprotrypsin signal sequence (SEQ ID NO: 35) and DNA sequences encoding each domain of human EphA4 were amplified by PCR and cloned into the pcDNA 3.4 vector having the DNA sequence , encoding MBP and a histidine tag (Invitrogen/LifeTechnologies), for constructing expression vectors for the extracellular region protein-MBP-His human EphA4, the ligand-binding domain protein-MBP-His EphA4, the 1-MBP-His domain protein of human fibronectin type III EphA4 and the 2-MBP-His domain protein of human fibronectin type III EphA4. The amino acid sequence of human EphA4 used in vector construction is shown at SEQ ID NO: 5, its extracellular region is shown at SEQ ID NO: 36, the ligand binding domain is shown at SEQ ID NO: 37, fibronectin type III domain 1 is shown at SEQ ID NO: 38 and fibronectin type III domain 2 is shown under SEQ ID NO: 39. The above expression vectors were transfected into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 expression system (Thermo SCIENTIFIC). After four days, the culture supernatant was collected and passed through a 0.45 μm filter (Millipore). Rough purification was performed using amylose resin (NEB) and buffer exchanged to PBS (Wako Pure Chemical) with a Zeba Spin Desalting column (Thermo SCIENTIFIC). The monomer fraction was differentially purified using Superdex 200 10/300 (GE Healthcare).

[0140]

For Antibody A, assessment of binding activity to various domains in human EphA4 was performed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with rabbit anti-6-His antibody (Bethyl Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing twice with 0.02% Tween 20/PBS (Nacalai Tesque), human EphA4 extracellular region protein-MBP-His, human EphA4 ligand-binding domain protein-MBP-His, fibronectin type III fibronectin 1-MBP-His domain protein EphA4 human and human fibronectin type III EphA4 2-MBP-His domain protein (final concentration 10 nM) were seeded into wells and incubated at room temperature for one hour. After washing three times, antibody A was added to the wells (final concentration 10 nM) and incubated at room temperature for one hour. Horseradish peroxidase-labeled goat anti-mouse IgG Fcγ fragment antibody (Jackson ImmunoResearch Laboratories) was added and incubated at room temperature for one hour. After washing five times, ™B solution (KPL) was added to the wells and after confirming a moderate amount of staining, an equal amount of stop solution (2N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells. Absorbance at 450 nm and 650 nm was read using a microplate reader (PerkinElmer).

[0141]

Antibody A had binding activity to the extracellular region (ECD) and ligand binding domain (LBD) of human EphA4 (Figure 8). There was no response to fibronectin type III domain 1 (FN1) or fibronectin type III domain 2 (FN2). Accordingly, Antibody A was found to specifically bind to the ligand binding domain of the extracellular region of human EphA4.

[0142]

Comparative Example 10: Effect of Anti-EphA4 Monoclonal Antibody on Increasing Spine Number in Hippocampal Neuron

Preparation of rat hippocampal neurons was carried out as described in Comparative Example 1 (B) above. Rat hippocampal neurons were injected with the EGFP gene using a nucleofector (Lonza), mixed with rat hippocampal neurons without gene injection, and seeded in a 24-well plate (Falcon) with a coverslip (Matsunami Glass Industries) coated with poly-L-lysine.

[0143]

Spine counting using hippocampal neurons was performed according to the steps below. EGFP-injected rat hippocampal neurons on day 13 of culture, seeded in a 24-well plate (Falcon) with a coverslip (Matsunami Glass Industries) coated with poly-L-lysine, were treated with control antibody (mouse IgG ₁ ; BioLegend) or antibody A (6.7 and 20 nM) for 24 hours. The coverslip was then transferred to 2% PFA (Wako Pure Chemical)/4% sucrose (Wako Pure Chemical)/PBS solution and left for 20 minutes to fix the cells. After removing the fixative solution and washing the cells three times with PBS, 0.25% Triton X-100 (Wako Pure Chemical)/PBS was added and the cells were permeabilized for 15 minutes. The solution was removed, the coverslip was transferred to 2% BSA (Sigma)/0.25% Triton X-100/Opti-MEM (GIBCO) and blocked for one hour, then left with anti-GFP antibody for 1.5 hours (Nacalai Tesque) to carry out the reaction. After removing the primary antibody solution and washing three times with PBS, the secondary antibody was left for one hour to react. After removing the secondary antibody solution and washing three times with PBS, Prolong Gold anti-fade reagent (Molecular probes) was added for fixation and observed with an LSM800 (ZEISS). The experiment described above was performed in triplicate and for each experiment, neurons were removed from two coverslips, spines on each dendrite were counted using Imaris® image analysis software (Bitplane), and the number of spines per 10 μm was calculated for each neuron.

[0144]

Antibody A increased the number of spines in the hippocampal neuron (Figure 9). From this result it is clear that Antibody A had activity in stabilizing spines in hippocampal neurons.

[0145]

Comparative Example 11: Effect of Anti-EphA4 Monoclonal Antibody on Inhibiting Tau Phosphorylation in Vivo

Evaluation of the effect on inhibition of tau phosphorylation in vivo using a tau transgenic mouse (rTg4510) was carried out according to the following steps. Tau transgenic mice (rTg4510) aged 20 to 26 weeks were injected subcutaneously with Antibody A or a control antibody produced by conventional methods by immunizing mice with dinitrophenol (mouse anti-dinitrophenol antibody) twice a week, each time at a dose of 100 mg/kg ( 10 ml/kg). 3.5 days after the last injection, anesthesia was performed with 2% isoflurane (Intervet) and a mixture of three types of anesthetics (4.0 mg/kg dormicum (Astellas Pharma), 0.3 mg/kg domitor (Nippon Zenyaku Kogyo) and 5.0 mg/kg vetorfal (Meiji Seika Pharma)), under anesthesia, perfusion was performed with PBS (Wako Pure Chemical) containing 3 units/ml heparin (Ajinomoto) and 1% phosphatase inhibitor cocktail (Nacalai Tesque), and the cerebral hemisphere was excised. The collected brain hemispheres were fixed at 4°C with shaking overnight in 2% paraformaldehyde (TAAB)/0.1 M phosphate buffer (Wako Pure Chemical). Hemisphere media was replaced with 20% sucrose (Wako Pure Chemical)/0.1 M phosphate buffer (Wako Pure Chemical) and subsequently with 25% sucrose/0.1 M phosphate buffer (Wako Pure Chemical) and then placed in Tissue-Tek OCT (Sakura Finetek Japan)/25% sucrose and frozen in liquid nitrogen. Sections were cut at 7 μm thickness using a CM1860 cryostat (Leica), mounted on a glass slide (Muto Pure Chemicals), air dried, and then placed in a sealed bag and stored at −80°C. The slide used for immunostaining was thawed, air dried, and then washed with PBS (Wako Pure Chemical), immersed in 1% BSA (Sigma)/10% normal donkey serum (Jackson ImmunoResearch Laboratories)/0.5 % Triton X-100 (Wako Pure Chemical)/PBS and blocked for one hour and then left overnight with anti-phosphorylated tau antibody AT8 (Fujirebio Europe NV) to react at normal temperatures. After washing three times with PBS, the secondary antibody was left for one hour to react. After washing three times with PBS, Prolong Gold anti-fluorescence reagent (molecular probes) was applied to the section and the slide was fixed and observed with an LSM700 (ZEISS). The area with a positive signal from AT8 in the stratum radiatum CA1 of the hippocampus was measured using ImageJ image analysis software, and the proportion of the area with an AT8-positive signal relative to the total area was calculated.

[0146]

Antibody A reduced the phosphorylated tau protein signal in the CA1 region of the hippocampus (Figure 10). From this result, it can be seen that Antibody A had activity in suppressing the progression of pathology in tau transgenic mice (rTg4510).

[0147]

Comparative Example 12 Epitope Mapping of EphA4 Ligand Binding Domain (EphA4-LBD) by X-Ray Crystal Structure Analysis

The production of A-Fab antibody was carried out according to the steps below. 101.1 mg of antibody A was dissolved in 0.1 M sodium phosphate buffer (pH 7.0) containing 30 mM L-cysteine and 2 mM EDTA at a concentration of 15 mg/ml. Papain (Sigma) was added to this antibody solution in an amount of 1/200 of the antibody and enzymatic digestion was carried out at 37°C for 18 hours. The enzymatic digestion solution of Antibody A was dialyzed against PBS and the precipitate was removed by centrifugation (the resulting precipitate was redissolved in PBS and mixed with the centrifugation supernatant). The following steps were then performed to remove impurities other than the A-Fab antibody.

1) Purification on a protein A column

This enzymatic digestion solution was applied to a 2 mL ProSep vA High Capacity column (Millipore) equilibrated with PBS, and the intermediate and wash fractions were isolated with PBS.

2) Affinity purification using anti-human IgG Fcγ antibody

An anti-human IgG Fcγ affinity column (Jackson ImmunoResearch Laboratories) covalently bound to NHS-activated Sepharose 4FF (GE Healthcare) was prepared according to the manual for that Sepharose. The solution isolated in step 1) above was loaded onto this affinity column and the intermediate solution and its wash solution with PBS were isolated.

3) Purification by gel filtration

The intermediate fraction obtained in the above step 2) was concentrated using an ultrafiltration membrane. Superose 12 (GE Healthcare) was equilibrated with PBS, concentrated sample applied, and separated and purified. A portion of the separated and purified fraction was analyzed by SDS-PAGE and the fraction containing high purity A-Fab antibody was isolated and pooled. The sample thus purified was considered to be A-Fab antibody.

[0148]

In order to obtain the complex of A-Fab antibody and EphA4-LBD antigen, EphA4-LBD was prepared (Qin H. et al., J. Biol. Chem., 283: 29473-29484 (2008)). Mix 0.68 µM EphA4-LBD (200 µM, 3.4 ml) and 0.45 µM A-Fab antibody (300 µM, 1.5 ml) so that EphA4-LBD has approximately 1.5 -fold molar ratio relative to the A-Fab antibody. The mixed solution was then applied to HILOAD 26/60 Superdex 75 prep grade (GE Healthcare) and eluted with chromatography buffer (25 mM Tris/HCl (pH 7.5), 100 mM NaCl). The fraction containing the complex was analyzed by SDS PAGE, high purity fractions were collected and concentrated to approximately 40.8 mg/ml and the resulting product was used for crystallization.

