Anti-EphA4 antibody

Abstract

[Problem] To provide an antibody that can bind specifically to EphA4 and detect EphA4 at high detection sensitivity and a method and a kit for detecting or quantifying EphA4 characterized by the antibody. [Solution] Provided are an antibody having a specific heavy chain CDR sequence and light chain CDR sequence and an antigen-binding fragment thereof, and a method and kit characterized by the same. Specifically, an antibody according to the present invention or antigen-binding fragment thereof includes: (a) a heavy chain including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 52, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 53, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 54, and a light chain including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 55, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 56, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 57; (b) a heavy chain including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 64, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 65, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 66, and a light chain including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 67, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 68, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 69; or (c) a heaving chain including a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 40, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 41, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 42, and a light chain including a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 43, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 44, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 45.

Description

Technical Field

[0001] The present disclosure relates to an antibody that binds to EphA4, a nucleic acid encoding the antibody, a vector containing the nucleic acid, a cell containing the vector, a method for producing the antibody, a method for detecting or quantifying EphA4 using the antibody, and a kit for detecting or quantifying EphA4.

Background Art

[0002] EphA4 is one of the receptor-type tyrosine kinase family and is a molecule that controls spines, which are small spike-like structures present on dendritic protrusions. Ephrin type A and type B are known as ligands for EphA4, and when EphA4 binds to its ligand ephrin, a detachment signal is induced, causing spine retraction. EphA4 is highly expressed in the hippocampus and cerebral cortex and its extracellular region is cleaved by matrix metalloproteinase (MMP) and ADAM (a disintegrin and metalloproteinase). The cleaved fragment of EphA4 is released extracellularly and is also present in plasma (Patent Document 1).

[0003] EphA4 has hitherto been suggested to be involved in the pathogenesis of Alzheimer's disease (hereinafter also referred to as "AD") (Non-Patent Documents 1 to 4). For example, EphA4 is known to be activated in AD patients and AD model mice (Non-Patent Documents 2 to 4), and in AD, the density of spines decreases and the degree is related to the clinical symptoms of AD (Non-Patent Document 5). Therefore, it is considered that abnormal activation of EphA4 may be one of the causes of the onset and progression of AD (Non-Patent Document 6). EphA4 in vivo has been suggested as a potential marker for detecting a given neurological disease such as AD, and an antibody that can detect EphA4 or its extracellular fragment in a biological sample with higher detection sensitivity than conventional antibodies is desired.

Prior Art Documents

[0004] [Patent Document 1] WO2012 / 147798A1 [Non-patent literature]

[0005] [Non-Patent Document 1] Rosenberger AF et al., Acta Neuropathol Commun. 2014 Jul 16;2:79 [Non-Patent Document 2] Fu AK et al., Proc Natl Acad Sci US A. 2014 Jul 8;111(27):9959-64 [Non-Patent Document 3] Vargas LM et al.,, PLoS One. 2014 Mar 21;9(3) [Non-Patent Document 4] Huang TY et al., J Exp Med. 2017 Dec 4;214(12):3669-3685. [Non-Patent Document 5] Boros et al., Ann Neurol. 2017 Oct;82(4):602-614 [Non-Patent Document 6] Vargas LM et al., Biochim Biophys Acta Mol Basis Dis. 2018 Apr;1864:1148-1159 [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure aims to provide an antibody that specifically binds to EphA4 and can detect EphA4 with high detection sensitivity. This disclosure also aims to provide a method for detecting or quantifying EphA4 using the aforementioned antibody. This disclosure further aims to provide a kit containing an anti-EphA4 antibody capable of specifically detecting or quantifying EphA4 with high detection sensitivity. [Means for solving the problem]

[0007] To solve the above problems, the inventors discovered that they could compose an antibody capable of specifically binding to EphA4 with particularly high detection sensitivity from a large number of scFv obtained from screening a rabbit antibody phage library, and thus completed an anti-EphA4 antibody.

[0008] Therefore, this disclosure encompasses the following features: [1] An anti-EphA4 antibody or its antigen-binding fragment, The aforementioned antibody is (a) Heavy chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 52; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 53; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 54; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 55; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 56; and A light chain containing the CDR3 light chain, which consists of the amino acid sequence shown in Sequence ID No. 57; (b) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO. 64; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 65; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 66; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 67; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 68; and A light chain containing the light chain CDR3, which consists of the amino acid sequence shown in Sequence ID No. 69; or (c) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 40; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 41; and A heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence shown in Sequence No. 42; and A light chain CDR1 consisting of the amino acid sequence shown in Sequence No. 43; A light chain CDR2 consisting of the amino acid sequence shown in Sequence No. 44; and A light chain comprising a light chain CDR3 consisting of the amino acid sequence shown in Sequence No. 45; Comprising, An anti-EphA4 antibody or an antigen-binding fragment thereof.

[0009] [2] The anti-EphA4 antibody or an antigen-binding fragment thereof according to [1], wherein The antibody is A heavy chain CDR1 consisting of the amino acid sequence shown in Sequence No. 52; A heavy chain CDR2 consisting of the amino acid sequence shown in Sequence No. 53; and A heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence shown in Sequence No. 54; and A light chain CDR1 consisting of the amino acid sequence shown in Sequence No. 55; A light chain CDR2 consisting of the amino acid sequence shown in Sequence No. 56; and A light chain comprising a light chain CDR3 consisting of the amino acid sequence shown in Sequence No. 57; Comprising, An anti-EphA4 antibody or an antigen-binding fragment thereof.

