Anti-EphA4 antibody

Abstract

[Problem] To provide: an anti-EphA4 antibody that can bind to EphA4 and promote the cleavage of EphA4; and a pharmaceutical composition containing said antibody as an active ingredient. [Solution] An anti-EphA4 antibody comprises a heavy chain including a heavy chain CDR1 of SEQ ID NO: 30, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 32, and a light chain including a light chain CDR1 of SEQ ID NO: 33, a light chain CDR2 of SEQ ID NO: 34, and a light chain CDR3 of SEQ ID NO: 35, or comprises a heavy chain including a heavy chain CDR1 of SEQ ID NO: 42, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 43, and a light chain including a light chain CDR1 of SEQ ID NO: 44, a light chain CDR2 of SEQ ID NO: 34, and a light chain CDR3 of SEQ ID NO: 35.

Description

[Technical Field]

[0001] The present disclosure relates to antibodies that bind to EphA4, nucleic acids encoding the antibodies, vectors containing the nucleic acids, cells containing the vectors, methods for producing the antibodies, and pharmaceutical compositions containing the antibodies. [Background technology]

[0002] EphA4 is a member of the receptor tyrosine kinase family. Ephrin type A and type B are known to be ligands of EphA4, and binding of EphA4 to its ligand, ephrin, induces a detachment signal. It has been suggested that EphA4 is involved in the pathology of Alzheimer's disease (hereinafter also referred to as "AD") (Non-Patent Documents 1 to 4), and it has been reported that inhibition of binding between EphA4 and ephrin inhibits the amyloid β (Aβ) signaling pathway in neurotransmission. (1-42) It has been reported that tau oligomer-mediated dysfunction is rescued (Patent Document 1). In AD, aggregates (neurofibrillary tangles) formed by hyperphosphorylated tau are thought to be involved in neuronal cell death (Non-Patent Document 5). It has also been reported that inhibition of tau phosphorylation suppresses neurodegeneration resulting in synaptic loss (Non-Patent Documents 6 and 7) and improves memory impairment and cognitive dysfunction (Non-Patent Documents 8 to 11). Reports suggest that activation of CDK5 is a factor in tau phosphorylation (Non-Patent Documents 12 and 13).

[0003] EphA4 is highly expressed in the hippocampus and cerebral cortex and is cleaved by matrix metalloproteinases (MMPs), ADAM (a disintegrin and metalloproteinase), and gamma-secretase in an activity-dependent manner. This EphA4 cleavage is known to stabilize spines, which are important structures for neuronal function (Non-Patent Document 14). Decreased spine density has been reported in AD (Non-Patent Document 15). Furthermore, a reduction in EphA4 cleavage fragments has been confirmed in AD NFT stages V and VI, suggesting that EphA4 cleavage is involved in the pathology of AD (Non-Patent Document 16). Furthermore, decreased spine density is known to correlate with cognitive impairment, a clinical symptom of AD (Non-Patent Documents 15 and 17). Furthermore, spine stabilization (increased spine density) has been reported to improve cognitive function in Alzheimer's disease models (Non-Patent Document 18), suggesting that spine stabilization may have therapeutic potential in AD.

[0004] KYL peptide, Compound 1, and the like are known as existing EphA4 inhibitors (Patent Document 2, Non-Patent Documents 19 and 20), but there have been no reports of antibodies that have the activity of promoting EphA4 cleavage. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2016 / 019280A1 [Patent Document 2] WO2012 / 156351A1 [Non-patent literature]

[0006] [Non-Patent Document 1] Vargas LM et al.,, PLoS One. 2014 Mar 21;9(3) [Non-patent document 2] Fu AK et al., Proc Natl Acad Sci US A. 2014 Jul 8;111(27):9959-64 [Non-licensed document 3] Rosenberger AF et al., Acta Neuropathol Commun. 2014 Jul 16;2:79

Non-licensed Document 4

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0007] An objective of the present disclosure is to provide an anti-EphA4 antibody that can bind to EphA4 and promote EphA4 cleavage, and a pharmaceutical composition containing the antibody as an active ingredient. [Means for solving the problem]

[0008] As a result of extensive research to achieve the above object, the present inventors have completed a desired anti-EphA4 antibody that can bind to EphA4 and promote EphA4 cleavage.

[0009] (1) An anti-EphA4 antibody, The anti-EphA4 antibody (a) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 30; (b) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31; and (c) a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 32; and (d) a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 33; (e) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (f) a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35 an anti-EphA4 antibody, or (g) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 42; (h) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31; and (i) a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 43; and (j) a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 44; (k) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35 an anti-EphA4 antibody comprising:

[0010] (2) The anti-EphA4 antibody according to (1), The anti-EphA4 antibody is a human antibody. Anti-EphA4 antibody.

[0011] (3) The anti-EphA4 antibody according to (1) or (2), The anti-EphA4 antibody specifically binds to EphA4 and promotes EphA4 cleavage. Anti-EphA4 antibody.

[0012] (4) The anti-EphA4 antibody according to any one of (1) to (3), The anti-EphA4 antibody specifically binds to EphA4 and inhibits binding of EphA4 to ephrin. Anti-EphA4 antibody.

[0013] (5) The anti-EphA4 antibody according to any one of (1) to (4), the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7, The light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8. Anti-EphA4 antibody.

[0014] (6) The anti-EphA4 antibody according to any one of (1) to (5), the heavy chain constant region and the light chain constant region comprise amino acid sequences derived from a human antibody; Anti-EphA4 antibody.

[0015] (7) The anti-EphA4 antibody according to (6), the heavy chain constant region is a human IgG constant region; Anti-EphA4 antibody.

[0016] (8) The anti-EphA4 antibody according to (7), the human IgG constant region is a human IgG2 constant region; Anti-EphA4 antibody.

[0017] (9) The anti-EphA4 antibody according to (8), The constant region of human IgG2 comprises the amino acid sequence set forth in SEQ ID NO: 15. Anti-EphA4 antibody.

[0018] (10) The anti-EphA4 antibody according to any one of (1) to (4), the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11; The light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12. Anti-EphA4 antibody.

[0019] (11) The anti-EphA4 antibody according to any one of (1) to (4) and (10), the heavy chain constant region and the light chain constant region comprise amino acid sequences derived from a human antibody; Anti-EphA4 antibody.

[0020] (12) The anti-EphA4 antibody according to (11), the heavy chain constant region is a human IgG constant region; Anti-EphA4 antibody.

[0021] (13) The anti-EphA4 antibody according to (12), the human IgG constant region is a human IgG constant region consisting of a combination of human IgG1 and human IgG2; Anti-EphA4 antibody.

[0022] (14) The anti-EphA4 antibody according to (13), The constant region of human IgG consisting of a combination of human IgG1 and human IgG2 comprises the amino acid sequence shown in SEQ ID NO: 16. Anti-EphA4 antibody.

[0023] (15) The anti-EphA4 antibody according to any one of (6) to (9) and (11) to (14), the light chain constant region is a human Igλ constant region; Anti-EphA4 antibody.

[0024] (16) The anti-EphA4 antibody according to (15), The constant region of the human Igλ comprises the amino acid sequence set forth in SEQ ID NO: 17. Anti-EphA4 antibody.

[0025] (17) An anti-EphA4 antibody, the anti-EphA4 antibody comprises a heavy chain and a light chain; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 20; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 21; The C-terminal lysine of the heavy chain may be deleted. Anti-EphA4 antibody.

[0026] (18) The anti-EphA4 antibody according to (17), the C-terminal lysine of the heavy chain is deleted; Anti-EphA4 antibody.

[0027] (19) An anti-EphA4 antibody, the anti-EphA4 antibody comprises a heavy chain and a light chain; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 26; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 27; The C-terminal lysine of the heavy chain may be deleted. Anti-EphA4 antibody.

[0028] (20) The anti-EphA4 antibody according to (19), the C-terminal lysine of the heavy chain is deleted; Anti-EphA4 antibody.

[0029] (21) An isolated nucleic acid encoding the anti-EphA4 antibody according to any one of (1) to (20).

[0030] (22) A vector containing the nucleic acid according to (21).

[0031] (23) A host cell containing the vector according to (22).

[0032] (24) A method for producing an anti-EphA4 antibody, comprising the step of culturing the host cell according to (23).

[0033] (25) A pharmaceutical composition comprising the anti-EphA4 antibody according to any one of (1) to (20).