[0149]

Crystallization of the complex was carried out using the sessile drop vapor diffusion method using a Hydra II Plus One System autocrystallization device (Matrix Technologies Corp., Ltd.). MRC-2 (Molecular Dimensions) was used as a plate. The reservoir solution composition was 100 mM HEPES (pH 7.5), 10% polyethylene glycol 8000 and 8% ethylene glycol, and this reservoir solution and the solution of the above complex were mixed so that the volume ratio was 1:1 in order to induce crystallization drops The resulting crystallization plate was left to settle at 20°C.

[0150]

After crystallization under the above conditions, crystals characterized by space group P212121, lattice constants a=71.0 Å, b=84.5 Å and c=116.1 Å were obtained. X-rays (1.0 Å) were applied to the resulting crystals to obtain diffraction data of 1.79 Å. Diffraction data were processed using HKL2000 (HKL Research Inc.), and phase determination was performed using the molecular displacement method. For the molecular replacement method, the PHASER program (version 2.5.0, McCoy A. J. et al., J. Appl. Cryst. 40: 658-674 (2007)) included in the CCP4 software package (Collaborative computational project number 4, [ CCP4] version 6.5.0, Acta Cryst D 67: 235-242 (2011). The crystal structure of EphA4-LBD (PDBID: 3CKH) and the crystal structure of IgG Fab region (PDBID: 2VXT (L chain) and 1FGN (H chain)) were used as a search model for the molecular replacement method. The molecular model was built using the COOT program (Emsley P. et al., Acta Cryst. D 60: 2126-2132n (2004)) to match the electron density obtained from a particular phase, and structure refinement was carried out using the REFMAC program ( Murshudov G.N., Acta Cryst. D 53: 240-255 (1997)).

The crystal structure of the complex with a resolution of 2.0 Å was obtained using structure calculations (R=0.212, Rfree=0.258).

[0151]

The crystal structure of the resulting A-Fab/EphA4-LBD antibody complex was analyzed using an interaction detection tool equipped with the MOE 2018.0101 computational chemistry system (Chemical Computing Group Inc.) and identified the amino acid residues on the EphA4-LBD that were in direct contact with the antibody A-Fab (Figure 11A). The amino acid residues identified were Glu51, Gly52, Ile59, Gln71, Cys73, Asn74, Val75, Met76, Glu77, Thr104, Arg106, Leu111, Pro112, Met115, Arg162, Met164, Cys191, Ala193, and Val195. Figure 11B shows the surface structure of EphA4-LBD generated using Maestro (version 11.0, Schrodinger, LLC). As a result, the present inventors concluded that the region in which these amino acid residues are present is the binding region of the A-Fab antibody in the EphA4-LBD.

[0152]

Example 1: Preparation of Humanized Antibody from Antibody A

Preparation of humanized anti-EphA4 antibody

The variable region of the humanized antibody was constructed. Based on the high degree of homology with respect to the framework region (FR) of Antibody A among the FRs of human antibodies IGHV3-33*03 (SEQ ID NO: 42) and JH6 (SEQ ID NO: 43) for the heavy chain and IGKV1-17*01 (SEQ ID NO: 40) and JK4 (SEQ ID NO: 41) for the light chain were selected as the FR for the humanized antibody. The amino acids in the FR that interact with the CDR amino acids were then predicted using a predictive model of the 3D structure of Mouse Antibody A and transplanted together with the CDR of Antibody A having the Y32F mutation in the heavy chain CDR1 (SEQ ID NOs: 44, 27, 28 and 29-31 ), and designed HK2-42 (SEQ ID NO: 45) as a heavy chain variable region of a humanized antibody, and designed L1-8 (SEQ ID NO: 46) as a light chain variable region of a humanized antibody. The amino acid sequences of the transplanted CDRs are shown in Table 2, and the nucleic acid sequences are shown in Table 3.

[0153]

Human IgG ₂ constant region (SEQ ID NO: 47) was used as the heavy chain constant region. Human Ig κ chain (SEQ ID NO: 48) was used as the light chain constant region. An expression vector (pcDNA 3.4) containing a gene sequence encoding the amino acid sequence of the humanized antibody was transfected into Expi293F cells (Gibco/ThermoFisher) using the Expi293 expression system (Gibco/ThermoFisher). As the gene sequence encoding the amino acid sequence of the humanized antibody, the nucleic acid sequence shown in SEQ ID NO: 55 was used as the heavy chain variable region, the nucleic acid sequence shown in SEQ ID NO: 56 was used as the light chain variable region, the sequence the nucleic acid sequence shown in SEQ ID NO: 57 was used as the heavy chain constant region, and the nucleic acid sequence shown in SEQ ID NO: 58 was used as the light chain constant region, respectively. The full-length heavy chain amino acid sequence (not including the signal sequence) of the humanized antibody was the amino acid sequence shown under SEQ ID NO: 59, the full-length light chain amino acid sequence (not including the signal sequence) was the amino acid sequence shown under SEQ ID NO: 60. The nucleic acid sequence encoding the full length of the heavy chain of the humanized antibody was the nucleic acid sequence shown under SEQ ID NO: 61, and the nucleic acid sequence encoding the full length of the light chain was the nucleic acid sequence shown under SEQ ID NO: : 62. The supernatant was collected and the humanized version of Antibody A (Antibody B) was purified using MabSelectSuRe (GE Healthcare).

[0154]

[Table 2] Antibody B CDR amino acid sequences Name Subsequence Heavy chain CDR1 RFGVH (SEQ ID NO: 44) Heavy chain CDR2 VIWRGGSTDYNAAFMS (SEQ ID NO. 27) Heavy chain CDR3 ESLFGVYYDYGYYSMDY (SEQ ID NO. 28) Light chain CDR1 RASQEISGYLS (SEQ ID NO. 29) Light chain CDR2 AASTLDS (SEQ ID NO. 30) Light chain CDR3 LQYASYPLT (SEQ ID NO. 31)

[Table 3] Antibody B CDR nucleic acid sequences Name Subsequence Heavy chain CDR1 AGATTTGGAGTGCAT (SEQ ID NO: 49) Heavy chain CDR2 GTGATCTGGAGGGGAGGATCCACCGACTACAACGCTGCTTTTATGAGC(SEQ ID NO. 50) Heavy chain CDR3 GAGAGCCTGTTCGGCGTGTACTATGACTACGGCTACTATTCTATGGATTAT (SEQ ID NO: 51) Light chain CDR1 CGCGCCTCCCAGGAGATCTCTGGCTACCTGTCC (SEQ ID NO. 52) Light chain CDR2 GCTGCCTCCACCCTGGACTCT (SEQ ID NO. 53) Light chain CDR3 CTGCAGTACGCTTCCTATCCACTGACC (SEQ ID NO. 54)

[0155]

Example 2: Affinity of Humanized Anti-EphA4 Monoclonal Antibody for Human EphA4

The binding affinity of antibody B prepared in Example 1 to human EphA4 was determined by surface plasmon resonance (SPR method) using Biacore T200 (GE Healthcare). First, anti-His antibody (GE Healthcare, 28-9950-56) was fixed to the CM5 sensor chip. Fixation was accomplished by the amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and ethanolamine was used for blocking (sensor chip and all fixation reagents were purchased from GE Healthcare ). It was diluted to 3.5 μg/ml with fixation buffer (10 mM sodium acetate, pH 4.5) and fixed to the sensor chip according to the protocol included with the Biacore T200. Human EphA4 extracellular region protein-SEAP-His10 was diluted in HBS-EP running buffer (GE Healthcare, BR-1001-88) and the solution was applied to the flow cell for 120 seconds for capture (capture amount was approximately 10 RU). Antibody B, serially diluted to a range of 100, 50, 25, 12.5, 6.3, 3.2, 1.6 and 0 nM with HBS-EP, was then applied to the sensor chip for 120 seconds and the curve was subsequently monitored binding reactions during addition (binding phase, 120 seconds) and after addition is complete (dissociation phase, 600 seconds). After completion of each tracking, 4 M MgCl ₂ (60 seconds, Wako Pure Chemical) was added to regenerate the sensor chip. The resulting binding reaction curve was fit to a 1:1 binding model using the BIA evaluation software supplied with the system, and the affinity (KD=kd/ka) for human EphA4 was calculated.

[0156]

The binding affinity of antibody B for human EphA4 (KD value) was 1.34×10 ^-9 M (Figure 12). Antibody B has been shown to exhibit almost the same affinity as Antibody A, which was the antibody before humanization.

[0157]

Example 3 EphA4 Cleavage Enhancing Activity of a Humanized Anti-EphA4 Monoclonal Antibody in Hippocampal Neurons

In the case of Antibody B prepared in Example 1, the evaluation of EphA4 cleavage enhancing activity using hippocampal neurons was carried out according to the steps below.

Rat hippocampal neurons seeded in a 96-well plate (Falcon) were treated with antibody B (2.0, 6.7 and 20 nM) and the γ-selectase inhibitory drug compound E (50 nM, Enzo Life Sciences) . After twenty-four hours, it was washed with PBS (Wako Pure Chemical), sample buffer with SDS (Laemmli sample buffer (Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) was added to extract cells, and boiled for 5 minutes. SDS-PAGE was performed on this sample, Western blotting was performed with an anti-EphA4 monoclonal antibody (Abnova), the band concentration was quantified, and the EphA4 C-terminal fragment/full-length EphA4 value was calculated.

[0158]

Antibody B enhanced the EphA4 cleavage response in hippocampal neurons in a concentration-dependent manner (Figure 13).

[0159]

Example 4 Inhibitory Activity of a Humanized Anti-EphA4 Monoclonal Antibody on Human EphA4 Ligand-Binding

For Antibody B obtained in Example 1, evaluation of the binding inhibitory activity between human EphA4 and human ligand was performed according to the steps below. Alkaline phosphatase antibody (Thermo SCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), human extracellular region protein-SEAP-His EphA4 (at a final concentration of 10 nM) was seeded into the wells and incubated at room temperature for one hour. After washing three times, ligand and serially diluted antibody B were added to each well (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 and 3000 nM ). It should be noted that biotinylated human Ephrin A5-Fc chimera (R&D Systems, final concentration 0.7 nM) and biotinylated human Ephrin B3-Fc chimera (R&D Systems, final concentration 2.3 nM) were used as ligands. After incubation at room temperature for one hour and washing three times, horseradish peroxidase-labeled streptavidin (GE Healthcare) was added and the mixture was incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells and incubated at room temperature for 2-5 minutes. An equal amount of stop solution (1N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells and the absorbance was read at 450 nm using a microplate reader (Molecular Devices or PerkinElmer).