[0010] [3] The anti-EphA4 antibody or an antigen-binding fragment thereof according to [2], wherein The antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence No. 10, and A light chain variable region consisting of the amino acid sequence shown in Sequence No. 11, Comprising, An anti-EphA4 antibody or an antigen-binding fragment thereof.

[0011] [4] The anti-EphA4 antibody or an antigen-binding fragment thereof according to [2], wherein The antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence No. 14, and The light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 15, including, Anti-EphA4 antibody or its antigen-binding fragment.

[0012] [5] [1] The anti-EphA4 antibody or its antigen-binding fragment, The aforementioned antibody is Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 64; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 65; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 66; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 67; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 68; and A light chain containing the CDR3 light chain, which consists of the amino acid sequence shown in Sequence ID No. 69; including, Anti-EphA4 antibody or its antigen-binding fragment.

[0013] [6] [5] The anti-EphA4 antibody or its antigen-binding fragment described therein The aforementioned antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 18, and The light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 19, including, Anti-EphA4 antibody or its antigen-binding fragment.

[0014] [7] [1] The anti-EphA4 antibody or its antigen-binding fragment, The aforementioned antibody is Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 40; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 41; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 42; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 43; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 44; and A light chain containing the CDR3 light chain, which consists of the amino acid sequence shown in Sequence ID No. 45; including, Anti-EphA4 antibody or its antigen-binding fragment.

[0015] [8] [7] The anti-EphA4 antibody or its antigen-binding fragment described therein The aforementioned antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 6, and The light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 7, including, Anti-EphA4 antibody or its antigen-binding fragment.

[0016] [9] An anti-EphA4 antibody or an antigen-binding fragment thereof, as described in any of [1] to [8], The heavy chain constant region of the antibody is the constant region of rabbit IgG. Anti-EphA4 antibody or its antigen-binding fragment.

[0017]

[10] [9] The anti-EphA4 antibody or its antigen-binding fragment described therein The constant region of the aforementioned rabbit IgG includes the amino acid sequence shown in SEQ ID NO: 22. Anti-EphA4 antibody or its antigen-binding fragment.

[0018]

[11] An anti-EphA4 antibody or an antigen-binding fragment thereof, as described in any of [1] to

[10] , The constant region of the light chain of the aforementioned antibody is the constant region of rabbit Igκ. Anti-EphA4 antibody or its antigen-binding fragment.

[0019]

[12]

[11] The anti-EphA4 antibody or its antigen-binding fragment described therein The constant region of the rabbit Igκ includes the amino acid sequence shown in SEQ ID NO: 23. Anti-EphA4 antibody or its antigen-binding fragment.

[0020]

[13] Anti-EphA4 antibody, The aforementioned antibody is A heavy chain containing the amino acid sequence shown in SEQ ID NO: 24, and Light chain containing the amino acid sequence shown in SEQ ID NO: 25 including, Anti-EphA4 antibody.

[0021]

[14] Anti-EphA4 antibody, The aforementioned antibody is A heavy chain containing the amino acid sequence shown in SEQ ID NO: 28, and Light chain containing the amino acid sequence shown in SEQ ID NO: 29 including, Anti-EphA4 antibody.

[0022]

[15] Anti-EphA4 antibody, The aforementioned antibody is A heavy chain containing the amino acid sequence shown in SEQ ID NO: 32, and Light chain containing the amino acid sequence shown in SEQ ID NO: 33 including, Anti-EphA4 antibody.

[0023]

[16] Anti-EphA4 antibody, The aforementioned antibody is A heavy chain containing the amino acid sequence shown in SEQ ID NO: 36, and Light chain containing the amino acid sequence shown in SEQ ID NO: 37 including, Anti-EphA4 antibody.

[0024]

[17] An anti-EphA4 antibody or an antigen-binding fragment thereof, as described in any of [1] to

[16] , The antibody or its antigen-binding fragment is labeled. Anti-EphA4 antibody or its antigen-binding fragment.

[0025]

[18] Isolated nucleic acids encoding an anti-EphA4 antibody or an antigen-binding fragment thereof, as described in any of [1] to

[16] .

[0026] A vector containing nucleic acids as described in

[19]

[18] .

[0027]

[20]

[19] Host cells containing the vector described above.

[0028] A method for producing an anti-EphA4 antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cells described in

[21]

[20] .

[0029]

[22] A method for producing an anti-EphA4 antibody or an antigen-binding fragment thereof, comprising the step of culturing a host cell containing an isolated nucleic acid encoding an anti-EphA4 antibody or an antigen-binding fragment thereof as described in any of [1] to

[16] .

[0030]

[23] A method for detecting or quantifying human EphA4 in a biological sample, This includes contacting the biological sample with an anti-EphA4 antibody or an antigen-binding fragment described in any of [1] to

[16] , method.