[0034] (26) The pharmaceutical composition according to (25), comprising at least one pharmaceutically acceptable carrier. [Effects of the Invention]

[0035] The present disclosure provides an anti-EphA4 antibody that can bind to EphA4 and promote EphA4 cleavage, a nucleic acid encoding the antibody, a vector containing the nucleic acid, a cell containing the vector, a method for producing the antibody, and a pharmaceutical composition containing the antibody as an active ingredient. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows the binding affinity of anti-EphA4 monoclonal antibodies (antibodies A and B) to human EphA4. [Figure 2] FIG. 2 shows the inhibitory activity of anti-EphA4 monoclonal antibodies (antibodies A and B) on human EphA4-human ligand binding. [Figure 3] FIG. 3 shows the selectivity of anti-EphA4 monoclonal antibodies (antibodies A and B) for each human Eph receptor. [Figure 4] FIG. 4 shows the reactivity of anti-EphA4 monoclonal antibodies (antibodies A and B) with mouse, rat, monkey, and human EphA4. [Figure 5] FIG. 5 shows the reactivity of anti-EphA4 monoclonal antibodies (antibodies A and B) with the human EphA4 extracellular domain (ECD), ligand-binding domain (LBD), fibronectin type III domain 1 (FN1), and fibronectin type III domain 2 (FN2). [Figure 6] FIG. 6 shows the EphA4 cleavage-promoting activity of anti-EphA4 monoclonal antibodies (antibodies A and B) in hippocampal neurons. [Figure 7]FIG. 7 shows the activity of anti-EphA4 monoclonal antibodies (antibodies A and B) to promote the cleavage of human EphA4 in hippocampal neurons. [Figure 8] FIG. 8 shows the increasing effect of anti-EphA4 monoclonal antibodies (antibodies A and B) on the number of spines in hippocampal neurons. DETAILED DESCRIPTION OF THE INVENTION

[0037] The regions identified or encoded by SEQ ID NOs used herein are as follows: JPEG0007796049000001.jpg169159

[0038] The present disclosure relates to anti-EphA4 antibodies that bind to EphA4. The anti-EphA4 antibody according to the present disclosure is an antibody capable of recognizing and binding to EphA4. As described below, the antibody may be an intact antibody or a synthetic antibody (e.g., a recombinant antibody, a chimeric antibody, a humanized antibody, etc.) as long as it has binding affinity to EphA4. Herein, EphA4 can be understood to refer to EphA4 derived from humans, mice, rats, and monkeys. EphA4 derived from humans, mice, rats, and monkeys can be obtained from public databases containing sequence information, such as Genbank, provided by the National Center for Biotechnology Information. Alternatively, the sequence information of the EphA4 gene can be obtained by designing primers based on the nucleotide sequence information of EphA4 from closely related animal species and cloning the primers from RNA extracted from the desired animal species. For example, the nucleotide sequence information of human, mouse, rat, and monkey EphA4 is registered in the database under Genbank Accession Nos. NM_004438.5, NM_007936.3, NM_001162411.1, and NM_001260870.1, respectively.

[0039] In one aspect, the anti-EphA4 antibody is an antibody that specifically binds to EphA4. The term "specific binding" is a term well known to those skilled in the art, and methods for determining the specific binding of an antibody or its antigen-binding fragment to an antigen or epitope are also well known. In one embodiment, "specific binding" is understood to mean that the anti-EphA4 antibody is capable of immunologically binding to EphA4 with greater binding affinity, avidity, more rapidly, and / or with a longer duration than the antibody binds 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" means binding to EphA4 with at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 In yet another embodiment, "specific binding" is understood to mean binding to EphA4 through an immunological reaction, but not substantially binding to other molecules of the Eph receptor family.

[0040] In one aspect, the anti-EphA4 antibody is an antibody that binds to the extracellular region of EphA4. In one embodiment, the anti-EphA4 antibody is an antibody that binds to the ligand-binding domain (LBD) of the extracellular region of EphA4.

[0041] In one embodiment, the anti-EphA4 antibody specifically binds to EphA4 and promotes EphA4 cleavage. In a specific embodiment, the anti-EphA4 antibody specifically binds to EphA4 and promotes cleavage of the EphA4 extracellular domain by matrix metalloproteinases (MMPs) or ADAMs (a disintegrin and metalloproteinase).

[0042] In one embodiment, the anti-EphA4 antibody can specifically bind to EphA4 and inhibit the binding of EphA4 to its ligand, ephrin.

[0043] In another embodiment, the anti-EphA4 antibody specifically binds to EphA4 and can increase the number of spines in hippocampal neurons or stabilize spines in hippocampal neurons.

[0044] In one embodiment, the present disclosure encompasses anti-EphA4 antibodies that specifically bind to at least one of human EphA4, mouse EphA4, rat EphA4, and monkey EphA4 and can inhibit binding to its ligand. In another embodiment, the present disclosure encompasses anti-EphA4 antibodies that specifically bind to two or more of human EphA4, mouse EphA4, rat EphA4, and monkey EphA4 and can inhibit binding to their ligand. In yet another embodiment, the present disclosure encompasses anti-EphA4 antibodies that specifically bind to all of human EphA4, mouse EphA4, rat EphA4, and monkey EphA4 and can inhibit binding to their ligand.

[0045] The antigen-binding properties (e.g., binding affinity and species cross-reactivity) of anti-EphA4 antibodies can be measured using methods known to those skilled in the art. For example, binding affinity can be measured using, but is not limited to, Biacore® biosensor, KinExA biosensor, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), flow cytometry, fluorescence quenching, fluorescence transfer, yeast display, and / or immunostaining. The neutralizing activity of anti-EphA4 antibodies against the binding of EphA4 to its ligand can be measured using, but is not limited to, Biacore® biosensor, ELISA, and / or flow cytometry.

[0046] The anti-EphA4 antibodies of the present disclosure may be monoclonal antibodies, so long as they bind to EphA4.

[0047] Anti-EphA4 antibodies according to the present disclosure may be of any class, such as IgG, IgA, or IgM (or subclasses thereof), and are not limited to a specific class. Immunoglobulins are classified into different classes based on the antibody amino acid sequence of the constant region of the heavy chain (also called H chain). There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Furthermore, there are two types of antibody light chains (also called L chains): λ chains and κ chains.

[0048] The anti-EphA4 antibody according to the present disclosure may be an IgG antibody, such as an IgG1 antibody or an IgG2 antibody, etc. Furthermore, the anti-EphA4 antibody according to the present disclosure may optionally be in the form of a monomer, a dimer, or a multimer.

[0049] The anti-EphA4 antibody according to the present disclosure may be a combination of IgG antibodies of different subclasses, for example, a human IgG consisting of a combination of IgG1 and IgG2 (in the present disclosure, human IgG 1 / 2 The antibody may be an antibody, etc.

[0050] The variable region of an antibody according to the present disclosure may refer to the variable region of the antibody light chain and / or the variable region of the antibody heavy chain, and the constant region of an antibody may refer to the constant region of the antibody light chain and / or the constant region of the antibody heavy chain. The heavy and light chain variable regions each consist of four framework regions (FRs) linked by three CDRs, also known as complementarity-determining regions. The CDRs in each chain are held in close proximity by the FRs and, together with the CDRs in the other chain, contribute to the formation of the antigen-binding site of the antibody. The Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda, MD), an approach based on interspecies sequence variability, is used to determine CDRs.

[0051] In one embodiment, an anti-EphA4 antibody of the present disclosure comprises a heavy chain constant region and / or a light chain constant region derived from a human antibody.

[0052] As used herein, a monoclonal antibody may refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical except for possible natural mutations. Monoclonal antibodies are directed against a single antigenic site and are highly specific. Furthermore, in contrast to typical polyclonal antibodies, which target different antigens or different epitopes, each monoclonal antibody targets a single epitope on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method.

[0053] In the present disclosure, a human antibody refers to an antibody whose variable and constant region sequences are derived from human immunoglobulin sequences, and also encompasses antibodies containing sequences into which desired modifications have been introduced, such as modifications that increase binding affinity for a target, decrease immunogenicity, or increase stability, as well as modifications that reduce heterogeneity of antibodies produced by non-human cells, so long as they are derived from human immunoglobulin sequences.