[0160]

Antibody B inhibited binding between human EphA4 and human ligand in a concentration-dependent manner, and IC ₅₀ values for binding of human Ephrin A5 and Ephrin B3 were approximately 4.9 nM and 1.6 nM, respectively. Accordingly, it was found that Antibody B strongly inhibited the binding between human EphA4 and the human ligand and exhibited almost the same inhibitory activity as Antibody A, which was the antibody before humanization (Figure 14).

[0161]

Example 5: Inhibitory Activity of Humanized Anti-EphA4 Monoclonal Antibody on Mouse Ligand-Binding Mouse EphA4

For Antibody B prepared in Example 1, evaluation of the binding inhibitory activity between mouse EphA4 and mouse ligand was performed according to the steps below. Alkaline phosphatase antibody (Thermo SCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.02% Tween 20/PBS (Thermo SCIENTIFIC), mouse extracellular region protein-SEAP-His EphA4 (final concentration 10 nM) was added to the wells and incubated as indicated at room temperature for one hour. After washing three times, ligand and serially diluted antibody B were added to each well (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 and 3000 nM ). It should be noted that biotinylated mouse Ephrin A1-Fc chimera (R&D Systems, final concentration 6 nM) and biotinylated mouse Ephrin B2-Fc chimera (R&D Systems, final concentration 2.5 nM) were used as ligands. After incubation at room temperature for one hour and washing three times, horseradish peroxidase-labeled streptavidin (GE Healthcare) was added and the mixture was incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells and incubated at room temperature for 2 minutes. An equal amount of stop solution (1N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells and the absorbance was read at 450 nm using a microplate reader (Molecular Devices or PerkinElmer).

[0162]

Antibody B inhibited binding between mouse EphA4 and mouse ligand in a concentration-dependent manner, and the IC ₅₀ values for binding of mouse Ephrin A1 and Ephrin B2 were approximately 8.7 nM and 4.2 nM, respectively. Accordingly, it was found that Antibody B strongly inhibited the binding between mouse EphA4 and the mouse ligand and exhibited almost the same inhibitory activity as Antibody A, which was the antibody before humanization (Figure 15).

[0163]

Example 6: Selectivity of Humanized Anti-EphA4 Monoclonal Antibody for the Human Eph Receptor

Similar to the method for producing the extracellular region-SEAP-His protein of mouse EphA4 described in comparative example 1, DNA sequences encoding the signal sequence and extracellular region of each human Eph receptor (EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10 , EphB1, EphB2, EphB3, EphB4, and EphB6) were amplified by RT-PCR from tissue-derived total RNA and cloned into the pENTR1A vector (Invitrogen/LifeTechnologies) having a DNA sequence encoding the SEAP protein and a histidine tag. DNA sequences encoding the signal sequence and extracellular region of each human Eph receptor, SEAP protein, and histidine tag were then transferred into the pcDNA 3.1_rfcB vector by LR reaction using the Gateway System (Invitrogen/Life Technologies) to construct vectors (referred to as “expression vector” Eph receptor extracellular region protein-SEAP-His") expressing a protein containing a SEAP protein and a His tag fused to the extracellular region of each human Eph receptor (referred to as "Eph receptor extracellular region protein-SEAP-His").

[0164]

Each of the human Eph receptor extracellular region-SEAP-His protein expression vectors were then introduced into Expi293F cells (Gibco/ThermoFisher) using the Expi293 expression system (Gibco/ThermoFisher). Five days after incubation (5% CO ₂ , 37°C), the culture supernatant was collected and centrifuged at room temperature at 1500 rpm for 5 minutes. The centrifuged supernatant was filtered through a 0.45 μm pore size filter (Millipore).

[0165]

In the case of Antibody B obtained in Example 1, human Eph receptor binding activity was assessed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with rabbit anti-6-His antibody (Bethyl Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), each of the human Eph receptor extracellular region-SEAP-His proteins (1 nM final concentration) was seeded into each well and incubated at room temperature for one hour. After washing three times, a solution of human IgG (100 μg/ml, Mitsubishi Pharma Corporation) and antibody B (10 μg/ml) was added to the wells and incubated at room temperature for one hour. Horseradish peroxidase-labeled donkey anti-human IgG antibody (Jackson ImmunoResearch Laboratories) was added to this and incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells, and after moderate staining was confirmed, an equal amount of stop solution (1 N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells ) and absorbance at 450 nm were read using a microplate reader (PerkinElmer).

[0166]

Antibody B, like Antibody A which was the pre-humanization antibody, was found to specifically bind to human EphA4 of the human Eph receptor family (FIG. 16).

[0167]

Example 7: Selectivity of Humanized Anti-EphA4 Monoclonal Antibody for the Mouse Eph Receptor

According to the method for producing the extracellular region protein-Fc-His EphA4 in accordance with Comparative Example 1, the DNA sequences encoding the signal sequence and the extracellular region of each mouse Eph receptor (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2 , EphB3, EphB4, and EphB6) were amplified by RT-PCR from tissue-derived total RNA and cloned into the pENTR1A vector (Invitrogen/LifeTechnologies) having a DNA sequence encoding the human IgG ₁ Fc region and a histidine tag. DNA sequences encoding the signal sequence and extracellular region of each mouse Eph receptor (EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4 and EphB6), Fc and histidine tag were then transferred into the vector pcDNA 3.1_rfcB by LR reaction using the Gateway System (Invitrogen/Life Technologies) to construct an extracellular region-Fc-His protein expression vector of each mouse Eph receptor. In constructing the mouse EphA2 extracellular region-Fc-His protein expression vector, DNA sequences encoding the signal sequence and extracellular region of mouse EphA2 were amplified by RT-PCR from total tissue-derived RNA and cloned into the pcDNA 3.1 vector having the DNA sequence , encoding Fc and a histidine tag, to construct an expression vector for the mouse extracellular region protein-Fc-His EphA2.

[0168]

Each of the mouse Eph receptor extracellular region-Fc-His protein expression vectors were then introduced into Expi293F cells (Gibco/ThermoFisher) using the Expi293 expression system (Gibco/ThermoFisher). After 5 days of culture (5% CO ₂ , 37°C, 120 rpm), the culture supernatant was collected and centrifuged at room temperature at 1500 rpm for 5 minutes. The centrifuged supernatant was filtered through a 0.45 μm pore size filter (Millipore).

[0169]

For Antibody B, mouse Eph receptor binding activity was assessed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with rabbit anti-6-His antibody (Bethyl Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), each of the mouse Eph receptor extracellular region-Fc-His proteins (1 nM final concentration) was seeded into each well and incubated at room temperature for one hour. After washing three times, a solution of human IgG (100 μg/ml, Sigma) and antibody B (10 μg/ml) was added to the wells and incubated at room temperature for one hour. Horseradish peroxidase-labeled goat anti-human kappa light chain (IBL) antibody was added and incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells, and after moderate staining was confirmed, an equal amount of stop solution (1 N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells ) and absorbance at 450 nm were read using a microplate reader (PerkinElmer).

[0170]

Among the mouse Eph receptor family, antibody B had specific binding activity only to mouse EphA4 (Figure 17).

[0171]

Example 8 Reactivity of Humanized EphA4 Monoclonal Antibody to Mouse, Rat, Monkey and Human EphA4

[0172]

For Antibody B, the assessment of binding activity to various EphA4s was performed according to the steps below.

Alkaline phosphatase antibody (Thermo SCIENTIFIC) was coated onto the wells of a 96-well plate (Nunc). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing three times with 0.05% Tween 20/PBS (Thermo SCIENTIFIC), mouse, rat, monkey and human extracellular region-SEAP-His EphA4 proteins (1 nM final concentration) were seeded into the wells and incubated at room temperature for one hour . After washing three times, a solution of human IgG (100 μg/ml, Mitsubishi Pharma Corporation) and antibodies B (0, 0.00013, 0.00064, 0.0032, 0.016, 0.08, 0.4, 2 and 10) were added to the wells. µg/ml) and incubated at room temperature for one hour. Horseradish peroxidase-labeled donkey anti-human IgG antibody (Jackson ImmunoResearch Laboratories) was added to this and incubated at room temperature for one hour. After washing three times, ™BZ solution (3,3',5,5'-tetramethylbenzidine, Sigma) was added to the wells, and after moderate staining was confirmed, an equal amount of stop solution (1 N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells ) and absorbance at 450 nm were read using a microplate reader (PerkinElmer).

[0173]

Antibody B had equivalent binding activity to all of mouse, rat, monkey and human EphA4 (Figure 18).

[0174]

Example 9 Reactivity of Humanized Anti-EphA4 Monoclonal Antibody to the Extracellular Region, Ligand Binding Domain, Type III Fibronectin Domain 1, Type III Fibronectin Domain 2 of Human EphA4

In the case of Antibody B prepared in Example 1, assessment of binding activity to various domains in human EphA4 was performed according to the steps below.

Wells of a 96-well plate (Nunc) were coated with rabbit anti-6-His antibody (Bethyl Laboratories). After incubation at 4°C overnight, wells were blocked at room temperature for one hour with 1% Block ACE (DS Pharma Biomedical). After washing twice with 0.02% Tween 20/PBS (Nacalai Tesque), human EphA4 extracellular region protein-MBP-His, human EphA4 ligand-binding domain protein-MBP-His, fibronectin type III fibronectin 1-MBP-His domain protein EphA4 human and human fibronectin type III EphA4 2-MBP-His domain protein (final concentration 10 nM) were seeded into wells and incubated at room temperature for one hour. After washing three times, antibody B was added to the wells (final concentration 10 nM) and incubated at room temperature for one hour. Horseradish peroxidase-labeled rabbit anti-human IgG Fcγ antibody (Jackson ImmunoResearch Laboratories) was added and incubated at room temperature for one hour. After washing five times, ™B solution (KPL) was added to the wells and after confirming a moderate amount of staining, an equal amount of stop solution (2N H ₂ SO ₄ , Wako Pure Chemical) was added to the wells. Absorbance at 450 nm and 650 nm was read using a microplate reader (PerkinElmer).