[0031] The method described in

[24]

[23] , The method wherein the human EphA4 is the N-terminal fragment of human EphA4.

[0032] The method described in

[25]

[23] or

[24] , A method wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

[0033] A method according to any one of

[23] to

[25] , The method wherein the method is ELISA.

[0034] A method by any of

[27]

[23] to

[26] , The method wherein the aforementioned method is a sandwich ELISA.

[0035]

[28] The method described in any of

[23] to

[27] , Furthermore, the method includes contacting the biological sample with the labeled anti-EphA4 antibody or its antigen-binding fragment described in

[17] , method.

[0036]

[29] A kit for detecting or quantifying human EphA4, The anti-EphA4 antibody or its antigen-binding fragment as described in any of [1] to

[17] , kit.

[0037] The kit described in

[30]

[29] , The aforementioned kit is a sandwich ELISA kit. The anti-EphA4 antibody or its antigen-binding fragment comprises at least two types, kit.

[0038] A kit as described in

[31]

[29] or

[30] , An anti-EphA4 antibody or its antigen-binding fragment, comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and An anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19, including, kit.

[0039] A kit as described in

[32]

[29] or

[30] , An anti-EphA4 antibody or its antigen-binding fragment, comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, An anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, including, kit.

[0040]

[33]

[29] or

[30] a kit as described above, An anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11, and An anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19, including, kit.

[0041] A kit as described in any of

[34]

[29] to

[33] , The aforementioned human EphA4 is the N-terminal fragment of human EphA4, kit.

[0042] A kit as described in any of

[35]

[29] to

[34] , The kit contains the N-terminal fragment of human EphA4, kit.

[0043]

[36] A kit according to any one of

[29] to

[35] for detecting or quantifying human EphA4 in a biological sample.

[0044]

[37]

[36] The kit described above, A kit in which the biological sample is blood, serum, plasma, or cerebrospinal fluid.

[0045]

[38] An anti-EphA4 antibody or antigen-binding fragment thereof, as described in any of [1] to

[17] , for use in the detection or quantification of human EphA4.

[0046]

[39] An anti-EphA4 antibody or its antigen-binding fragment according to any one of [1] to

[17] , for use in the detection or quantification of the N-terminal fragment of human EphA4. [Effects of the Invention]

[0047] According to this disclosure, an antibody is provided that specifically binds to EphA4 and can detect EphA4 with high detection sensitivity. The disclosure also provides a kit containing an anti-EphA4 antibody capable of specifically detecting EphA4 with high detection sensitivity. [Brief explanation of the drawing]