[0054] Anti-EphA4 antibodies according to the present disclosure also include antibodies that have been appropriately modified (e.g., modified antibodies, or partial substitutions, additions, and / or deletions of the antibody amino acid sequence) while retaining the functions of the human antibodies (or to add or improve the functions of the antibodies). More specifically, antibodies in which the amino acid sequence of the constant region has been modified to modify the effector function of the antibody are also within the scope of the present disclosure. For example, antibodies in which valine (Val) at position 234 of the EU numbering of a human IgG2 antibody has been substituted with alanine (Ala) and glycine (Gly) at position 237 has been substituted with alanine (Ala) to reduce antibody-dependent cellular cytotoxicity (ADCC) activity and / or antibody-dependent cellular phagocytosis (ADCP) activity are also within the scope of the present disclosure. Furthermore, bispecific antibodies (Kontermann (2012), mAbs 4, 182-97) that have both an antigen-binding site having the CDR sequence of an anti-EphA4 antibody according to the present disclosure and an antigen-binding site that binds to a different antigen are also within the scope of the present disclosure.

[0055] Human antibodies can be produced by methods known in the art. For example, the human antibodies of the present disclosure can be produced as recombinant antibodies by obtaining antibodies with binding affinity to EphA4 from known phage-display human antibody libraries (naive libraries, synthetic libraries, etc.), determining the sequences of their variable regions, and combining them with desired human constant region sequences and introducing them into non-human animal cells.

[0056] Alternatively, human antibodies can be produced by immunizing any of a number of non-human transgenic animals (containing some or all of the human immunoglobulin heavy and light chain loci in their genomes) with an EphA4 antigen. In one embodiment, the non-human animal containing human immunoglobulin genes is an animal with a human immunoglobulin "minilocus" (e.g., GenPharm International, Inc.). In some embodiments, human antibodies can be produced using XenoMouse® mice (Abgenix, Inc., Fremont, CA), HuMAb-Mouse® mice (Medarex, Inc.), VelocImmune® mice (Regeneron Pharmaceuticals, Inc.), AlivaMab mice (Ablexis, LLC), or the like.

[0057] The anti-EphA4 antibodies of the present disclosure may be modified, if desired, to alter (a) the three-dimensional structure of the amino acid sequence in the modified region, such as a sheet or helix conformation; (b) the charge or hydrophobicity state of the molecule at the target site; or (c) the effect of the modification on maintaining the volume of the side chains, or the modification may result in no apparent observable changes.

[0058] Modifications of the anti-EphA4 antibodies of the present disclosure may be achieved, for example, by substitution, deletion, or addition of constituent amino acid residues.

[0059] As used herein, the term "amino acid" is used 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. In light of this broad definition, those skilled in the art will understand that the term "amino acids" as used herein includes, for example, L-amino acids, D-amino acids, chemically modified amino acids such as amino acid variants and amino acid derivatives, amino acids that do not form proteins in vivo, such as norleucine, β-alanine, and ornithine, and chemically synthesized compounds that have the properties of amino acids known to those skilled in the art. Examples of unnatural amino acids include 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-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), amino acids with an extra methylene in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (such as cysteic acid).

[0060] Naturally occurring amino acid residues can be divided into the following groups, for example, based on common 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.

[0061] Non-conservative substitutions in the amino acid sequence constituting an antibody may be made by exchanging an amino acid belonging to one of these groups for an amino acid belonging to another group. More conservative substitutions may be made by exchanging an amino acid belonging to one of these groups for another amino acid from the same group. Similarly, deletions or substitutions may be made as appropriate in the amino acid sequence.

[0062] Modifications of amino acids constituting antibodies may be post-translational modifications such as sugar glycosylation, acetylation, or phosphorylation. Antibodies may be glycosylated at conserved positions in their constant regions. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine ​​(where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in an antibody defines a potential glycosylation site. O-linked glycosylation can involve the attachment of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (e.g., serine or threonine), or, optionally, to 5-hydroxyproline or 5-hydroxylysine. Those skilled in the art can appropriately select glycosylation conditions (for example, the type of host cell or cell culture medium, pH, etc., when glycosylation is carried out using a biological technique) depending on the purpose.

[0063] The anti-EphA4 antibodies of the present disclosure may be further modified by other modification methods, either alone or in combination, based on common technical knowledge known to those skilled in the art.

[0064] The anti-EphA4 antibodies of the present disclosure can be produced by methods well known to those skilled in the art. For example, a nucleic acid encoding an anti-EphA4 antibody of the present disclosure may be incorporated into an expression vector, the expression vector may be introduced into a host cell, and the host cell may be cultured to produce the antibody. Thus, the present disclosure encompasses a nucleic acid encoding an anti-EphA4 antibody, a vector containing the nucleic acid, a host cell containing the vector, and a method for producing an anti-EphA4 antibody, the method comprising culturing the host cell.

[0065] Nucleic acids encoding anti-EphA4 antibodies according to the present disclosure may contain DNA encoding a signal sequence, or may contain DNA encoding a signal sequence at the 5'-end of the DNA encoding the heavy chain variable region and the DNA encoding the light chain variable region. A signal sequence is an amino acid residue present at the N-terminus of a protein that is necessary for a secretory protein or an integral membrane protein to pass through a lipid bilayer after synthesis on the ribosome. In the present disclosure, a signal sequence is not particularly limited as long as it has this function. Signal sequences that may be contained in anti-EphA4 antibodies according to the present disclosure include signal sequences derived from humans, mice, rats, rabbits, donkeys, goats, horses, chickens, dogs, cats, yeast, etc.

[0066] The anti-EphA4 antibodies of the present disclosure may be isolated or purified according to methods known to those skilled in the art.

[0067] As used herein, "isolated" or "purified" means artificially separated or purified from a natural state. When a molecule or composition occurs in nature, it is "isolated" or "purified" when it has been altered or removed from its original environment, or both. Examples of isolation or purification methods include electrophoretic, molecular biological, immunological, or chromatographic techniques, and specific examples include, but are not limited to, ion exchange chromatography, hydrophobic chromatography, reverse-phase HPLC chromatography, isoelectric focusing, or alkaline extraction.

[0068] In one embodiment, the anti-EphA4 antibody comprises the following CDRs: (a) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 30; (b) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31; (c) a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 32; (d) a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 33; (e) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (f) a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35.

[0069] In one embodiment, the anti-EphA4 antibody comprises the following CDRs: (g) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 42; (h) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31; (i) a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 43; (j) a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 44; (k) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (l) A light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 35.

[0070] In one embodiment, the anti-EphA4 antibody is a human antibody.

[0071] In another embodiment, the anti-EphA4 antibody comprises a heavy chain and a light chain, and the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7 or 11, and / or the light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8 or 12. In this embodiment, the variable region of the heavy chain and / or the variable region of the light chain may comprise an amino acid sequence in which one or more amino acids have been substituted, added, and / or deleted in the amino acid sequence set forth in SEQ ID NO: 7 or 11 and / or the amino acid sequence set forth in SEQ ID NO: 8 or 12. Here, "multiple" is not limited as long as it maintains binding affinity to EphA4 and promotes EphA4 cleavage, but may be 2 to 15 or 2 to 10, for example, 9, 8, 7, 6, 5, 4, 3, or 2, or within 10% of the number of amino acids in the amino acid sequence, for example, within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0072] In a specific embodiment, the anti-EphA4 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0073] In a specific embodiment, the anti-EphA4 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11, and the light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12.

[0074] In one embodiment, the heavy chain of the anti-EphA4 antibody comprises a human IgG2 constant region. In a specific embodiment, the human IgG2 constant region comprises the amino acid sequence of SEQ ID NO:15.

[0075] In another embodiment, the heavy chain of the anti-EphA4 antibody comprises a constant region of human IgG consisting of a combination of human IgG1 and IgG2. In a specific embodiment, the constant region of human IgG consisting of a combination of human IgG1 and IgG2 comprises the amino acid sequence of SEQ ID NO: 16.

[0076] In one embodiment, the light chain of the anti-EphA4 antibody comprises the constant region of human Igλ. In a specific embodiment, the constant region of human Igλ comprises the amino acid sequence of SEQ ID NO:17.

[0077] In one embodiment, the heavy chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO:20, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO:21.

[0078] In another embodiment, the heavy chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 20, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 21, and the C-terminal lysine of the heavy chain may be deleted.

[0079] In a specific embodiment, the heavy chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO: 20, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO: 21, with the C-terminal lysine of the heavy chain deleted.

[0080] In one embodiment, the heavy chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO:26, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO:27.

[0081] In another embodiment, the heavy chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 26, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 27, and the C-terminal lysine of the heavy chain may be deleted.

[0082] In a specific embodiment, the heavy chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO: 26, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence set forth in SEQ ID NO: 27, with the C-terminal lysine of the heavy chain deleted.