[0175]

Antibody B had binding activity to the extracellular region (ECD) and ligand binding domain (LBD) of human EphA4 (Figure 19). There was no response to fibronectin type III domain 1 (FN1) or fibronectin type III domain 2 (FN2). Accordingly, antibody B was found to bind specifically to the ligand binding domain of the extracellular region of human EphA4.

[0176]

Example 10: Effect of Humanized Anti-EphA4 Monoclonal Antibody to Increase the Spine Number of a Hippocampal Neuron

Preparation of rat hippocampal neurons was carried out as described in Comparative Example 1 (B). Rat hippocampal neurons were injected with the EGFP gene using a nucleofector (Lonza) and seeded in a 24-well plate (Falcon) with a coverslip (Matsunami Glass Industries) coated with poly-L-lysine.

[0177]

Spine counting using hippocampal neurons was performed according to the steps below. EGFP-injected rat hippocampal neurons on day 13 of culture, seeded in a 24-well plate (Falcon) with a coverslip (Matsunami Glass Industries) coated with poly-L-lysine, were treated with control antibody (human _IgG2 ; Sigma) or antibody B (6.7 and 20 nM) for 24 hours. The coverslip was then transferred to 2% PFA (Wako Pure Chemical)/4% sucrose (Wako Pure Chemical)/PBS solution and left for 20 minutes to fix the cells. After removing the fixative solution and washing the cells three times with PBS, 0.25% Triton X-100 (Wako Pure Chemical)/PBS was added and the cells were permeabilized for 15 minutes. The solution was removed, the coverslip was transferred to 2% BSA (Sigma)/0.25% Triton X-100/OPTI-MEM (GIBCO) and blocked for one hour, then left with anti-GFP antibody for 1.5 hours (Nacalai Tesque) to carry out the reaction. After removing the primary antibody solution and washing three times with PBS, the secondary antibody was left for one hour to react. After removing the secondary antibody solution and washing three times with PBS, Prolong Gold anti-fade reagent (Molecular probes) was added for fixation and observed with an LSM800 (ZEISS). The experiment described above was performed in triplicate and for each experiment, neurons were removed from two coverslips, spines on each dendrite were counted using Imaris® image analysis software (Bitplane), and the number of spines per 10 μm was calculated for each neuron.

[0178]

Antibody B increased the number of spines in the hippocampal neuron (Figure 20). From the above result, it is clear that Antibody B had activity in stabilizing spines in hippocampal neurons.

[0179]

Example 11: Human EphA4 Cleavage Enhancing Activity of a Humanized Anti-EphA4 Monoclonal Antibody

In the case of Antibody B prepared in Example 1, the activity to enhance human EphA4 binding cleavage was assessed according to the steps below.

Preparation of rat hippocampal neurons was carried out as described in Comparative Example 1 (B). Rat hippocampal neurons were injected with the human EphA4-HA protein expression vector using a nucleofector (Lonza) and seeded into a 96-well plate (Falcon) coated with poly-L-lysine. Seeded rat hippocampal neurons were treated with antibody B (6.7, 20 and 67 nM) and the γ-selectase inhibitory drug compound E (50 nM, Enzo Life Sciences). After approximately twenty-four hours, it was washed with PBS (Wako Pure Chemical), sample buffer with SDS (Laemmli sample buffer (Bio-Rad) and 5% 2-mercaptoethanol (Bio-Rad)) was added to extract cells, and boiled in within 5 minutes. SDS-PAGE was performed on this sample, Western blotting was performed with a rat monoclonal anti-HA antibody (Roche), the band concentration was quantified, and the EphA4 C-terminal fragment/full-length EphA4 value was calculated.

[0180]

Antibody B enhanced the human EphA4 cleavage response in hippocampal neurons (Figure 21).

[0181]

Example 12. Involvement of MMPs and ADAMs in relation to the effect of a humanized monoclonal antibody to EphA4 on increasing the number of spines in a hippocampal neuron

Preparation of rat hippocampal neurons was carried out as described in Comparative Example 1 (B). A subset of rat hippocampal neurons were injected with the EGFP gene using a nucleofector (Lonza) and seeded into a 24-well plate (Falcon) with a cover glass (Matsunami Glass Industries) coated with poly-L-lysine.

[0182]

Spine counting using hippocampal neurons was performed according to the steps below. EGFP-injected rat hippocampal neurons on day 13 of culture, seeded in a 24-well plate (Falcon) with a coverslip (Matsunami Glass Industries) coated with poly-L-lysine, were treated with control antibody (human _IgG2 ; Sigma) or antibody B (20 nM), as well as DMSO (Sigma) or the MMP and ADAM inhibitor GM6001 (2.5 μM, MedChemExpress) for 24 hours. The coverslip was then transferred to 2% PFA (Wako Pure Chemical)/4% sucrose (Wako Pure Chemical)/PBS solution and left for 20 minutes to fix the cells. After removing the fixative solution and washing the cells three times with PBS, 0.25% Triton X-100 (Wako Pure Chemical)/PBS was added and the cells were permeabilized for 15 minutes. 0.25% Triton X-100/PBS was removed, the coverslip was transferred to 2% BSA (Sigma)/0.25% Triton X-100/OPTI-MEM (GIBCO) and blocked for one hour, followed by Anti-GFP antibody (Nacalai Tesque) was left for 1.5 hours to allow the reaction to occur. After removing the primary antibody solution and washing three times with PBS, the secondary antibody was left for one hour to react. After removing the secondary antibody solution and washing three times with PBS, Prolong Gold anti-fade reagent (Molecular probes) was added for fixation and observed with an LSM800 (ZEISS). The experiment described above was performed in triplicate and for each experiment, neurons were removed from two coverslips, spines on each dendrite were counted using Imaris® image analysis software (Bitplane), and the number of spines per 10 μm was calculated for each neuron.

[0183]

The increase in spine number in a hippocampal neuron by Antibody B was inhibited by co-treatment with GM6001 (Figure 22). From the above result, it can be seen that Antibody B had spine stabilizing activity through MMP and ADAM in hippocampal neurons.

[0184]

Example 13 Effect of Humanized Anti-EphA4 Monoclonal Antibody on Inhibiting Tau Phosphorylation in Vivo

Evaluation of the effect on inhibition of tau phosphorylation in vivo using a tau transgenic mouse (rTg4510) was carried out according to the following steps. Tau transgenic mice (rTg4510) aged 20 to 26 weeks were subcutaneously injected twice weekly with antibody B at a dose of 100 mg/kg (10 ml/kg). The control group was administered subcutaneously PBS (Wako Pure Chemical) at a dose of 10 ml/kg. 3.5 days after the last administration, mice were anesthetized using an inhaled anesthetic of 2-2.5% isoflurane (Intervet) and a mixture of three types of anesthetics (4.0 mg/kg Dormicum (Astellas Pharma), 0.3 mg/kg domitor (Nippon Zenyaku Kogyo) and 5.0 mg/kg vetorfal (Meiji Seika Pharma)), under anesthesia, perfusion was performed with PBS (Wako Pure Chemical) containing 3 units/ml heparin (Ajinomoto) and 1% phosphatase inhibitor cocktail ( Nacalai Tesque), and the cerebral hemispheres of mice were excised. The collected brain hemispheres were fixed at 4°C with shaking overnight and immersed in 2% paraformaldehyde (TAAB)/0.1 M phosphate buffer (Wako Pure Chemical). Hemisphere media was replaced with 10% sucrose (Wako Pure Chemical)/0.1 M phosphate buffer (Wako Pure Chemical) and subsequently with 20% sucrose/0.1 M phosphate buffer (Wako Pure Chemical) and then placed into Tissue-Tek OCT (Sakura Finetek Japan)/20% sucrose compound and frozen using an aluminum block cooled with liquid nitrogen. 7-μm-thick sections of the cerebral hemisphere were obtained using a CM1860 cryostat (Leica). Sections were mounted on a silane-coated glass slide (Muto Pure Chemicals), air dried, and then placed in a sealed bag and stored at −80°C. The slide used for immunostaining was removed from 80°C, air dried and then washed with PBS (Wako Pure Chemical), immersed in 1% BSA (Sigma)/10% normal donkey serum (Jackson ImmunoResearch Laboratories). /0.5% Triton X-100 (Wako Pure Chemical)/PBS and were blocked for one hour and then left overnight with anti-phosphorylated tau antibody AT8 (Fujirebio Europe NV) to react at normal temperatures. After washing three times with PBS, the secondary antibody was left for one hour to react. After washing three times with PBS, Prolong Gold anti-fluorescence reagent (molecular probes) was applied to the section and the slide was fixed and observed with an LSM700 (ZEISS). The area with a positive signal from AT8 in the stratum radiatum of CA1 of the hippocampus was measured using Metamorph image analysis software, and the proportion of the area with an AT8-positive signal relative to the total area was calculated.

[0185]

Antibody B reduced the phosphorylated tau protein signal in the CA1 region of the hippocampus (Figure 23). From this result, it is clear that antibody B had activity in suppressing the progression of pathology in tau transgenic mice (rTg4510).

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LIST OF SEQUENCES

<110> EISAI AR & D MANAGEMENT CO., LTD.