[0048] [Figure 1] Figure 1 shows the results of evaluating the binding activity of each anti-human EphA4 monoclonal antibody prepared in Example 1 against each human Eph receptor family. [Figure 2] Figure 2 shows the results of evaluating the binding activity of each anti-human EphA4 monoclonal antibody prepared in Example 1 against various EphA4 types. [Figure 3] Figure 3 shows the results of evaluating the binding activity of each anti-human EphA4 monoclonal antibody prepared in Example 1 to each region of human EphA4. [Figure 4] Figure 4 shows the signal-to-noise ratio (SN) for the human EphA4 extracellular region in a sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1. (Signal obtained when the EphA4 extracellular region was seeded at 10 ng / mL) - (Signal obtained when the EphA4 extracellular region was seeded at 0 ng / mL). [Figure 5] Figure 5 shows the S / N ratio ((signal obtained when seeding 10 ng / mL of EphA4 extracellular region) / (signal obtained when seeding 0 ng / mL of EphA4 extracellular region)) for the evaluation of the human EphA4 extracellular region in a sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1 against the human EphA4 extracellular region. [Figure 6]Figure 6 shows the signal-to-noise ratio (SN) for the human EphA4 extracellular region in a sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1. (Signal obtained when 1 ng / mL of EphA4 extracellular region was seeded) - (Signal obtained when 0 ng / mL of EphA4 extracellular region was seeded). [Figure 7] Figure 7 shows the S / N ratio ((signal obtained when seeding 1 ng / mL of EphA4 extracellular region) / (signal obtained when seeding 0 ng / mL of EphA4 extracellular region)) for the evaluation of the human EphA4 extracellular region in a sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1. [Figure 8] Figure 8 shows representative binding reaction curves for the KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies to human EphA4. [Figure 9] Figure 9 shows the binding specificity of the KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies to each human Eph receptor family. [Figure 10] Figure 10 shows the reactivity results of the KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies against mouse, rat, rabbit, monkey, and human EphA4. [Figure 11] Figure 11 shows the reactivity of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies against the extracellular domain (ECD), ligand-binding domain (LBD), fibronectin type III domain 1 (FN1), fibronectin type III domain 2 (FN2), and maltose-binding protein (MBP) of human EphA4. [Figure 12] Figure 12 shows the reactivity results for the extracellular domain of human EphA4 in a sandwich ELISA using antibody KPEP11_10 and HRP-labeled antibody KPEP11_18. [Figure 13] Figure 13 shows the results of a sandwich ELISA using antibody KPEP11_10 and HRP-labeled antibody KPEP11_18 for the reactivity of EphA4 N-terminal fragment in cerebrospinal fluid. [Figure 14] Figure 14 shows the reactivity results for the human EphA4 extracellular region in a sandwich ELISA constructed using the antibody KPEP11_18 and the HRP-labeled antibody KPEP11_10. [Figure 15] Figure 15 shows the reactivity results for the EphA4 N-terminal fragment in cerebrospinal fluid in a sandwich ELISA constructed using the antibody KPEP11_18 and the HRP-labeled antibody KPEP11_10. [Figure 16] Figure 16 shows the reactivity results for the human EphA4 extracellular domain in a sandwich ELISA constructed using the antibody KPEP11_10 and the HRP-labeled antibody KPEP11_04. [Figure 17] Figure 17 shows the reactivity results for the EphA4 N-terminal fragment in cerebrospinal fluid in a sandwich ELISA constructed using the antibody KPEP11_10 and the HRP-labeled antibody KPEP11_04. [Figure 18] Figure 18 shows the reactivity results for the human EphA4 extracellular region in a sandwich ELISA constructed using the antibody KPEP11_18 and the HRP-labeled antibody KPEP11_08. [Figure 19] Figure 19 shows the reactivity results for the EphA4 N-terminal fragment in cerebrospinal fluid in a sandwich ELISA constructed using the antibody KPEP11_18 and the HRP-labeled antibody KPEP11_08. [Figure 20] Figure 20 shows the results of evaluating the reactivity to the extracellular domain of human EphA4 in sandwich ELISAs constructed using antibody KPEP11_10, HRP-labeled antibody KPEP11_04, HRP-labeled antibody KPEP11_18, and HRP-labeled EphA4 antibody (Sino or R&D). [Figure 21]Figure 21 shows the results of evaluating the reactivity to the EphA4 N-terminal fragment in human plasma using a sandwich ELISA constructed with antibody KPEP11_10, HRP-labeled antibody KPEP11_04, HRP-labeled antibody KPEP11_18, and HRP-labeled EphA4 antibody (Sino or R&D). [Figure 22] Figure 22 shows the results of evaluating the reactivity of a sandwich ELISA constructed using antibody KPEP11_10, HRP-labeled antibody KPEP11_04, HRP-labeled antibody KPEP11_18, and HRP-labeled EphA4 antibody (Sino or R&D) to the EphA4 N-terminal fragment in human cerebrospinal fluid. [Figure 23] Figure 23 shows the results of evaluating the reactivity to the extracellular domain of human EphA4 in sandwich ELISAs constructed using antibody KPEP11_18, HRP-labeled antibody KPEP11_08, HRP-labeled antibody KPEP11_10, and HRP-labeled EphA4 antibody (Sino or R&D). [Figure 24] Figure 24 shows the results of evaluating the reactivity to the EphA4 N-terminal fragment in human plasma using a sandwich ELISA constructed with antibody KPEP11_18, HRP-labeled antibody KPEP11_08, HRP-labeled antibody KPEP11_10, and HRP-labeled EphA4 antibody (Sino or R&D). [Figure 25] Figure 25 shows the results of evaluating the reactivity of a sandwich ELISA constructed using antibody KPEP11_18, HRP-labeled antibody KPEP11_08, HRP-labeled antibody KPEP11_10, and HRP-labeled EphA4 antibody (Sino or R&D) to the EphA4 N-terminal fragment in human cerebrospinal fluid. [Figure 26] Figure 26 shows the results of a correlation analysis performed based on the quantitative results of EphA4 N-terminal fragments in human cerebrospinal fluid analyzed by LC-MS and the quantitative results of EphA4 N-terminal fragments in human cerebrospinal fluid analyzed by ELISA analysis 1. [Figure 27]Figure 27 shows the results of a correlation analysis performed based on the quantitative results of EphA4 N-terminal fragments in human cerebrospinal fluid analyzed by LC-MS and the quantitative results of EphA4 N-terminal fragments in human cerebrospinal fluid analyzed by ELISA analysis 2. [Modes for carrying out the invention]

[0049] The regions identified or coded by the sequence numbers used herein are as follows: JPEG0007867505000001.jpg189166

[0050] JPEG0007867505000002.jpg191166JPEG0007867505000003.jpg58166

[0051] This disclosure relates to an anti-EphA4 antibody that binds to EphA4. The anti-EphA4 antibody relating to this disclosure is an antibody capable of specifically recognizing and binding to EphA4. The anti-EphA4 antibody may be an intact antibody or a synthetic antibody (e.g., recombinant antibody, chimeric antibody, humanized antibody, etc.) as long as it has binding affinity to EphA4. In this specification, EphA4 can be understood to refer to EphA4 derived from humans, mice, rats, rabbits, or monkeys. EphA4 derived from humans, mice, rats, rabbits, and monkeys can be obtained from public databases where sequence information is registered, such as Genbank provided by the National Center for Biotechnology Information. In addition, it is possible to obtain sequence information of the EphA4 gene by designing primers based on the nucleotide sequence information of EphA4 from closely related animal species and cloning from RNA extracted from the desired animal species. For example, the nucleotide sequence information for human, mouse, rat, rabbit, and monkey EphA4 is registered in the database as Genbank Accession No. NM_004438.5, NM_007936.3, NM_001162411.1, XM_002712496.3, and NM_001260870.1, respectively.