[0083] In another embodiment, the anti-EphA4 antibody may have a deletion of the lysine located at the C-terminus (carboxy-terminus) of the heavy chain, for example, to reduce heterogeneity of antibodies produced by antibody-producing cells (U.S. Patent Application Publication No. 2010 / 0297697 and Liu H et al., MAbs. 2014 Sep-Oct;6(5):1145-1154). In the present disclosure, anti-EphA4 antibodies having a deletion of the C-terminal lysine of the heavy chain also include anti-EphA4 antibodies in which the C-terminal lysine of the heavy chain has been deleted by genetic modification and anti-EphA4 antibodies in which the C-terminal lysine of the heavy chain has been cleaved after translation by carboxypeptidase or the like. Furthermore, in the present disclosure, anti-EphA4 antibodies in which the C-terminal lysine of the heavy chain has been deleted include not only anti-EphA4 antibodies in which the C-terminal lysine has been deleted in both heavy chains, but also anti-EphA4 antibodies in which the C-terminal lysine has been deleted in only one heavy chain.

[0084] In one aspect, the present disclosure relates to an isolated nucleic acid encoding an anti-EphA4 antibody. An isolated nucleic acid encoding an anti-EphA4 antibody refers to one or more nucleic acid molecules encoding the heavy chain and / or light chain of the anti-EphA4 antibody. In one embodiment, the nucleic acid of the present disclosure encodes the heavy chain of the anti-EphA4 antibody. In another embodiment, the nucleic acid of the present disclosure encodes the light chain of the anti-EphA4 antibody. In yet another embodiment, the nucleic acid of the present disclosure encodes the heavy chain and the light chain of the anti-EphA4 antibody. The nucleic acid of the present disclosure also includes a first nucleic acid molecule encoding the heavy chain of the anti-EphA4 antibody and a second nucleic acid molecule encoding the light chain of the anti-EphA4 antibody.

[0085] In another aspect, the present disclosure relates to a vector comprising an isolated nucleic acid encoding an anti-EphA4 antibody. A vector according to the present disclosure refers to one or more vectors comprising an isolated nucleic acid encoding an anti-EphA4 antibody. In one embodiment, a vector according to the present disclosure comprises a nucleic acid encoding the heavy chain of an anti-EphA4 antibody and a nucleic acid encoding the light chain of an anti-EphA4 antibody. In another embodiment, a vector according to the present disclosure comprises nucleic acids encoding the heavy and light chains of an anti-EphA4 antibody. In yet another embodiment, a vector according to the present disclosure comprises a first vector comprising a nucleic acid encoding the heavy chain of an anti-EphA4 antibody and a second vector comprising a nucleic acid encoding the light chain of an anti-EphA4 antibody. The vector according to the present disclosure may be, but is not limited to, a plasmid, cosmid, virus, phage, or the like. For example, viral vectors according to the present disclosure include retroviral, lentiviral, adenoviral, adeno-associated viral, or herpes simplex viral vectors.

[0086] In yet another aspect, the present disclosure also includes host cells containing the vectors of the present disclosure, and methods for producing anti-EphA4 antibodies, comprising the step of culturing the host cells. Host cells of the present disclosure may be, but are not limited to, Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, NSO cells, etc. In one embodiment, the method for producing anti-EphA4 antibodies comprises the steps of culturing the host cells and recovering the anti-EphA4 antibodies secreted from the host cells (or the culture medium of the host cells).

[0087] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an anti-EphA4 antibody. The pharmaceutical composition of the present disclosure can be prepared according to known methods, such as those described in the Japanese Pharmacopoeia (JP), the United States Pharmacopoeia (USP), or the European Pharmacopoeia (EP).

[0088] The anti-EphA4 antibodies of the present disclosure may be useful for treating Alzheimer's disease. That is, in one embodiment, the present disclosure encompasses a pharmaceutical composition for treating Alzheimer's disease, comprising an anti-EphA4 antibody. In another embodiment, the present disclosure encompasses a method for treating Alzheimer's disease, comprising administering a therapeutically effective amount of an anti-EphA4 antibody to a subject with Alzheimer's disease. In another embodiment, the present disclosure encompasses the use of an anti-EphA4 antibody for the manufacture of a therapeutic agent for Alzheimer's disease. In another embodiment, the present disclosure encompasses an anti-EphA4 antibody for use in treating Alzheimer's disease.

[0089] The anti-EphA4 antibodies of the present disclosure can be used alone or in combination with other agents or compositions in a therapeutic regimen. For example, the anti-EphA4 antibodies of the present disclosure can be administered simultaneously or at different times with another agent. Such combined therapy includes coadministration (two or more agents in the same or separate formulations) and separate administration (e.g., simultaneously or sequentially). When two or more agents are administered separately, administration of the anti-EphA4 antibodies of the present disclosure can precede or follow the administration of the concomitant therapeutic regimen.

[0090] There are no limitations on the subjects to which the pharmaceutical composition of the present disclosure can be administered, and it can be used, for example, for humans or non-human mammals (monkeys, mice, rats, rabbits, cows, horses, goats, etc.).

[0091] The method of administering the pharmaceutical composition according to the present disclosure to a subject (administration route, dosage, number of doses per day, timing of administration, etc.) is not limited and can be appropriately determined by a person skilled in the art (e.g., a physician) depending on the subject's health condition, the severity of the disease, the type of concomitant drug, etc.

[0092] Those skilled in the art will understand that the present disclosure may be implemented by appropriately combining any one or more of the aspects described herein, unless technically inconsistent.Furthermore, those skilled in the art will understand that the present disclosure would preferably be implemented by appropriately combining any preferred or advantageous aspects described herein, unless technically inconsistent.

[0093] The documents cited in this specification should be considered to be expressly incorporated herein by reference in their entirety, and a person skilled in the art would understand, in accordance with the context of this specification, that the relevant disclosure contents of those documents can be incorporated as part of this specification without departing from the spirit and scope of the present disclosure.

[0094] The references cited herein are provided solely for the purpose of disclosing relevant art prior to the filing date of the present application and should not be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements in these references are based on information then available to the applicant and do not constitute any admission that the contents of such statements are accurate.

[0095] The terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0096] As used herein, unless the context clearly dictates otherwise, the term "comprises" intends the presence of stated items (such as components, steps, elements or numbers), and does not exclude the presence of other items (such as components, steps, elements or numbers). The term "consist of" encompasses embodiments described with the terms "consist of" and / or "consist essentially of."

[0097] As used herein, the term "neutralizing activity" refers to the activity of inhibiting the binding of EphA4 to its ligand, and / or the activity of inhibiting signal transduction, cellular molecular expression responses, or functional changes induced in the human body by EphA4 binding to its ligand.

[0098] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0099] Although terms such as "first," "second," etc. are used to describe various elements, it is understood that these elements should not be limited by these terms themselves. These terms are used only to distinguish one element from another. For example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the present disclosure.

[0100] In this specification, numerical values ​​used to indicate component contents, numerical ranges, etc. should be understood to be modified by the term "about" unless otherwise specified. For example, "4°C" is understood to mean "about 4°C" unless otherwise specified, and it is natural that a person skilled in the art would be able to reasonably understand this level in accordance with common technical knowledge and the meaning of this specification.

[0101] Unless the context clearly indicates otherwise, as used in this specification and claims, each aspect appearing in the singular may also be in the plural, and vice versa, unless technically inconsistent.

[0102] The present disclosure will be described in more detail below with reference to examples. However, the present disclosure can be embodied in various forms and should not be construed as being limited to the examples described herein. Those skilled in the relevant technical field can implement the present disclosure with various modifications, additions, deletions, substitutions, etc. without changing the spirit or scope of the present disclosure. [Example]

[0103] Example 1: Generation of human anti-EphA4 monoclonal antibodies To produce monoclonal antibodies that bind to human EphA4 (GenBank Accession No. NP_004429.1, SEQ ID NO: 1), a protein in which the extracellular region of human EphA4 (positions 20 to 547) (SEQ ID NO: 2) was fused with secreted alkaline phosphatase (SEAP) and a histidine tag (hereinafter referred to as "human EphA4 extracellular region-SEAP-His protein"; SEQ ID NO: 3), a protein in which the extracellular region of human EphA4 was fused with the Fc region (Fc) of human IgG1 and a histidine tag (hereinafter referred to as "human EphA4 extracellular region-Fc-His protein"; SEQ ID NO: 4), and a protein in which the extracellular region of human EphA4 was fused with maltose-binding protein (MBP) and a histidine tag (hereinafter referred to as "human EphA4 extracellular region-MBP-His protein"; SEQ ID NO: 5) were prepared by the following steps.