<120> Technical field

<130>ESAP1901F

<150> JP2019-122982

<151> 2019-07-01

<160> 62

<170> PatentIn version 3.5

<210> 1

<211> 986

<212> PROTEIN

<213> Mus musculus

<400> 1

Met Ala Gly Ile Phe Tyr Phe Ile Leu Phe Ser Phe Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr

20 25 30

Leu Leu Asp Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser

35 40 45

Pro Leu Glu Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn

50 55 60

Thr Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Met Glu Ala Ser Gln

65 70 75 80

Asn Asn Trp Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg

85 90 95

Val Tyr Ile Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro

100 105 110

Gly Val Met Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu

115 120 125

Ser Asp Asn Asp Lys Glu Arg Phe Ile Arg Glu Ser Gln Phe Gly Lys

130 135 140

Ile Asp Thr Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly

145 150 155 160

Asp Arg Ile Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu

165 170 175

Ser Lys Lys Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile

180 185 190

Ala Leu Val Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val

195 200 205

Arg Asn Leu Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser

210 215 220

Ser Leu Val Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys

225 230 235 240

Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Asn Cys Leu Cys Asn Ala Gly His Glu Glu Gln Asn Gly Glu

260 265 270

Cys Gln Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala

275 280 285

Ser Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala

290 295 300

Thr Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile

325 330 335

Ser Asn Val Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln

340 345 350

Asn Thr Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys

355 360 365

Cys Gly Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val

370 375 380

His Tyr Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile

385 390 395 400

Thr Asp Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val

405 410 415

Asn Gly Val Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val

420 425 430

Thr Val Thr Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln

435 440 445

Ala Lys Glu Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro

450 455 460

Asp Arg Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu

465 470 475 480

Lys Asp Gln Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg

485 490 495

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

500 505 510

Val Arg Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu

515 520 525

Glu Val Thr Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala

530 535 540

Asn Ser Thr Val Leu Leu Val Ser Val Ser Gly Ser Val Val Leu Val

545 550 555 560

Val Ile Leu Ile Ala Ala Phe Val Ile Ser Arg Arg Arg Ser Lys Tyr

565 570 575

Ser Lys Ala Lys Gln Glu Ala Asp Glu Glu Lys His Leu Asn Gln Gly

580 585 590

Val Arg Thr Tyr Val Asp Pro Phe Thr Tyr Glu Asp Pro Asn Gln Ala

595 600 605

Val Arg Glu Phe Ala Lys Glu Ile Asp Ala Ser Cys Ile Lys Ile Glu

610 615 620

Lys Val Ile Gly Val Gly Glu Phe Gly Glu Val Cys Ser Gly Arg Leu

625 630 635 640

Lys Val Pro Gly Lys Arg Glu Ile Cys Val Ala Ile Lys Thr Leu Lys

645 650 655

Ala Gly Tyr Thr Asp Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser

660 665 670

Ile Met Gly Gln Phe Asp His Pro Asn Ile Ile His Leu Glu Gly Val

675 680 685

Val Thr Lys Cys Lys Pro Val Met Ile Ile Thr Glu Tyr Met Glu Asn

690 695 700

Gly Ser Leu Asp Ala Phe Leu Arg Lys Asn Asp Gly Arg Phe Thr Val

705 710 715 720

Ile Gln Leu Val Gly Met Leu Arg Gly Ile Gly Ser Gly Met Lys Tyr

725 730 735

Leu Ser Asp Met Ser Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile

740 745 750

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Met Ser

755 760 765

Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg Gly Gly

770 775 780

Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr Arg Lys

785 790 795 800

Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met Trp Glu

805 810 815

Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp

820 825 830

Val Ile Lys Ala Ile Glu Glu Gly Tyr Arg Leu Pro Pro Pro Met Asp

835 840 845

Cys Pro Ile Ala Leu His Gln Leu Met Leu Asp Cys Trp Gln Lys Glu

850 855 860

Arg Ser Asp Arg Pro Lys Phe Gly Gln Ile Val Asn Met Leu Asp Lys

865 870 875 880

Leu Ile Arg Asn Pro Asn Ser Leu Lys Arg Thr Gly Ser Glu Ser Ser

885 890 895

Arg Pro Asn Thr Ala Leu Leu Asp Pro Ser Ser Pro Glu Phe Ser Ala

900 905 910

Val Val Ser Val Gly Asp Trp Leu Gln Ala Ile Lys Met Asp Arg Tyr

915 920 925

Lys Asp Asn Phe Thr Ala Ala Gly Tyr Thr Thr Leu Glu Ala Val Val

930 935 940

His Met Ser Gln Asp Asp Leu Ala Arg Ile Gly Ile Thr Ala Ile Thr

945 950 955 960

His Gln Asn Lys Ile Leu Ser Ser Val Gln Ala Met Arg Thr Gln Met

965 970 975

Gln Gln Met His Gly Arg Met Val Pro Val

980 985

<210> 2

<211> 528

<212> PROTEIN

<213> Mus musculus

<400> 2

Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp

1 5 10 15

Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu

20 25 30

Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile

35 40 45

Arg Thr Tyr Gln Val Cys Asn Val Met Glu Ala Ser Gln Asn Asn Trp

50 55 60

Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile

65 70 75 80

Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met

85 90 95

Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn

100 105 110

Asp Lys Glu Arg Phe Ile Arg Glu Ser Gln Phe Gly Lys Ile Asp Thr

115 120 125

Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile

130 135 140

Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys

145 150 155 160

Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val

165 170 175

Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu

180 185 190

Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val

195 200 205

Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro

210 215 220

Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn

225 230 235 240

Cys Leu Cys Asn Ala Gly His Glu Glu Gln Asn Gly Glu Cys Gln Ala

245 250 255

Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys Ala

260 265 270

Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys

275 280 285

Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met

290 295 300

Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val

305 310 315 320

Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly

325 330 335

Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala

340 345 350

Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr

355 360 365

Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp Leu

370 375 380

Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val

385 390 395 400

Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val Thr

405 410 415

Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu

420 425 430

Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro

435 440 445

Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln

450 455 460

Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp

465 470 475 480

Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala

485 490 495

Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr

500 505 510

Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala Asn Ser Thr

515 520 525

<210> 3

<211> 1030

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 3

Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp

1 5 10 15

Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu

20 25 30

Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile

35 40 45

Arg Thr Tyr Gln Val Cys Asn Val Met Glu Ala Ser Gln Asn Asn Trp

50 55 60

Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile

65 70 75 80

Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met

85 90 95

Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn

100 105 110

Asp Lys Glu Arg Phe Ile Arg Glu Ser Gln Phe Gly Lys Ile Asp Thr

115 120 125

Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile

130 135 140

Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys

145 150 155 160

Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val

165 170 175

Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu

180 185 190

Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val

195 200 205

Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro

210 215 220

Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn

225 230 235 240

Cys Leu Cys Asn Ala Gly His Glu Glu Gln Asn Gly Glu Cys Gln Ala

245 250 255

Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Ser Cys Ala

260 265 270

Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys

275 280 285

Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met

290 295 300

Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val

305 310 315 320

Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly

325 330 335

Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala

340 345 350

Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr

355 360 365

Pro Gln Gln Asn Gly Leu Lys Thr Thr Arg Val Ser Ile Thr Asp Leu

370 375 380

Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val

385 390 395 400

Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val Thr

405 410 415

Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu

420 425 430

Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro

435 440 445

Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln

450 455 460

Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp

465 470 475 480

Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala

485 490 495

Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr

500 505 510

Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala Asn Ser Thr

515 520 525

Ala Ala Ala Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn

530 535 540

Arg Glu Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala

545 550 555 560

Gln Thr Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly

565 570 575

Val Ser Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp

580 585 590

Lys Leu Gly Pro Glu Ile Pro Leu Ala Met Asp Arg Phe Pro Tyr Val

595 600 605

Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly

610 615 620

Ala Thr Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr

625 630 635 640

Ile Gly Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg

645 650 655

Gly Asn Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys

660 665 670

Ser Val Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala

675 680 685

Gly Thr Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp

690 695 700

Val Pro Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln

705 710 715 720

Leu Ile Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys

725 730 735

Tyr Met Phe Arg Met Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr

740 745 750

Ser Gln Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp

755 760 765

Leu Ala Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu

770 775 780

Met Gln Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe

785 790 795 800

Glu Pro Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr Leu Asp

805 810 815

Pro Ser Leu Met Glu Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg

820 825 830

Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile Asp His

835 840 845

Gly His His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met

850 855 860

Phe Asp Asp Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp

865 870 875 880

Thr Leu Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly

885 890 895

Gly Tyr Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys

900 905 910

Ala Arg Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro

915 920 925

Gly Tyr Val Leu Lys Asp Gly Ala Arg Pro Asp Val Thr Glu Ser Glu

930 935 940

Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu

945 950 955 960

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

965 970 975

Ala His Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val

980 985 990

Met Ala Phe Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala

995 1000 1005

Pro Pro Ala Gly Thr Thr Asp Ala Ala His Pro Gly His His His

1010 1015 1020

His His His His His His

1025 1030

<210> 4

<211> 19

<212> PROTEIN

<213> Mus musculus

<400> 4

Met Ala Gly Ile Phe Tyr Phe Ile Leu Phe Ser Phe Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala

<210> 5

<211> 986

<212> PROTEIN

<213> Homo sapiens

<400> 5

Met Ala Gly Ile Phe Tyr Phe Ala Leu Phe Ser Cys Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr

20 25 30

Leu Leu Asp Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser

35 40 45

Pro Leu Glu Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn

50 55 60

Thr Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln

65 70 75 80

Asn Asn Trp Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg

85 90 95

Val Tyr Ile Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro

100 105 110

Gly Val Met Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu

115 120 125

Ser Asp Asn Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys

130 135 140

Ile Asp Thr Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly

145 150 155 160

Asp Arg Ile Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu

165 170 175

Ser Lys Lys Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile

180 185 190

Ala Leu Val Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val

195 200 205

Arg Asn Leu Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser

210 215 220

Ser Leu Val Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys

225 230 235 240

Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Asn Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu

260 265 270

Cys Gln Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala

275 280 285

Thr Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala

290 295 300

Thr Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile

325 330 335

Ser Asn Val Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln

340 345 350

Asn Thr Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys

355 360 365

Cys Gly Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val

370 375 380

His Tyr Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr Lys Val Ser Ile

385 390 395 400

Thr Asp Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val

405 410 415

Asn Gly Val Ser Lys Tyr Asn Pro Asn Pro Asp Gln Ser Val Ser Val

420 425 430

Thr Val Thr Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln

435 440 445

Ala Lys Glu Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro

450 455 460

Asp Arg Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu

465 470 475 480

Lys Asp Gln Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg

485 490 495

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

500 505 510

Val Arg Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu

515 520 525

Glu Val Thr Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala

530 535 540

Asn Ser Thr Val Leu Leu Val Ser Val Ser Gly Ser Val Val Leu Val

545 550 555 560

Val Ile Leu Ile Ala Ala Phe Val Ile Ser Arg Arg Arg Ser Lys Tyr

565 570 575

Ser Lys Ala Lys Gln Glu Ala Asp Glu Glu Lys His Leu Asn Gln Gly

580 585 590

Val Arg Thr Tyr Val Asp Pro Phe Thr Tyr Glu Asp Pro Asn Gln Ala

595 600 605

Val Arg Glu Phe Ala Lys Glu Ile Asp Ala Ser Cys Ile Lys Ile Glu

610 615 620

Lys Val Ile Gly Val Gly Glu Phe Gly Glu Val Cys Ser Gly Arg Leu

625 630 635 640

Lys Val Pro Gly Lys Arg Glu Ile Cys Val Ala Ile Lys Thr Leu Lys

645 650 655

Ala Gly Tyr Thr Asp Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser

660 665 670

Ile Met Gly Gln Phe Asp His Pro Asn Ile Ile His Leu Glu Gly Val

675 680 685

Val Thr Lys Cys Lys Pro Val Met Ile Ile Thr Glu Tyr Met Glu Asn

690 695 700

Gly Ser Leu Asp Ala Phe Leu Arg Lys Asn Asp Gly Arg Phe Thr Val

705 710 715 720

Ile Gln Leu Val Gly Met Leu Arg Gly Ile Gly Ser Gly Met Lys Tyr

725 730 735

Leu Ser Asp Met Ser Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile

740 745 750

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Met Ser

755 760 765

Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg Gly Gly

770 775 780

Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr Arg Lys

785 790 795 800

Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met Trp Glu

805 810 815

Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp

820 825 830

Val Ile Lys Ala Ile Glu Glu Gly Tyr Arg Leu Pro Pro Pro Met Asp

835 840 845

Cys Pro Ile Ala Leu His Gln Leu Met Leu Asp Cys Trp Gln Lys Glu

850 855 860

Arg Ser Asp Arg Pro Lys Phe Gly Gln Ile Val Asn Met Leu Asp Lys

865 870 875 880

Leu Ile Arg Asn Pro Asn Ser Leu Lys Arg Thr Gly Thr Glu Ser Ser

885 890 895

Arg Pro Asn Thr Ala Leu Leu Asp Pro Ser Ser Pro Glu Phe Ser Ala

900 905 910

Val Val Ser Val Gly Asp Trp Leu Gln Ala Ile Lys Met Asp Arg Tyr

915 920 925

Lys Asp Asn Phe Thr Ala Ala Gly Tyr Thr Thr Leu Glu Ala Val Val

930 935 940

His Val Asn Gln Glu Asp Leu Ala Arg Ile Gly Ile Thr Ala Ile Thr

945 950 955 960

His Gln Asn Lys Ile Leu Ser Ser Val Gln Ala Met Arg Thr Gln Met

965 970 975

Gln Gln Met His Gly Arg Met Val Pro Val

980 985

<210> 6

<211> 528

<212> PROTEIN

<213> Homo sapiens

<400> 6

Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp

1 5 10 15

Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu

20 25 30

Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile

35 40 45

Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln Asn Asn Trp

50 55 60

Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile

65 70 75 80

Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met

85 90 95

Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn

100 105 110

Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys Ile Asp Thr

115 120 125

Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile

130 135 140

Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys

145 150 155 160

Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val

165 170 175

Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu

180 185 190

Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val

195 200 205

Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro

210 215 220

Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn

225 230 235 240

Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu Cys Gln Ala

245 250 255

Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Thr Cys Ala

260 265 270

Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys

275 280 285

Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met

290 295 300

Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val

305 310 315 320

Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly

325 330 335

Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala

340 345 350

Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr

355 360 365

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

370 375 380

Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val

385 390 395 400

Ser Lys Tyr Asn Pro Asn Pro Asp Gln Ser Val Ser Val Thr Val Thr

405 410 415

Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu

420 425 430

Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro

435 440 445

Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln

450 455 460

Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp

465 470 475 480

Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala

485 490 495

Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr

500 505 510

Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala Asn Ser Thr

515 520 525

<210> 7

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223>ad29S

<400> 7

acatcactcc gt 12

<210> 8

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> ad29AS

<400> 8

acggagtgat gtccgtcgac gtatctctgc gttgatactt cagcgtagct 50

<210> 9

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 9

agctacgctg aagtatcaac gcagag 26

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 10

gccagtggat agactgatgg 20

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 11

gatggataca gttggtgcag c 21

<210> 12

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 12

Met Ala Val Leu Val Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

Val Leu Ser

<210> 13

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 13

Gln Val Gln Leu Lys Gln Ser Gly Pro Ser Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys Glu Ser Leu Phe Gly Val Tyr Tyr Asp Tyr Gly Tyr Tyr Ser Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 14

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 14

Met Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Thr Arg Cys

20

<210> 15

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 15

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 Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser 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 Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 16

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 16

atggctgtcc tggtgctgct cctctgcctg gtgacattcc caagctgtgt cctgtcc 57

<210> 17

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 17

caggtgcagc tgaagcagtc aggacctagc ctagtgcagc cctcacagag cctgtccata 60

acctgcacag tctctggttt ctcattaact aggtatggtg tacactgggt tcgccagtct 120

caggaaagg gtctggagtg gctgggagtg atttggagag gtggaagcac agactacaat 180

gcagctttca tgtccagact gagcatcacc aaggacaact ccaagagcca agttttcttt 240

aaaatgaaca gtctgcaagc tgatgacact gccatatact actgtgccaa agaaagccta 300

tttggggtct actatgatta cgggtactat tctatggact actggggtca aggaacctca 360

gtcaccgtct cctca 375

<210> 18

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 18

atggacatga gggttcctgc tcacgttttt ggcttcttgt tgctctggtt tccaggtacc 60

agatgt 66

<210> 19

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 19

gacatccaaa tgacccagtc tccatcctcc ttatctgcct ctctggggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt atccgctcac gttcggtgct 300

gggaccaagc tggagctgaa a 321

<210> 20

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 20

gcgaagcttg ccgccaccat ggctgtcctg gtgctgctcc 40

<210> 21

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 21

gcgaagcttg ccgccaccat ggacatgagg gttcctgctc acg 43

<210> 22

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 22

gcggaattca tcatttacca gggagagtggg agaggc 36

<210> 23

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic primer

<400> 23

cgcgaattca ctaacactca ttcctgttga agctcttgac 40

<210> 24

<211> 324

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 24

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 25

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 25

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 26

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 26

Arg Tyr Gly Val His

15

<210> 27

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 27

Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met Ser

1 5 10 15

<210> 28

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 28

Glu Ser Leu Phe Gly Val Tyr Tyr Asp Tyr Gly Tyr Tyr Ser Met Asp

1 5 10 15

Tyr

<210> 29

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 29

Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser

1 5 10

<210> 30

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 30

Ala Ala Ser Thr Leu Asp Ser

15

<210> 31

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic peptide

<400> 31

Leu Gln Tyr Ala Ser Tyr Pro Leu Thr

15

<210> 32

<211> 986

<212> PROTEIN

<213> Macaca mulatta

<400> 32

Met Ala Gly Ile Phe Tyr Phe Ala Leu Phe Ser Cys Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr

20 25 30

Leu Leu Asp Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser

35 40 45

Pro Leu Glu Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn

50 55 60

Thr Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln

65 70 75 80

Asn Asn Trp Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg

85 90 95

Val Tyr Ile Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro

100 105 110

Gly Val Met Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu

115 120 125

Ser Asp Asn Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys

130 135 140

Ile Asp Thr Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly

145 150 155 160

Asp Arg Ile Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu

165 170 175

Ser Lys Lys Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile

180 185 190

Ala Leu Val Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val

195 200 205

Arg Asn Leu Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser

210 215 220

Ser Leu Val Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys

225 230 235 240

Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Asn Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu

260 265 270

Cys Gln Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala

275 280 285

Thr Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala

290 295 300

Thr Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile

325 330 335

Ser Asn Val Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln

340 345 350

Asn Thr Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys

355 360 365

Cys Gly Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val

370 375 380

His Tyr Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr Lys Val Ser Ile

385 390 395 400

Thr Asp Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val

405 410 415

Asn Gly Val Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val

420 425 430

Thr Val Thr Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln

435 440 445

Ala Lys Glu Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro

450 455 460

Asp Arg Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu

465 470 475 480

Lys Asp Gln Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg

485 490 495

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

500 505 510

Val Arg Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu

515 520 525

Glu Val Thr Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala

530 535 540

Asn Ser Thr Val Leu Leu Val Ser Val Ser Gly Ser Val Val Leu Val

545 550 555 560

Val Ile Leu Ile Ala Ala Phe Val Ile Ser Arg Arg Arg Ser Lys Tyr

565 570 575

Ser Lys Ala Lys Gln Glu Ala Asp Glu Glu Lys His Leu Asn Gln Gly

580 585 590

Val Arg Thr Tyr Val Asp Pro Phe Thr Tyr Glu Asp Pro Asn Gln Ala

595 600 605

Val Arg Glu Phe Ala Lys Glu Ile Asp Ala Ser Cys Ile Lys Ile Glu

610 615 620

Lys Val Ile Gly Val Gly Glu Phe Gly Glu Val Cys Ser Gly Arg Leu

625 630 635 640

Lys Val Pro Gly Lys Arg Glu Ile Cys Val Ala Ile Lys Thr Leu Lys

645 650 655

Ala Gly Tyr Thr Asp Lys Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser

660 665 670

Ile Met Gly Gln Phe Asp His Pro Asn Ile Ile His Leu Glu Gly Val

675 680 685

Val Thr Lys Cys Lys Pro Val Met Ile Ile Thr Glu Tyr Met Glu Asn

690 695 700

Gly Ser Leu Asp Ala Phe Leu Arg Lys Asn Asp Gly Arg Phe Thr Val

705 710 715 720

Ile Gln Leu Val Gly Met Leu Arg Gly Ile Gly Ser Gly Met Lys Tyr

725 730 735

Leu Ser Asp Met Ser Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile

740 745 750

Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Met Ser

755 760 765

Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg Gly Gly

770 775 780

Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr Arg Lys

785 790 795 800

Phe Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met Trp Glu

805 810 815

Val Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp

820 825 830

Val Ile Lys Ala Ile Glu Glu Gly Tyr Arg Leu Pro Pro Pro Met Asp

835 840 845

Cys Pro Ile Ala Leu His Gln Leu Met Leu Asp Cys Trp Gln Lys Glu

850 855 860

Arg Ser Asp Arg Pro Lys Phe Gly Gln Ile Val Asn Met Leu Asp Lys

865 870 875 880

Leu Ile Arg Asn Pro Asn Ser Leu Lys Arg Thr Gly Thr Glu Ser Ser

885 890 895

Arg Pro Asn Thr Ala Leu Leu Asp Pro Ser Ser Pro Glu Phe Ser Ala

900 905 910

Val Val Ser Val Gly Asp Trp Leu Gln Ala Ile Lys Met Asp Arg Tyr

915 920 925

Lys Asp Asn Phe Thr Ala Ala Gly Tyr Thr Thr Leu Glu Ala Val Val

930 935 940

His Val Asn Gln Glu Asp Leu Ala Arg Ile Gly Ile Thr Ala Ile Thr

945 950 955 960

His Gln Asn Lys Ile Leu Ser Ser Val Gln Ala Met Arg Thr Gln Met

965 970 975

Gln Gln Met His Gly Arg Met Val Pro Val

980 985

<210> 33

<211> 528

<212> PROTEIN

<213> Macaca mulatta

<400> 33

Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr Leu Leu Asp

1 5 10 15

Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser Pro Leu Glu

20 25 30

Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn Thr Pro Ile

35 40 45

Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln Asn Asn Trp

50 55 60

Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg Val Tyr Ile

65 70 75 80

Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro Gly Val Met

85 90 95

Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu Ser Asp Asn

100 105 110

Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys Ile Asp Thr

115 120 125

Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly Asp Arg Ile

130 135 140

Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu Ser Lys Lys

145 150 155 160

Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile Ala Leu Val

165 170 175

Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val Arg Asn Leu

180 185 190

Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser Ser Leu Val

195 200 205

Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys Asp Val Pro

210 215 220

Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro Ile Gly Asn

225 230 235 240

Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu Cys Gln Ala

245 250 255

Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala Thr Cys Ala

260 265 270

Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala Thr Ser Cys

275 280 285

Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala Ala Ser Met

290 295 300

Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile Ser Asn Val

305 310 315 320

Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln Asn Thr Gly

325 330 335

Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys Cys Gly Ala

340 345 350

Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val His Tyr Thr

355 360 365

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

370 375 380

Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val Asn Gly Val

385 390 395 400

Ser Lys Tyr Asn Pro Ser Pro Asp Gln Ser Val Ser Val Thr Val Thr

405 410 415

Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu

420 425 430

Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro

435 440 445

Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln

450 455 460

Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp

465 470 475 480

Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala

485 490 495

Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr

500 505 510

Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala Asn Ser Thr

515 520 525

<210> 34

<211> 19

<212> PROTEIN

<213> Homo sapiens

<400> 34

Met Ala Gly Ile Phe Tyr Phe Ala Leu Phe Ser Cys Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala

<210> 35

<211> 15

<212> PROTEIN

<213> Homo sapiens

<400> 35

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala

1 5 10 15

<210> 36

<211> 547

<212> PROTEIN

<213> Homo sapiens

<400> 36

Met Ala Gly Ile Phe Tyr Phe Ala Leu Phe Ser Cys Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr

20 25 30

Leu Leu Asp Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser

35 40 45

Pro Leu Glu Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn

50 55 60

Thr Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln

65 70 75 80

Asn Asn Trp Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg

85 90 95

Val Tyr Ile Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro

100 105 110

Gly Val Met Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu

115 120 125

Ser Asp Asn Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys

130 135 140

Ile Asp Thr Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly

145 150 155 160

Asp Arg Ile Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu

165 170 175

Ser Lys Lys Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile

180 185 190

Ala Leu Val Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val

195 200 205

Arg Asn Leu Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser

210 215 220

Ser Leu Val Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys

225 230 235 240

Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Asn Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu

260 265 270

Cys Gln Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala

275 280 285

Thr Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala

290 295 300

Thr Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro Leu Asn Leu Ile

325 330 335

Ser Asn Val Asn Glu Thr Ser Val Asn Leu Glu Trp Ser Ser Pro Gln

340 345 350

Asn Thr Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val Val Cys Lys Lys

355 360 365

Cys Gly Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys Gly Ser Gly Val

370 375 380

His Tyr Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr Lys Val Ser Ile

385 390 395 400

Thr Asp Leu Leu Ala His Thr Asn Tyr Thr Phe Glu Ile Trp Ala Val

405 410 415

Asn Gly Val Ser Lys Tyr Asn Pro Asn Pro Asp Gln Ser Val Ser Val

420 425 430

Thr Val Thr Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln

435 440 445

Ala Lys Glu Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro

450 455 460

Asp Arg Pro Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu

465 470 475 480

Lys Asp Gln Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg

485 490 495

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

500 505 510

Val Arg Ala Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu

515 520 525

Glu Val Thr Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala

530 535 540

Asn Ser Thr

545

<210> 37

<211> 328

<212> PROTEIN

<213> Homo sapiens

<400> 37

Met Ala Gly Ile Phe Tyr Phe Ala Leu Phe Ser Cys Leu Phe Gly Ile

1 5 10 15

Cys Asp Ala Val Thr Gly Ser Arg Val Tyr Pro Ala Asn Glu Val Thr

20 25 30

Leu Leu Asp Ser Arg Ser Val Gln Gly Glu Leu Gly Trp Ile Ala Ser

35 40 45

Pro Leu Glu Gly Gly Trp Glu Glu Val Ser Ile Met Asp Glu Lys Asn

50 55 60

Thr Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Met Glu Pro Ser Gln

65 70 75 80

Asn Asn Trp Leu Arg Thr Asp Trp Ile Thr Arg Glu Gly Ala Gln Arg

85 90 95

Val Tyr Ile Glu Ile Lys Phe Thr Leu Arg Asp Cys Asn Ser Leu Pro

100 105 110

Gly Val Met Gly Thr Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Tyr Glu

115 120 125

Ser Asp Asn Asp Lys Glu Arg Phe Ile Arg Glu Asn Gln Phe Val Lys

130 135 140

Ile Asp Thr Ile Ala Ala Asp Glu Ser Phe Thr Gln Val Asp Ile Gly

145 150 155 160

Asp Arg Ile Met Lys Leu Asn Thr Glu Ile Arg Asp Val Gly Pro Leu

165 170 175

Ser Lys Lys Gly Phe Tyr Leu Ala Phe Gln Asp Val Gly Ala Cys Ile

180 185 190

Ala Leu Val Ser Val Arg Val Phe Tyr Lys Lys Cys Pro Leu Thr Val

195 200 205

Arg Asn Leu Ala Gln Phe Pro Asp Thr Ile Thr Gly Ala Asp Thr Ser

210 215 220

Ser Leu Val Glu Val Arg Gly Ser Cys Val Asn Asn Ser Glu Glu Lys

225 230 235 240

Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Asn Cys Leu Cys Asn Ala Gly His Glu Glu Arg Ser Gly Glu

260 265 270

Cys Gln Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu Ser Thr Asp Ala

275 280 285

Thr Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val Trp Glu Gly Ala

290 295 300

Thr Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala Asp Asn Asp Ala

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro

325

<210> 38

<211> 236

<212> PROTEIN

<213> Homo sapiens

<400> 38

Pro Leu Thr Val Arg Asn Leu Ala Gln Phe Pro Asp Thr Ile Thr Gly

1 5 10 15

Ala Asp Thr Ser Ser Leu Val Glu Val Arg Gly Ser Cys Val Asn Asn

20 25 30

Ser Glu Glu Lys Asp Val Pro Lys Met Tyr Cys Gly Ala Asp Gly Glu

35 40 45

Trp Leu Val Pro Ile Gly Asn Cys Leu Cys Asn Ala Gly His Glu Glu

50 55 60

Arg Ser Gly Glu Cys Gln Ala Cys Lys Ile Gly Tyr Tyr Lys Ala Leu

65 70 75 80

Ser Thr Asp Ala Thr Cys Ala Lys Cys Pro Pro His Ser Tyr Ser Val

85 90 95

Trp Glu Gly Ala Thr Ser Cys Thr Cys Asp Arg Gly Phe Phe Arg Ala

100 105 110

Asp Asn Asp Ala Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro

115 120 125

Leu Asn Leu Ile Ser Asn Val Asn Glu Thr Ser Val Asn Leu Glu Trp

130 135 140

Ser Ser Pro Gln Asn Thr Gly Gly Arg Gln Asp Ile Ser Tyr Asn Val

145 150 155 160

Val Cys Lys Lys Cys Gly Ala Gly Asp Pro Ser Lys Cys Arg Pro Cys

165 170 175

Gly Ser Gly Val His Tyr Thr Pro Gln Gln Asn Gly Leu Lys Thr Thr

180 185 190

Lys Val Ser Ile Thr Asp Leu Leu Ala His Thr Asn Tyr Thr Phe Glu

195 200 205

Ile Trp Ala Val Asn Gly Val Ser Lys Tyr Asn Pro Asn Pro Asp Gln

210 215 220

Ser Val Ser Val Thr Val Thr Thr Asn Gln Ala Ala

225 230 235

<210> 39

<211> 112

<212> PROTEIN

<213> Homo sapiens

<400> 39

Thr Asn Gln Ala Ala Pro Ser Ser Ile Ala Leu Val Gln Ala Lys Glu

1 5 10 15

Val Thr Arg Tyr Ser Val Ala Leu Ala Trp Leu Glu Pro Asp Arg Pro

20 25 30

Asn Gly Val Ile Leu Glu Tyr Glu Val Lys Tyr Tyr Glu Lys Asp Gln

35 40 45

Asn Glu Arg Ser Tyr Arg Ile Val Arg Thr Ala Ala Arg Asn Thr Asp

50 55 60

Ile Lys Gly Leu Asn Pro Leu Thr Ser Tyr Val Phe His Val Arg Ala

65 70 75 80

Arg Thr Ala Ala Gly Tyr Gly Asp Phe Ser Glu Pro Leu Glu Val Thr

85 90 95

Thr Asn Thr Val Pro Ser Arg Ile Ile Gly Asp Gly Ala Asn Ser Thr

100 105 110

<210> 40

<211> 96

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 40

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 Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly 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 His Asn Ser Tyr Pro Pro

85 90 95

<210> 41

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 41

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 42

<211> 98

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys

<210> 43

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 43

Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr

1 5 10 15

Val Ser

<210> 44

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 44

Arg Phe Gly Val His

15

<210> 45

<211> 125

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 45

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Arg Phe

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Glu Ser Leu Phe Gly Val Tyr Tyr Asp Tyr Gly Tyr Tyr Ser Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 46

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 46

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 Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp 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 Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 326

<212> PROTEIN

<213> Homo sapiens

<400> 47

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Ala Ala Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 48