[0052] In one aspect of this disclosure, EphA4 includes the amino acid sequence shown in Sequence ID No. 1 or an amino acid sequence in which one or more amino acids are substituted, added, or deleted from said amino acid sequence. Here, “more than” is not limited as long as it retains the same functional characteristics as the original sequence, but is between 2 and 100, for example, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids, or the number of amino acids in the amino acid sequence is within 10%, for example, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, or within 1%.

[0053] In this disclosure, the term “specific binding” is 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 or its antigen-binding fragment can bind to EphA4 by immunological reaction with greater binding affinity and binding activity, more rapidly, and / or for a longer duration than it would to bind to other target molecules. In another embodiment, “specific binding” means that the antibody binds to EphA4 at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least 10 -10 This can be demonstrated by antibodies with a KD of M or less. In yet another embodiment, "specific binding" is understood to be binding to EphA4 by immunological reaction but substantially not binding to other family molecules of the Eph receptor (e.g., EphA1, EphA2, EphA3, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6).

[0054] The term "antigen-binding fragment" is not particularly limited as long as it is a fragment of an anti-EphA4 antibody that maintains specific binding to EphA4, but examples include Fab, Fab', F(ab')2, Fv, scFv, etc.

[0055] Methods for measuring the binding properties (e.g., binding affinity and species cross-reactivity) of an anti-EphA4 antibody or its antigen-binding fragment to an antigen may be those known to those skilled in the art. For example, binding affinity may be measured using, but is not limited to, Biacore® biosensors, KinExA biosensors, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), flow cytometry, fluorescence quenching, fluorescence transfer, yeast display, and / or immunostaining.

[0056] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure may be any class, such as IgG, IgA, or IgM (or their subclasses), and is not limited to any particular class. Immunoglobulins are classified into different classes based on the antibody amino acid sequence of the constant region of the heavy chain (sometimes called the H chain). There are five main classes of immunoglobulins: 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 corresponding constant regions of the heavy chains of different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. In addition, there are two types of light chains (sometimes called L chains) of antibodies: λ chains and κ chains.

[0057] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure may be an IgG antibody, and the anti-EphA4 antibody relating to this disclosure may, as applicable, be in the form of a monomer, dimer, or polymer.

[0058] The variable region of the antibody or its antigen-binding fragment relating to this disclosure may mean the variable region of the antibody light chain and / or the variable region of the antibody heavy chain, and the constant region of the antibody may mean the constant region of the antibody light chain and / or the constant region of the antibody heavy chain. The variable regions of the heavy chain and light chain 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 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. Techniques for determining CDR include, but are not limited to, (1) an approach based on interspecies sequence variability (e.g., Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda MD); and (2) an approach based on crystal structure studies of antigen-antibody complexes (Al-lazikani et al., 1997 J. Molec. Biol. 273:927-948). These approaches, or other approaches, may be used in combination.

[0059] In the anti-EphA4 antibody or its antigen-binding fragment according to this disclosure, heavy chain sequences and / or light chain sequences derived from humans, mice, rats, rabbits, or monkeys may be used, for example, but are not limited thereto. In one embodiment, the anti-EphA4 antibody or its antigen-binding fragment according to this disclosure has rabbit-derived heavy chain sequences and light chain sequences.

[0060] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure may be modified as desired. The modification of the anti-EphA4 antibody or its antigen-binding fragment may be a modification that changes (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 hydrophobic state of the molecule at the target site; or (c) the effect of the modification on maintaining the volume of the side chain, or it may be a modification in which these changes are not readily observable.

[0061] Modification of the anti-EphA4 antibody or its antigen-binding fragment according to this disclosure may be achieved, for example, by substitution, deletion, or addition of constituent amino acid residues.

[0062] In this specification, the term "amino acid" is used in its broadest sense and includes not only natural 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 understand, considering this broad definition, that the amino acids used herein include, for example, L-amino acids; D-amino acids; chemically modified amino acids such as amino acid mutants and amino acid derivatives; amino acids that do not form protein building blocks in living organisms, such as norleucine, β-alanine, and ornithine; and chemically synthesized compounds that possess the properties of amino acids known to those skilled in the art. Examples of unnatural amino acids include α-methyl amino 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-hydroxyhistidine, homohistidine, α-fluoromethylhistidine, and α-methylhistidine), amino acids with an extra methylene group in their side chain ("homo" amino acids), and amino acids in which a carboxylic acid functional group amino acid in the side chain is replaced by a sulfonic acid group (such as cysteic acid).

[0063] Naturally occurring amino acid residues can be classified into the following groups, for example, based on their general side-chain properties: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Asn, Gln, Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0064] Non-conservative substitutions in the amino acid sequences constituting antibodies may be made by exchanging an amino acid belonging to one of these groups with an amino acid belonging to another group. More conservative substitutions may be made by exchanging an amino acid belonging to one of these groups with another amino acid of the same group. Similarly, deletions or substitutions of amino acid sequences may be made as appropriate.