[0104] First, we constructed the pcDNA3.1-human EphA4 extracellular domain-SEAP-His expression vector, the pcDNA3.1-human EphA4 extracellular domain-Fc-His expression vector, and the pcDNA3.4-human EphA4 extracellular domain-MBP-His expression vector. DNA sequences encoding the human EphA4 signal sequence (SEQ ID NO: 6) and extracellular domain were amplified by RT-PCR using total RNA derived from human brain. The amplified DNA was then cloned into the SalI / NotI sites of the pENTR1A vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding SEAP and a histidine tag, or the pENTR1A vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding Fc and a histidine tag. Next, DNA sequences encoding the human EphA4 signal sequence, extracellular domain, SEAP, and histidine tag or the human EphA4 signal sequence, extracellular domain, Fc, and histidine tag were transferred to the pcDNA3.1_rfcB vector by LR reaction using the Gateway System (Invitrogen / LifeTechnologies) to construct the pcDNA3.1-human EphA4 extracellular domain-SEAP-His and pcDNA3.1-human EphA4 extracellular domain-Fc-His expression vectors. For the pcDNA3.4-human EphA4 extracellular domain-MBP-His expression vector, DNA sequences encoding the human EphA4 signal sequence and extracellular domain were amplified by PCR and cloned into the pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding MBP and a histidine tag to construct the human EphA4 extracellular domain-MBP-His protein expression vector. Each of the above expression vectors was transfected into Expi293F cells (Thermo Scientific) using the Expi293 Expression System (Thermo Scientific). After 4 days, the culture medium was collected and clarified to remove the cells. Purification was performed using TALON resin (TaKaRa), and the buffer was replaced with PBS (FUJIFILM Wako Pure) by dialysis.

[0105] Screening was performed using human EphA4 protein and a fully human antibody synthetic phage library to obtain human antibody fragments (scFv) that specifically bind to human EphA4. Human EphA4 extracellular domain-SEAP-His protein was captured using Dynabeads magnetic beads (Thermo Scientific) or nickel plates (Pierce), and the fully human antibody synthetic phage library was added. After 1 or 2 hours, unbound phage were removed by a series of washing cycles using PBS-Tween (0.1% v / v) or PBS. Bound phage particles were eluted and then amplified via infection in E. coli TG1 host cells. Infected TG1 cells were harvested, plated, and incubated at 30°C. This panning process was repeated two more times using the amplified phage.

[0106] After three rounds of panning, single colonies from TG1 cells infected with the enriched phages were inoculated into the medium in a 96-well plate. Expression of the FLAG-tagged scFv was induced by adding IPTG, and the plates were cultured overnight at 30°C with shaking. The TG1 cells were spun down, and wells reactive to human EphA4 were selected using the E. coli culture supernatant containing the scFv.

[0107] Reactivity to human EphA4 was assessed by ELISA using human EphA4 extracellular region-Fc-His protein or human EphA4 extracellular region-MBP-His protein according to the following procedure. Anti-FLAG antibody (SIGMA) was coated onto wells of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with 2% skim milk (BD) at room temperature for 2 hours. After washing three times with 0.02% Tween 20 / PBS (Nacalai Tesque), E. coli culture supernatant containing human EphA4 extracellular region-Fc-His protein or human EphA4 extracellular region-MBP-His protein (final concentration 20 nM) and scFv was added to each well and incubated for 2 hours at room temperature. After washing three times, horseradish peroxidase-conjugated anti-His antibody (MBL) was added and incubated for 1 hour at room temperature. After washing five times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 15-20 minutes at room temperature. An equal volume of stop solution (1N H2SO4, FUJIFILM Wako Pure) was added to the wells, and the absorbance at 450 nm was read using a microplate reader (Thermo Scientific). Human EphA4-specific human antibody fragments were selected as a result of screening, and the gene sequence of each fragment was determined by sequencing.

[0108] The resulting human antibody fragment (scFv) was converted to an IgG format by subcloning DNA sequences encoding the variable regions into vectors expressing the heavy and light chain constant regions of the antibody, respectively. Using the Expi293 expression system (ThermoFisher), an expression vector (pcDNA3.4) containing the gene sequence encoding the human anti-EphA4 monoclonal antibody was transfected into Expi293F cells (ThermoFisher). The supernatant was collected, and human anti-EphA4 monoclonal antibodies were isolated using MabSelectSuRe (Cytiva). The resulting human anti-EphA4 monoclonal antibodies were narrowed down to candidates based on their EphA4 cleavage-promoting activity, EphA4 specificity, phosphorylation status of downstream molecules of EphA4 signaling, and immunogenicity. Immunogenicity was assessed using EpiScreen® (Abzena). In this series of evaluations, antibodies in which the heavy and light chains of human anti-EphA4 monoclonal antibodies were recombined and antibodies in which mutations were introduced into the CDRs were also produced, and a total of more than 300 human anti-EphA4 monoclonal antibodies were evaluated.

[0109] The cleavage-promoting activity of the obtained human anti-EphA4 monoclonal antibody against EphA4 was evaluated using rat hippocampal neurons. Rat hippocampal neurons were prepared as follows: 18-day-pregnant rat fetuses (Charles River Japan) were removed, and the heads were incised to remove the brains. The hippocampal regions were dissected under a stereomicroscope and then placed in digestion solution (137 mM NaCl (Wako Pure Chemicals), 5 mM KCl (Wako Pure Chemicals), 7 mM NaHPO (Wako Pure Chemicals), 25 mM Hepes (DOJINDO), 0.5 mg / mL DNase (Sigma), 0.25% trypsin (Life Technologies)) and shaken at 37°C for 10 minutes. The solution was removed, and 20% fetal bovine serum / Hanks buffer (Sigma) was added. After removing the solution and washing twice with Hanks buffer, the hippocampal tissue was pipetted in Hanks buffer to prepare a cell suspension. The cells were seeded onto a poly-L-lysine-coated 96-well dish (Falcon) containing culture medium (Neurobasal medium (Life technologies), 1× B-27 supplement (Life technologies), 0.5 mM L-glutamine (Life technologies)).

[0110] EphA4 cleavage-promoting activity was assessed using hippocampal neurons as follows. Rat hippocampal neurons seeded in 96-well dishes (Falcon) were treated with anti-EphA4 monoclonal antibody (20 nM) and the γ-secretase inhibitor Compound E (50 nM, Enzo Life Sciences). 24 hours later, the neurons were washed with PBS (Wako Pure Chemical Industries, Ltd.) and harvested in SDS sample buffer (Laemmli sample buffer (Bio-Rad) with 5% 2-mercaptoethanol (Bio-Rad)). The cells were then boiled for 5 minutes. SDS-PAGE and Western blotting analysis or analysis using the automated Western system Jess (Protein Simple) were performed using anti-EphA4 monoclonal antibody (Abnova). Band intensities were quantified, and the ratio of EphA4 C-terminal fragments to full-length EphA4 was calculated.

[0111] The above-mentioned evaluation of EphA4 cleavage-promoting activity yielded two human anti-EphA4 monoclonal antibodies, antibody A and antibody B, which possess the activity of promoting EphA4 cleavage. Antibody A and antibody B did not induce phosphorylation of downstream molecules of EphA4 signaling and were low in immunogenicity (reducing T cell proliferation and IL-2 production by 10% or less).