<211> 107

<212> PROTEIN

<213> Homo sapiens

<400> 48

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 49

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 49

agatttggag tgcat 15

<210> 50

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 50

gtgatctgga ggggaggatc caccgactac aacgctgctt ttatgagc 48

<210> 51

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 51

gagagcctgt tcggcgtgta ctatgactac ggctactatt ctatggatta t 51

<210> 52

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 52

cgcgcctccc aggagatctc tggctacctg tcc 33

<210> 53

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 53

gctgcctcca ccctggactc t 21

<210> 54

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 54

ctgcagtacg cttcctatcc actgacc 27

<210> 55

<211> 375

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 55

caggtgcagc tggtggagag cggagaggagga gtggtgcagc ctggcaggtc cctgaggctg 60

agctgtgccg tgtccggctt cagcctgaca agatttggag tgcattgggt gcgccaggct 120

ccaggcaagg gactggagtg ggtggccgtg atctggaggg gaggatccac cgactacaac 180

gctgctttta tgagccggct gacaatctct aaggataact ccaagaatac cgtgtatctg 240

cagatgaact ccctgagggc tgaggacacc gccgtgtact attgcgccaa ggagagcctg 300

ttcggcgtgt actatgacta cggctactat tctatggatt attggggcca gggcaccaca 360

gtgacagtgt cctcc 375

<210> 56

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 56

gatatccaga tgacacagtc cccttccagc ctgtctgcct ccgtgggcga cagagtgacc 60

atcacatgtc gcgcctccca ggagatctct ggctacctgt cctggctgca gcagaagcca 120

ggcaaggctc ccaagcgcct gatctatgct gcctccaccc tggactctgg agtgccttcc 180

aggttcagcg gctctcggtc cggcacagag tacaccctga caatctcttc cctgcagcct 240

gaggatttcg ccacctacta ttgcctgcag tacgcttcct atccactgac ctttggcggc 300

ggcacaaagg tggagatcaa g 321

<210> 57

<211> 978

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 57

gcctccacca agggaccatc cgtgttccca ctggccccat cttcccggag cacatctgag 60

tccaccgccg ctctgggctg tctggtgaag gattacttcc ctgagccagt gacagtgtct 120

tggaactccg gcgctctgac atccggcgtg cacacctttc ctgccgtgct gcagagctct 180

ggcctgtaca gcctgtccag cgtggtgacc gtgccctctt ccaatttcgg cacccagaca 240

tatacctgca acgtggacca taagccttcc aatacaaagg tggataagac cgtggagaga 300

aagagctgcg tggagtgtcc accttgccca gctccaccag ccgctgcccc tagcgtgttc 360

ctgtttcctc caaagccaaa ggacacactg atgatctctc gcacacccga ggtgacctgc 420

gtggtggtgg acgtgtccca cgaggatcca gaggtgcagt ttaactggta cgtggatggc 480

gtggaggtgc ataatgctaa gaccaagccc agggaggagc agttcaactc cacatttcgg 540

gtggtgagcg tgctgaccgt ggtgcaccag gactggctga acggcaagga gtataagtgt 600

aaggtgagca ataagggcct gcccgcccct atcgagaaga caatctctaa gaccaagggc 660

cagcctagag agccacaggt gtacaccctg cccccttctc gcgaggagat gacaaagaac 720

caggtgtccc tgacctgcct ggtgaagggc ttctatccta gcgacatcgc tgtggagtgg 780

gagtctaatg gccagccaga gaacaattac aagaccacac cacccatgct ggacagcgat 840

ggctctttct ttctgtattc taagctgaca gtggataagt ccaggtggca gcagggcaac 900

gtgtttagct gctctgtgat gcatgaggcc ctgcacaatc attacaccca gaagtccctg 960

agcctgtctc caggcaag 978

<210> 58

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 58

aggacagtgg ccgctccatc cgtgttcatc tttcccccta gcgacgagca gctgaagagc 60

ggcaccgcct ctgtggtgtg cctgctgaac aatttctacc ccagggaggc caaggtgcag 120

tggaaggtgg ataacgctct gcagagcggc aattctcagg agtccgtgac cgagcaggac 180

agcaaggatt ctacatattc cctgagctct accctgacac tgagcaaggc cgattacgag 240

aagcacaagg tgtatgcttg cgaggtgacc catcagggcc tgtccagccc agtgacaaag 300

tcttttaaca ggggcgagtg t 321

<210> 59

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 59

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ser Leu Thr Arg Phe

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Glu Ser Leu Phe Gly Val Tyr Tyr Asp Tyr Gly Tyr Tyr Ser Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Arg Ser Thr Ser

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu

210 215 220

Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Ala Ala

225 230 235 240

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe

290 295 300

Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 60

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 60

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 Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp 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 Ala Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 61

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 61

caggtgcagc tggtggagag cggagaggagga gtggtgcagc ctggcaggtc cctgaggctg 60

agctgtgccg tgtccggctt cagcctgaca agatttggag tgcattgggt gcgccaggct 120

ccaggcaagg gactggagtg ggtggccgtg atctggaggg gaggatccac cgactacaac 180

gctgctttta tgagccggct gacaatctct aaggataact ccaagaatac cgtgtatctg 240

cagatgaact ccctgagggc tgaggacacc gccgtgtact attgcgccaa ggagagcctg 300

ttcggcgtgt actatgacta cggctactat tctatggatt attggggcca gggcaccaca 360

gtgacagtgt cctccgcctc caccaaggga ccatccgtgt tcccactggc cccatcttcc 420

cggagcacat ctgagtccac cgccgctctg ggctgtctgg tgaaggatta cttccctgag 480

ccagtgacag tgtcttggaa ctccggcgct ctgacatccg gcgtgcacac ctttcctgcc 540

gtgctgcaga gctctggcct gtacagcctg tccagcgtgg tgaccgtgcc ctcttccaat 600

ttcggcaccc agacatatac ctgcaacgtg gaccataagc cttccaatac aaaggtggat 660

aagaccgtgg agagaaagag ctgcgtggag tgtccacctt gcccagctcc accagccgct 720

gcccctagcg tgttcctgtt tcctccaaag ccaaaggaca cactgatgat ctctcgcaca 780

cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg atccagaggt gcagtttaac 840

tggtacgtgg atggcgtgga ggtgcataat gctaagacca agcccaggga ggagcagttc 900

aactccacat ttcgggtggt gagcgtgctg accgtggtgc accaggactg gctgaacggc 960

aaggagtata agtgtaaggt gagcaataag ggcctgcccg cccctatcga gaagacaatc 1020

tctaagacca agggccagcc tagagagcca caggtgtaca ccctgccccc ttctcgcgag 1080

gagatgacaa agaaccaggt gtccctgacc tgcctggtga agggcttcta tcctagcgac 1140

atcgctgtgg agtgggagtc taatggccag ccagagaaca attacaagac cacaccaccc 1200

atgctggaca gcgatggctc tttctttctg tattctaagc tgacagtgga taagtccagg 1260

tggcagcagg gcaacgtgtt tagctgctct gtgatgcatg aggccctgca caatcattac 1320

acccagaagt ccctgagcct gtctccaggc aag 1353

<210> 62

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 62

gatatccaga tgacacagtc cccttccagc ctgtctgcct ccgtgggcga cagagtgacc 60

atcacatgtc gcgcctccca ggagatctct ggctacctgt cctggctgca gcagaagcca 120

ggcaaggctc ccaagcgcct gatctatgct gcctccaccc tggactctgg agtgccttcc 180

aggttcagcg gctctcggtc cggcacagag tacaccctga caatctcttc cctgcagcct 240

gaggatttcg ccacctacta ttgcctgcag tacgcttcct atccactgac ctttggcggc 300

ggcacaaagg tggagatcaa gaggacagtg gccgctccat ccgtgttcat ctttccccct 360

agcgacgagc agctgaagag cggcaccgcc tctgtggtgt gcctgctgaa caatttctac 420

cccagggagg ccaaggtgca gtggaaggtg gataacgctc tgcagagcgg caattctcag 480

gagtccgtga ccgagcagga cagcaaggat tctacatatt ccctgagctc taccctgaca 540

ctgagcaagg ccgattacga gaagcacaag gtgtatgctt gcgaggtgac ccatcagggc 600

ctgtccagcc cagtgacaaa gtcttttaac aggggcgagt gt 642

<---

Claims (37)

1. Anti-EphA4 antibody, where anti-EphA4 antibody contains: (a) a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 44; (b) a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 27; (c) a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 28; (d) a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 29; (e) a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 30; And (f) A light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 31. 2. Anti-EphA4 antibody according to claim 1, wherein the anti-EphA4 antibody is humanized. 3. Anti-EphA4 antibody according to claim 1 or 2, wherein the anti-EphA4 antibody specifically binds to EphA4 and enhances the cleavage of EphA4. 4. An anti-EphA4 antibody according to any one of claims 1 to 3, wherein the anti-EphA4 antibody specifically binds to EphA4 and inhibits the binding between EphA4 and ephrin. 5. Anti-EphA4 antibody according to any one of claims 1-4, where the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 45, and the light chain variable region consists of the amino acid sequence SEQ ID NO: 46. 6. Anti-EphA4 antibody according to any one of claims 1 to 5, wherein the heavy chain constant region and the light chain constant region contain amino acid sequences derived from a human antibody. 7. Anti-EphA4 antibody according to claim 6, where the heavy chain constant region is the human IgG constant region. 8. Anti-EphA4 antibody according to claim 7, where the human IgG constant region is the human IgG ₂ constant region. 9. Anti-EphA4 antibody according to claim 8, where The human IgG ₂ constant region contains the amino acid sequence SEQ ID NO: 47. 10. Anti-EphA4 antibody according to any one of claims 6 to 9, wherein the light chain constant region is the human Igκ constant region. 11. Anti-EphA4 antibody according to claim 10, where The human Igκ constant region contains the amino acid sequence SEQ ID NO: 48. 12. Anti-EphA4 antibody, where anti-EphA4 antibody contains a heavy chain containing the amino acid sequence of SEQ ID NO: 59, and light chain containing the amino acid sequence SEQ ID NO: 60. 13. Anti-EphA4 according to claim 12, where The C-terminal lysine of the heavy chain has been removed. 14. An isolated nucleic acid encoding an anti-EphA4 antibody according to any one of claims 1 to 13. 15. An expression vector containing the nucleic acid according to claim 14. 16. A host cell for producing an anti-EphA4 antibody according to any one of claims 1 to 13, containing a vector according to claim 15. 17. A method for producing anti-EphA4 antibodies, comprising the step of culturing a host cell according to claim 16. 18. Pharmaceutical composition for suppressing phosphorylation of tau protein, where the composition contains an anti-EphA4 antibody according to any one of claims 1 to 13.