[0065] Modifications of the amino acids constituting an antibody may include post-translational modifications such as glycosylation, acetylation, or phosphorylation by sugars. Antibodies can be glycosylated at conserved positions within their constant region. Antibody glycosylation is usually either N-linked or O-linked. N-linked glycosylation means the attachment of a 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 other than proline) are recognition sequences for enzymatically attaching a carbohydrate moiety to the asparagine side chain. The presence of any of these tripeptide sequences in an antibody indicates the presence of a potential glycosylation site. O-linked glycosylation may involve attachment of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (e.g., serine or threonine), and may optionally be attachment to 5-hydroxyproline or 5-hydroxylysine. Those skilled in the art can appropriately select the conditions for glycosylation (for example, the type of host cell and cell culture medium, pH, etc., when glycosylation is performed using biological methods) according to their purpose.

[0066] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure may be further modified, alone or in combination, by other modification methods based on common technical knowledge known to those skilled in the art.

[0067] The anti-EphA4 antibody or its antigen-binding fragment according to this disclosure can be produced by methods well known to those skilled in the art. For example, the antibody or its antigen-binding fragment may be produced by incorporating the nucleic acid encoding the anti-EphA4 antibody or its antigen-binding fragment according to this disclosure into an expression vector, introducing the expression vector into a host cell, and culturing the host cell. Accordingly, this disclosure includes a nucleic acid encoding the anti-EphA4 antibody or its antigen-binding fragment, a vector containing the nucleic acid, a host cell containing the vector, and a method for producing the anti-EphA4 antibody or its antigen-binding fragment, comprising the steps of culturing the host cell.

[0068] The nucleic acid encoding the anti-EphA4 antibody or its antigen-binding fragment according to this disclosure may have DNA encoding a signal sequence, DNA encoding a heavy chain variable region, and DNA encoding a signal sequence at the 5' end of the DNA encoding the light chain variable region. The signal sequence is an amino acid residue located at the N-terminus of a protein that is necessary for secreted proteins and membrane-bound proteins to pass through the lipid bilayer after being synthesized on ribosomes, and is not particularly limited in this disclosure as long as it has this function. Examples of signal sequences that the anti-EphA4 antibody or its antigen-binding fragment according to this disclosure may contain include signal sequences derived from humans, mice, rats, rabbits, donkeys, goats, horses, birds, dogs, cats, yeast, etc.

[0069] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure may be isolated or purified according to methods known to those skilled in the art.

[0070] In this specification, “isolated” or “purified” means that a molecule or composition has been artificially isolated or purified from its natural state. If a molecule or composition occurs naturally, it is “isolated” or “purified” when it has been altered, removed from its original environment, or both. Examples of isolation or purification methods include, but are not limited to, electrophoretic, molecular biological, immunological, or chromatographic methods, specifically, ion-exchange chromatography, hydrophobic chromatography, reverse-phase HPLC chromatography, isoelectric focusing, or alkaline extraction.

[0071] In one embodiment, the anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure comprises the following CDR: Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 40; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 41; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 42; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 43; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 44; and A light chain containing the light chain CDR3, which has the amino acid sequence shown in Sequence ID No. 45.

[0072] In another embodiment, the anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure comprises the following CDR: Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 52; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 53; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 54; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 55; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 56; and A light chain containing the CDR3 light chain, which consists of the amino acid sequence shown in Sequence ID No. 57;

[0073] In yet another embodiment, the anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure comprises the following CDR: Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 64; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 65; and A heavy chain containing the heavy chain CDR3, which consists of the amino acid sequence shown in SEQ ID NO: 66; and Light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 67; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 68; and A light chain containing the light chain CDR3, which has the amino acid sequence shown in Sequence ID No. 69.

[0074] In one embodiment, the anti-EphA4 antibody or its antigen-binding fragment includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0075] In one embodiment, the anti-EphA4 antibody or its antigen-binding fragment includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0076] In another embodiment, the anti-EphA4 antibody or its antigen-binding fragment includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15.

[0077] In yet another embodiment, the anti-EphA4 antibody or its antigen-binding fragment includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19.

[0078] In this disclosure, the variable regions of the heavy chain and / or the variable regions of the light chain may have an amino acid sequence in which one or more amino acids are substituted, added and / or deleted from the original sequence. Here, “more” is not limited insofar as it maintains binding affinity to EphA4 and promotes cleavage of EphA4, but is between 2 and 15, or between 2 and 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%.

[0079] In one embodiment, the heavy chain of the anti-EphA4 antibody or its antigen-binding fragment according to the Disclosure comprises the constant region of rabbit IgG. In a particular embodiment, the constant region of rabbit IgG comprises the amino acid sequence of SEQ ID NO: 22.

[0080] In one embodiment, the light chain of the anti-EphA4 antibody or its antigen-binding fragment according to the Disclosure comprises the constant region of rabbit Igκ. In a particular embodiment, the constant region of rabbit Igκ comprises the amino acid sequence of SEQ ID NO: 23.

[0081] In one embodiment, the heavy chain of the anti-EphA4 antibody according to the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 24, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 25.

[0082] In one embodiment, the heavy chain of the anti-EphA4 antibody according to the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 29.

[0083] In one embodiment, the heavy chain of the anti-EphA4 antibody according to the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 32, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 33.