[0112] The genes encoding the full-length heavy and light chains of antibody A and antibody B were fully synthesized using GenScript. The amino acid sequence of the heavy chain variable region of antibody A is the amino acid sequence shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 8. As the gene sequence encoding the amino acid sequence of antibody A, the nucleic acid sequence shown in SEQ ID NO: 9 was used for the heavy chain variable region, and the nucleic acid sequence shown in SEQ ID NO: 10 was used for the light chain variable region. The amino acid sequence of the heavy chain variable region of antibody B is the amino acid sequence shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 12. As the gene sequence encoding the amino acid sequence of antibody B, the nucleic acid sequence shown in SEQ ID NO: 13 was used for the heavy chain variable region, and the nucleic acid sequence shown in SEQ ID NO: 14 was used for the light chain variable region. The heavy chain constant region of antibody A was the constant region of human IgG2 (SEQ ID NO: 15), and the heavy chain constant region of antibody B was the constant region of human IgG1, in which CH1 and hinge region are human IgG1 and CH2 and CH3 are human IgG2. 1 / 2The constant region of antibody A (SEQ ID NO: 16) was used. Human Igλ (SEQ ID NO: 17) was used as the light chain constant region of antibody A. As gene sequences encoding the amino acid sequence of antibody A, the nucleic acid sequence shown in SEQ ID NO: 18 was used for the heavy chain constant region, and the nucleic acid sequence shown in SEQ ID NO: 19 was used for the light chain constant region. The amino acid sequence of the full-length heavy chain (excluding the signal sequence) of antibody A is the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence of the full-length light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 21. The nucleic acid sequence encoding the full-length heavy chain of antibody A is the nucleic acid sequence shown in SEQ ID NO: 22, and the nucleic acid sequence encoding the full-length light chain is the nucleic acid sequence shown in SEQ ID NO: 23. As gene sequences encoding the amino acid sequence of antibody B, the nucleic acid sequence shown in SEQ ID NO: 24 was used for the heavy chain constant region, and the nucleic acid sequence shown in SEQ ID NO: 25 was used for the light chain constant region. The amino acid sequence of the full-length heavy chain (excluding the signal sequence) of antibody B is the amino acid sequence shown in SEQ ID NO: 26, and the amino acid sequence of the full-length light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 27. The nucleic acid sequence encoding the full-length heavy chain of antibody B is shown in SEQ ID NO: 28, and the nucleic acid sequence encoding the full-length light chain is shown in SEQ ID NO: 29. Using the Expi293 Expression System (ThermoFisher) or the GS System (Lonza), expression vectors (pcDNA3.4 or pEE6.4 and pEE12.4) containing gene sequences encoding the amino acid sequences of antibody A and antibody B were transfected into Expi293F cells (ThermoFisher) or CHOK1SV cells (Lonza). The supernatants were collected, and human anti-EphA4 monoclonal antibodies, antibody A and antibody B, were purified using MabSelectSuRe (Cytiva).

[0113] The CDRs of Antibody A and Antibody B were determined according to the Kabat definition for identifying CDRs (Kabat numbering system). The amino acid and nucleic acid sequences of the CDRs of Antibody A are shown in Tables 1 and 2, respectively. The amino acid and nucleic acid sequences of the CDRs of Antibody B are shown in Tables 3 and 4, respectively. [Table 1] [Table 2] [Table 3] [Table 4]

[0114] Example 2: Binding affinity of human anti-EphA4 monoclonal antibodies to human EphA4 The binding affinity of antibody A and antibody B to human EphA4 was determined by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva). First, an anti-His antibody (Cytiva, 28-9950-56) was immobilized on a CM5 sensor chip. Immobilization was performed by the amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), followed by blocking with ethanolamine (sensor chips and immobilization reagents were all manufactured by Cytiva). The antibody was diluted to 3 μg / mL in immobilization buffer (10 mM sodium acetate, pH 4.5) and immobilized on the sensor chip according to the protocol provided with the Biacore T200. Human EphA4 extracellular domain-SEAP-His10 was diluted in running buffer HBS-EP+ (Cytiva, BR-1001-69) and pumped onto the flow cell for 120 seconds (approximately 6 RU capture volume). Antibody A and antibody B, serially diluted at 100, 50, 25, 12.5, 6.3, 3.2, 1.6, and 0 nM in HBS-EP+, were then applied to the sensor chip for 120 seconds. Binding curves were recorded sequentially during the binding phase (120 seconds) and after the end of the loading phase (600 seconds dissociation phase). After each measurement, the sensor chip was regenerated by adding 3 M MgCl2 (60 seconds). The resulting binding curves were subjected to fitting analysis using a 1:1 binding model using the BIA evaluation software provided with the system, and the binding affinity (KD = kd / ka) for human EphA4 was calculated. The above experiments were performed three times and the average values ​​were calculated for each parameter.

[0115] The binding affinity (KD value) of antibody A and antibody B for human EphA4 was 4.67 × 10 -10 M, 1.56 x 10 -10 The binding affinity of antibody A to human EphA4 was M (Fig. 1). Antibody A and antibody B had similar binding affinity to human EphA4. Representative binding reaction curves are shown in Fig. 1 as examples.

[0116] Example 3: Human EphA4-human ligand binding inhibitory activity of human anti-EphA4 monoclonal antibodies Antibody A and antibody B were evaluated for their inhibitory activity against the binding of human EphA4 to human ligands as follows: Anti-alkaline phosphatase antibody (Thermo Scientific) was coated onto the wells of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with 1% BlockAce (KAC) overnight at 4°C. After washing three times with 0.02% Tween 20 / PBS, human EphA4 extracellular domain-SEAP-His protein (final concentration 10 nM) was seeded into the wells and incubated at room temperature for 1 hour. After washing three times, the ligand and serially diluted antibody A or antibody B (0, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000 nM) were added to the wells. The ligands used were biotinylated human EphrinA5-Fc chimera (R&D Systems, final concentration 0.7 nM) and biotinylated human EphrinB3-Fc chimera (R&D Systems, final concentration 2.3 nM). After incubation at room temperature for 1 hour and washing three times, horseradish peroxidase-conjugated streptavidin (GE Healthcare) was added and incubated at room temperature for 1 hour. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 15–20 minutes at room temperature. An equal volume of stop solution (2N H2SO4, FUJIFILM Wako Pure) was added to the wells, and the absorbance at 450 nm was read using a microplate reader (Thermo Scientific).

[0117] Antibody A and antibody B inhibited the binding of human EphA4 to human ligands in a concentration-dependent manner, and the IC of antibody A for binding to human EphrinA5 and EphrinB3 was 50 The IC values ​​for antibody B were 3.9 nM and 3.0 nM, respectively, for human EphrinA5 and EphrinB3. 50 The values ​​were 4.8 nM and 4.1 nM, respectively. Therefore, it was found that antibody A and antibody B inhibit the binding of human EphA4 to its human ligand (FIG. 2).

[0118] Example 4: Selectivity of human anti-EphA4 monoclonal antibodies for human Eph receptors The binding activity of antibody A and antibody B to human Eph receptors was evaluated as follows: rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto the wells of a 96-well plate (Nunc). After incubation at room temperature for 1 hour or overnight at 4°C, the wells were blocked with 1% BlockAce (KAC) for 1 hour at room temperature. After washing three times with 0.02% Tween 20 / PBS, human Eph receptor extracellular domain-His protein (Creative Biomart, final concentration 1 nM) was seeded into each well and incubated for 1 hour at room temperature. After washing three times, antibody A and antibody B (10 μg / mL) were added and incubated for 1 hour at room temperature. After washing three times, horseradish peroxidase-conjugated rabbit anti-human IgG Fcγ fragment antibody (Jackson ImmunoResearch Laboratories) was added and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 3-5 minutes at room temperature. An equal volume of stop solution (2N H2SO4, FUJIFILM Wako Pure) was added to the wells, and the absorbance at 450 nm was read using a microplate reader (Thermo Scientific).

[0119] Antibody A and antibody B were found to bind specifically to human EphA4 among the human Eph receptor family (FIG. 3).

[0120] Example 5: Reactivity of human anti-EphA4 monoclonal antibodies to mouse, rat, monkey, and human EphA4 Mouse, rat, monkey, and human EphA4 extracellular domain-SEAP-His proteins were produced according to the following steps. Genes encoding SEAP-His and the mouse, rat, monkey, and human EphA4 extracellular domain were synthesized using Genscript. First, the synthesized gene fragment encoding SEAP-His was cloned into the pcDNA3.4 vector (Invitrogen / LifeTechnologies). The synthesized gene fragments encoding the mouse, rat, monkey, and human EphA4 extracellular domain were each cloned into the constructed pcDNA3.4-SEAP-His expression vector to construct mouse, rat, monkey, and human EphA4 extracellular domain-SEAP-His expression vectors. The amino acid sequence of human EphA4 used in vector construction is shown as SEQ ID NO: 1, its extracellular domain as SEQ ID NO: 2, the amino acid sequence of monkey EphA4 as SEQ ID NO: 51, its extracellular domain as SEQ ID NO: 52, the amino acid sequence of rat EphA4 as SEQ ID NO: 53, its extracellular domain as SEQ ID NO: 54, and the amino acid sequence of mouse EphA4 as SEQ ID NO: 55, its extracellular domain as SEQ ID NO: 56. Various EphA4 extracellular domain-SEAP-His proteins were prepared using human EphA4 extracellular domain-SEAP-His protein expression vectors, monkey EphA4 extracellular domain-SEAP-His protein expression vectors, rat EphA4 extracellular domain-SEAP-His protein expression vectors, and mouse EphA4 extracellular domain-SEAP-His protein expression vectors. The above expression vectors were transfected into Expi293F cells (Thermo Scientific) using the Expi293 expression system (Thermo Scientific). After 4 days, the culture medium was collected, the cells were removed, and the medium was clarified. Purification was performed using TALON resin (TaKaRa), and the buffer was replaced with PBS (FUJIFILM Wako Pure) by dialysis.

[0121] The binding activity of antibody A and antibody B to various EphA4 receptors was evaluated as follows: Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto the wells of a 96-well plate (Nunc). After incubation at room temperature for 1 hour, the wells were blocked overnight at 4°C with 1% BlockAce (KAC). After washing three times with 0.02% Tween 20 / PBS, mouse, rat, monkey, and human EphA4 extracellular domain-SEAP-His proteins (final concentration 1 nM) were seeded into the wells and incubated at room temperature for 1 hour. After washing three times, antibody A and antibody B (0, 0.00013, 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, and 10 μg / mL) were added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-conjugated rabbit anti-human IgG Fcγ fragment antibody (Jackson ImmunoResearch Laboratories) was added and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 3-5 minutes at room temperature. An equal volume of stop solution (2N H2SO4, FUJIFILM Wako Pure) was added to the wells, and the absorbance at 450 nm was read using a microplate reader (Thermo Scientific).

[0122] Antibody A and antibody B had comparable binding activity to mouse, rat, monkey, and human EphA4 (FIG. 4).

[0123] Example 6: Reactivity of human anti-EphA4 monoclonal antibodies to the human EphA4 extracellular domain, ligand-binding domain, fibronectin type III domain 1, and fibronectin type III domain 2 Proteins composed of the extracellular domain (ECD), ligand-binding domain (LBD), fibronectin type III domain 1 (FN1), or fibronectin type III domain 2 (FN2) of human EphA4 fused with maltose-binding protein (MBP) and a histidine tag (hereafter referred to as "human EphA4 extracellular domain-MBP-His protein," "human EphA4 ligand-binding domain-MBP-His protein," "human EphA4 fibronectin type III domain 1-MBP-His protein," and "human EphA4 fibronectin type III domain 2-MBP-His protein") were prepared as follows: First, the pcDNA3.4-human EphA4 extracellular domain, ligand-binding domain, fibronectin type III domain 1, or fibronectin type III domain 2-MBP-His expression vector was constructed. First, DNA sequences encoding the human EphA4 signal sequence (sequence number 6) or the preprotrypsin signal sequence (sequence number 57) and each domain of human EphA4 were amplified by PCR and cloned into the pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding MBP with an AAA or G4S linker and a histidine tag to construct expression vectors for human EphA4 extracellular region-MBP-His protein, human EphA4 ligand-binding domain-MBP-His protein, human EphA4 fibronectin type III domain 1-MBP-His protein, and human EphA4 fibronectin type III domain 2-MBP-His protein. The amino acid sequence of human EphA4 used in vector construction is shown as SEQ ID NO: 1, its extracellular region as SEQ ID NO: 2, the ligand-binding domain as SEQ ID NO: 58, fibronectin type III domain 1 as SEQ ID NO: 59, fibronectin type III domain 2 as SEQ ID NO: 60, and MBP and histidine tag (MBP-His protein) as SEQ ID NO: 61. The above expression vector was transfected into Expi293F cells (Thermo Scientific) using the Expi293 expression system (Thermo Scientific). After 4 days, the culture medium was collected and clarified to remove the cells.Human EphA4 extracellular domain-MBP-His protein and human EphA4 ligand-binding domain-MBP-His protein were purified using TALON resin (TaKaRa) and the buffer was replaced with PBS (FUJIFILM Wako Pure) by dialysis. Human EphA4 fibronectin type III domain 1-MBP-His protein and human EphA4 fibronectin type III domain 2-MBP-His protein were purified using amylose resin (NEB) and the monomer fraction was fractionated and purified using AKTA Explore 10s / Superdex200 10 / 300 GL (Cytiva).

[0124] The binding activity of antibody A, antibody B, and human IgG (Sigma) to various EphA4 receptors was evaluated as follows: Rabbit anti-6-His antibody (Bethyl Laboratories) was coated onto the wells of a 96-well plate (Nunc). After incubation at room temperature for 1 hour, the wells were blocked overnight at 4°C with 1% BlockAce (KAC). After washing three times with 0.02% Tween 20 / PBS, human EphA4 extracellular domain, human EphA4 ligand-binding domain, human EphA4 fibronectin type III domain 1, human EphA4 fibronectin type III domain 2-MBP-His protein, or MBP-His protein (final concentration 1 nM) was seeded into the wells and incubated at room temperature for 1 hour. After washing three times, antibody A and antibody B, human IgG (10 nM), were added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-conjugated rabbit anti-human IgG Fcγ fragment antibody (Jackson ImmunoResearch Laboratories) was added and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 3-5 minutes at room temperature. An equal volume of stop solution (2N H2SO4, FUJIFILM Wako Pure) was added to the wells, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Thermo Scientific).

[0125] Antibody A and Antibody B had binding activity to the extracellular domain (ECD) and ligand-binding domain (LBD) of human EphA4 (Figure 5). They did not react with fibronectin type III domain 1 (FN1) or fibronectin type III domain 2 (FN2). Therefore, it was demonstrated that Antibody A and Antibody B specifically bind to the ligand-binding domain of the extracellular domain of human EphA4.

[0126] Example 7: EphA4 cleavage-promoting activity of human anti-EphA4 monoclonal antibodies in hippocampal neurons Rat hippocampal neurons were prepared as follows. Embryos were removed from 18-day-old pregnant rats (Charles River Japan), and the heads were incised to remove the brains. The hippocampal regions were dissected under a stereomicroscope and then placed in digestion solution (137 mM NaCl (Wako Pure Chemicals), 5 mM KCl (Wako Pure Chemicals), 7 mM NaHPO (Wako Pure Chemicals), 25 mM Hepes (DOJINDO), 0.5 mg / mL DNase (Sigma), 0.25% trypsin (Thermo Fisher Scientific)) and shaken at 37°C for 10 minutes. The solution was removed, and 20% fetal bovine serum in Hanks buffer (Sigma) was added. After removing the solution and washing twice with Hanks buffer, the hippocampal tissue was pipetted in Hanks buffer to prepare a cell suspension. The cells were then suspended in culture medium (Neurobasal medium (Thermo Fisher Scientific), 1x B-27 supplement (Thermo Fisher Scientific), 0.5 mM L-glutamine (Thermo Fisher Scientific)) and seeded onto poly-L-lysine-coated 96-well dishes (Falcon).

[0127] The EphA4 cleavage-promoting activity of antibody A and antibody B obtained in Example 1 was evaluated using hippocampal neurons according to the following procedure. Rat hippocampal neurons seeded in a 96-well dish (Falcon) were treated for 24 hours with the γ-secretase inhibitor Compound E (50 nM, Enzo Life Sciences), solvent (PBS (FUJIFILM Wako Pure)), human IgG, antibody A, or antibody B (2.0, 6.7, or 20 nM). After washing with PBS (FUJIFILM Wako Pure), cells were harvested with SDS sample buffer (Laemmli sample buffer (Bio-Rad), 2.5% 2-mercaptoethanol (Bio-Rad)) and boiled for 5 minutes. The samples were analyzed using the automated Western system Jess. An anti-EphA4 monoclonal antibody (Abnova) was used for analysis. The signals of the EphA4 C-terminal fragment and full-length EphA4 were quantified, and the value of EphA4 C-terminal fragment / (full-length EphA4+EphA4 C-terminal fragment) was calculated.

[0128] Antibody A and antibody B promoted EphA4 cleavage in hippocampal neurons in a concentration-dependent manner (Figure 6).