[0084] In one embodiment, the heavy chain of the anti-EphA4 antibody according to the present disclosure comprises the amino acid sequence shown in SEQ ID NO: 36, and the light chain of the anti-EphA4 antibody comprises the amino acid sequence shown in SEQ ID NO: 37.

[0085] In one embodiment, the anti-EphA4 antibody or its antigen-binding fragment according to the Disclosure is labeled. In the Disclosure, “labeled” means a detectable compound or composition that is directly or indirectly bound to the antibody or its antigen-binding fragment. The label may be detectable by itself or by combination with another specific binding pair, for example, in the case of enzymatic labeling, it may produce a detectable signal by acting on or reacting with a substrate compound or composition.

[0086] In another embodiment, for example, to reduce heterogeneity of antibodies produced by antibody-producing cells (see U.S. Patent Application Publication No. 2010 / 0297697 and Liu H et al., MAbs. 2014 Sep-Oct;6(5):1145-1154), the anti-EphA4 antibody may have a deletion of the lysine located at the C-terminus (carboxyl terminus) of the heavy chain. In this disclosure, anti-EphA4 antibodies in which the C-terminal lysine of the heavy chain is deleted 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, etc. Furthermore, in this disclosure, anti-EphA4 antibodies in which the C-terminal lysine of the heavy chain is deleted include not only anti-EphA4 antibodies in which the C-terminal lysine is deleted in both heavy chains, but also anti-EphA4 antibodies in which the C-terminal lysine is deleted in only one of the heavy chains.

[0087] In one embodiment, the disclosure relates to an isolated nucleic acid encoding an anti-EphA4 antibody or its antigen-binding fragment. The isolated nucleic acid encoding an anti-EphA4 antibody or its antigen-binding fragment refers to one or more nucleic acid molecules encoding the heavy chain and / or light chain of the anti-EphA4 antibody or its antigen-binding fragment. In one embodiment, the nucleic acid relating to the disclosure encodes the heavy chain of the anti-EphA4 antibody or its antigen-binding fragment. In another embodiment, the nucleic acid relating to the disclosure encodes the light chain of the anti-EphA4 antibody or its antigen-binding fragment. In yet another embodiment, the nucleic acid relating to the disclosure encodes the heavy and light chains of the anti-EphA4 antibody or its antigen-binding fragment. The nucleic acid relating to the disclosure also includes a first nucleic acid molecule encoding the heavy chain of the anti-EphA4 antibody or its antigen-binding fragment, and a second nucleic acid molecule encoding the light chain of the anti-EphA4 antibody or its antigen-binding fragment.

[0088] In another embodiment, the disclosure relates to a vector comprising an isolated nucleic acid encoding an anti-EphA4 antibody or its antigen-binding fragment. The vectors of the disclosure refer to one or more vectors comprising an isolated nucleic acid encoding an anti-EphA4 antibody or its antigen-binding fragment. In one embodiment, the vector of the disclosure is a vector comprising a nucleic acid encoding the heavy chain of an anti-EphA4 antibody or its antigen-binding fragment and a nucleic acid encoding the light chain of an anti-EphA4 antibody or its antigen-binding fragment. In another embodiment, the vector of the disclosure is a vector comprising nucleic acids encoding the heavy and light chains of an anti-EphA4 antibody or its antigen-binding fragment. In yet another embodiment, the vector of the disclosure comprises a first vector comprising a nucleic acid encoding the heavy chain of an anti-EphA4 antibody or its antigen-binding fragment, and a second vector comprising a nucleic acid encoding the light chain of an anti-EphA4 antibody or its antigen-binding fragment. The vectors of the disclosure may be plasmids, cosmids, viruses, phages, etc., but are not limited to these. For example, viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or herpes simplex virus vectors, among others, which are included in the vectors related to this disclosure.

[0089] In yet another embodiment, the disclosure also includes a host cell containing the vector relating to the disclosure, and a method for producing an anti-EphA4 antibody or its antigen-binding fragment, comprising the step of culturing the host cell. The host cell relating to the disclosure may be, but is not limited to, Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, NS0 cells, etc. In one embodiment, the method for producing an anti-EphA4 antibody or its antigen-binding fragment includes the steps of culturing the host cell and recovering the anti-EphA4 antibody or its antigen-binding fragment secreted from the host cell (or the culture medium of the host cell).

[0090] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure, characterized by the above CDR, binds to any N-terminal region of EphA4. In this disclosure, the “N-terminal region” of EphA4 refers to the extracellular region (ECD) of EphA4, or the N-terminal region obtained when EphA4 is cleaved by a matrix metalloproteinase (MMP) or ADAM (a disintegrin and metalloproteinase). In human EphA4, the ECD is defined as having the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are substituted, added, and / or deleted from said amino acid sequence. Here, "multiple" means 2 to 15, or 2 to 10, for example 9, 8, 7, 6, 5, 4, 3, or 2, or the number of amino acids in the amino acid sequence is 10% or less, for example 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0091] The anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure specifically binds to EphA4 and can detect EphA4 with high detection sensitivity. Accordingly, this disclosure relates, in another embodiment, to a method for detecting or quantifying EphA4 in a biological sample using the anti-EphA4 antibody or its antigen-binding fragment relating to this disclosure (hereinafter also referred to as the method relating to this disclosure).