[0129] Example 8: Human EphA4 cleavage-promoting activity of human anti-EphA4 monoclonal antibodies First, a pCAHA vector containing a DNA sequence encoding human EphA4 and a DNA sequence encoding an HA tag was synthesized by GenScript Japan Co., Ltd., and then the DNA sequence encoding human EphA4 was inserted into the SalI / NotI site of the pCAHA vector to construct the pCA-human EphA4-HA expression vector. Rat hippocampal neurons were prepared as follows. Embryos were removed from 18-day-old pregnant rats (Charles River Japan), and the heads were incised to remove the brains. The hippocampal regions were dissected under a stereomicroscope and then placed in digestion solution (137 mM NaCl (Wako Pure Chemicals), 5 mM KCl (Wako Pure Chemicals), 7 mM NaHPO (Wako Pure Chemicals), 25 mM Hepes (DOJINDO), 0.5 mg / mL DNase (Sigma), 0.25% trypsin (Thermo Fisher Scientific)) and shaken at 37°C for 10 minutes. The solution was removed, and 20% fetal bovine serum in Hanks buffer (Sigma) was added. After removing the solution and washing twice with Hanks buffer, the hippocampal tissue was pipetted in Hanks buffer to prepare a cell suspension.

[0130] The cleavage-promoting activity of Antibody A and Antibody B obtained in Example 1 against human EphA4 was evaluated according to the following procedure. Rat hippocampal neurons were transfected with the pCA-human EphA4-HA expression vector using Nucleofector (Lonza) and suspended in culture medium (Neurobasal medium (Thermo Fisher Scientific), 1x B-27 supplement (Thermo Fisher Scientific), 0.5 mM L-glutamine (Thermo Fisher Scientific)) and then seeded onto poly-L-lysine-coated 96-well dishes (Falcon). The seeded rat hippocampal neurons were treated overnight with the γ-secretase inhibitor Compound E (50 nM, Enzo Life Sciences), solvent (PBS (Wako Pure Chemical Industries), human IgG, Antibody A, or Antibody B (6.7, 20, or 67 nM)). After washing with PBS, cells were harvested with SDS sample buffer (Laemmli sample buffer (Bio-Rad), 5% 2-mercaptoethanol (Bio-Rad)) and boiled for 5 minutes. SDS-PAGE was performed using this sample, followed by Western blotting using a rat anti-HA monoclonal antibody (Roche). Band intensities were quantified, and the ratio of human EphA4 C-terminal fragment to (full-length human EphA4 + human EphA4 C-terminal fragment) was calculated.

[0131] Antibody A and antibody B promoted human EphA4 cleavage in hippocampal neurons (FIG. 7).

[0132] Example 9: Increasing effect of human anti-EphA4 monoclonal antibody on spine density in hippocampal neurons Rat hippocampal neurons were prepared as described in Example 7. The EGFP gene was introduced into rat hippocampal neurons using Nucleofector (Lonza), and the neurons were mixed with non-transfected neurons and seeded onto a poly-L-lysine-coated 24-well plate (Falcon) with a cover glass (Matsunami Glass Industry).

[0133] Spine counting using hippocampal neurons was performed as follows. Rat hippocampal neurons on day 15 of culture were seeded on poly-L-lysine-coated 24-well plates (Falcon) containing cover slips (Matsunami Glass Industry). They were treated with a control antibody (human IgG2; Sigma), antibody A, or antibody B (6.7 or 20 nM) for 24 hours. The cover slips were then transferred to 2% PFA (Wako Pure Chemical Industries) / 4% sucrose (Wako Pure Chemical Industries) / PBS and left to stand for 20 minutes to fix the cells. The cover slips were then removed from the fixative, transferred to PBS, and washed three times. After that, the cells were permeabilized with 0.25% Triton X-100 (Wako Pure Chemical Industries) / PBS for 15 minutes. Coverslips were transferred to 2% BSA (Sigma) / 0.25% Triton X-100 / OPTI-MEM (GIBCO) and blocked for 1 hour. After incubation, anti-GFP antibody (Nacalai Tesque) and anti-Math2 antibody (Abcam) were added at room temperature for 1 hour and 1 hour 30 minutes. The primary antibody solution was removed, and the coverslips were washed three times with PBS. The secondary antibody was then added at room temperature for 1 hour in the dark. The secondary antibody solution was removed, and the coverslips were washed three times with PBS. The coverslips were then mounted with Prolong Gold antifade reagent (Molecular probes). Images were then captured and analyzed using an LSM800 (ZEISS). This experiment was performed three times. Math2-positive pyramidal cell-like neurons were extracted from two coverslips per experiment. The spines on each dendrite were counted using image analysis software Imaris® (Bitplane) to calculate the number of spines per 10 μm for each neuron.

[0134] Antibody A and antibody B increased the spine density of hippocampal neurons (Figure 8). These results indicate that antibody A and antibody B have the activity of stabilizing spines in hippocampal neurons.

Claims

1. 1. An anti-EphA4 antibody, The anti-EphA4 antibody (a) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 30; (b) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31; and (c) a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 32; and (d) a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 33; (e) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (f) a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35 an anti-EphA4 antibody, comprising or (g) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 42; (h) a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31; and (i) a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 43; and (j) a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 44; (k) a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35 An anti-EphA4 antibody comprising:

2. 2. The anti-EphA4 antibody of claim 1, the anti-EphA4 antibody is a human antibody; Anti-EphA4 antibody.

3. 3. The anti-EphA4 antibody of claim 1 or 2, The anti-EphA4 antibody specifically binds to EphA4 and promotes cleavage of EphA4. Anti-EphA4 antibody.

4. The anti-EphA4 antibody of any one of claims 1 to 3, The anti-EphA4 antibody specifically binds to EphA4 and inhibits the binding of EphA4 to ephrin. Anti-EphA4 antibody.

5. The anti-EphA4 antibody of any one of claims 1 to 4, the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO:7, The light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO:

8. Anti-EphA4 antibody.

6. The anti-EphA4 antibody of any one of claims 1 to 5, the heavy chain constant region and the light chain constant region comprise amino acid sequences derived from a human antibody; Anti-EphA4 antibody.

7. 7. The anti-EphA4 antibody of claim 6, the heavy chain constant region is a human IgG constant region; Anti-EphA4 antibody.

8. 8. The anti-EphA4 antibody of claim 7, The constant region of the human IgG is human IgG ２ is the constant region of Anti-EphA4 antibody.

9. 9. The anti-EphA4 antibody of claim 8, The human IgG ２ The constant region of comprises the amino acid sequence set forth in SEQ ID NO: 15, Anti-EphA4 antibody.

10. The anti-EphA4 antibody of any one of claims 1 to 4, the heavy chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11; The light chain comprises a variable region consisting of the amino acid sequence set forth in SEQ ID NO:

12. Anti-EphA4 antibody.

11. The anti-EphA4 antibody of any one of claims 1 to 4 and claim 10, the heavy chain constant region and the light chain constant region comprise amino acid sequences derived from a human antibody; Anti-EphA4 antibody.

12. 12. The anti-EphA4 antibody of claim 11, the heavy chain constant region is a human IgG constant region; Anti-EphA4 antibody.

13. 13. The anti-EphA4 antibody of claim 12, The constant region of the human IgG has a CH1 region and a hinge region of human IgG. １ and the CH2 and CH3 regions are derived from human IgG ２ is a human IgG constant region derived from Anti-EphA4 antibody.

14. 14. The anti-EphA4 antibody of claim 13, The human IgG １ and human IgG ２ The constant region of human IgG consisting of the combination of the above comprises the amino acid sequence shown in SEQ ID NO:

16. Anti-EphA4 antibody.

15. The anti-EphA4 antibody according to any one of claims 6 to 9 and 11 to 14, the light chain constant region is a human Igλ constant region; Anti-EphA4 antibody.

16. 16. The anti-EphA4 antibody of claim 15, The constant region of the human Igλ comprises the amino acid sequence set forth in SEQ ID NO:

17. Anti-EphA4 antibody.

17. 1. An anti-EphA4 antibody, the anti-EphA4 antibody comprises a heavy chain and a light chain; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 20; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 21; The C-terminal lysine of the heavy chain may be deleted. Anti-EphA4 antibody.

18. 18. The anti-EphA4 antibody of claim 17, the C-terminal lysine of the heavy chain is deleted; Anti-EphA4 antibody.

19. 1. An anti-EphA4 antibody, the anti-EphA4 antibody comprises a heavy chain and a light chain; the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:26; and the light chain comprises the amino acid sequence set forth in SEQ ID NO:27; The C-terminal lysine of the heavy chain may be deleted. Anti-EphA4 antibody.

20. 20. The anti-EphA4 antibody of claim 19, the C-terminal lysine of the heavy chain is deleted; Anti-EphA4 antibody.

21. An isolated nucleic acid encoding the anti-EphA4 antibody of any one of claims 1 to 20.

22. A vector comprising the nucleic acid of claim 21.

23. A host cell comprising the vector of claim 22.

24. A method for producing an anti-EphA4 antibody, comprising culturing the host cell of claim 23.

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