[0092] In the method relating to this disclosure, the "EphA4" to be measured includes not only the full-length EphA4 but also a fragment consisting of the N-terminal region of EphA4 (hereinafter also referred to simply as "EphA4 N-terminal fragment" or "N-terminal fragment of EphA4").

[0093] In the method relating to this disclosure, the biological sample is not particularly limited as long as it is a sample that may contain full-length EphA4 or the N-terminal fragment of EphA4. Examples include biologically derived liquid components (also called body fluids) such as blood, serum, plasma, cerebrospinal fluid (CSF), urine, saliva, tears, and sweat. In one embodiment, the biological sample is blood, serum, plasma, or cerebrospinal fluid. Furthermore, the biological sample is not limited to biological samples derived from humans, but also includes biological samples from animals other than humans. Examples of such animals, but are not limited to, include mice, rats, rabbits, and monkeys.

[0094] The method relating to this disclosure includes contacting a biological sample with the anti-EphA4 antibody relating to this disclosure or an antigen-binding fragment thereof. Detection or quantification of EphA4 can be carried out using immunoassays well known in the art.

[0095] The method relating to this disclosure may include contacting a biological sample with the anti-EphA4 antibody or its antigen-binding fragment (first antibody) relating to this disclosure, and then further contacting the biological sample with a labeled anti-EphA4 antibody or its antigen-binding fragment (second antibody). In this method, the first antibody and the labeled second antibody may be different antibodies.

[0096] The immunoassay uses a detectably labeled anti-EphA4 antibody or its antigen-binding fragment, or a detectably labeled anti-EphA4 antibody or its antigen-binding fragment (secondary antibody). Depending on the antibody labeling method, immunoassays are classified into enzyme immunoassays (EIA or ELISA), radioimmunoassays (RIA), fluorescent immunoassays (FIA), fluorescence-polarized immunoassays (FPIA), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassays (ECLIA), etc., and any of these can be used in the method relating to this disclosure.

[0097] In the ELISA method, enzymes such as peroxidase and alkaline phosphatase are used, while in the RIA method, 125 I, 131 I, 35 S, 3 In the FPIA method, radioactive substances such as H can be used. In the CLIA method, fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansilchloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials can be used. In the CLIA method, enzymes such as luciferase and β-galactosidase, luminescent substrates that are converted into luminescent substances by each enzyme, and antibodies labeled with luminescent substances such as luciferin and aequorin can be used. In addition, antibodies labeled with nanoparticles such as gold colloid and quantum dots can also be detected.

[0098] Furthermore, in immunoassays, EphA4 can be detected and measured by labeling an anti-EphA4 antibody or its antigen-binding fragment with biotin and then binding it to avidin or streptavidin labeled with an enzyme or the like.

[0099] In the ELISA method, for example, the sandwich method can be used. An anti-EphA4 antibody or its antigen-binding fragment is immobilized on a solid support, and a biological sample that has been appropriately treated is added and reacted. Then, another enzyme-labeled anti-EphA4 antibody or its antigen-binding fragment is added and reacted. After washing, the sample is reacted with an enzyme substrate, color development occurs, and the absorbance is measured to quantify EphA4 or the N-terminal fragment of EphA4.

[0100] For enzymes that are peroxidases, the enzyme substrate can be 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB or TMBZ), o-phenylenediamine (OPD), etc. For alkaline phosphatases, p-nitrophenyl phosphate (NPP), etc., can be used.

[0101] Furthermore, among the immunoassays mentioned above, agglutination methods can be cited as a simple way to detect trace amounts of protein. An example of an agglutination method is latex agglutination, in which latex particles are bound to an antibody.

[0102] When latex particles are bound with an anti-EphA4 antibody or its antigen-binding fragment and mixed with a biological sample, the antibody-bound latex particles will aggregate if EphA4 is present. Therefore, by irradiating the sample with near-infrared light and measuring the absorbance (turbidimetric method) or scattered light (turbidimetric method), the concentration of the aggregates can be determined.

[0103] In one embodiment, the method relating to this disclosure is a method for detecting or quantifying human EphA4 in a biological sample.

[0104] In one embodiment, the method according to this disclosure is a method for detecting or quantifying the N-terminal fragment of human EphA4 in a biological sample.

[0105] In one embodiment, the method according to the Disclosure uses a sandwich ELISA with a combination of the antibody or antigen-binding fragment according to the Disclosure for the detection or quantification of human EphA4.

[0106] This disclosure also relates, in another embodiment, to a kit comprising the anti-EphA4 antibody or its antigen-binding fragment for detecting or quantifying EphA4 (hereinafter also referred to as the Kit of this Disclosure). The Kit of this Disclosure may include any reagents or instruments that can be used to detect or quantify EphA4, or instructions for using the Kit.

[0107] In the kit relating to this disclosure, the "EphA4" to be measured includes not only full-length EphA4 but also the N-terminal fragment of EphA4.

[0108] In one embodiment, the kit relating to the present disclosure relates to a kit for detecting or quantifying human EphA4.

[0109] In one embodiment, the kit relating to the present disclosure relates to a kit for detecting or quantifying the N-terminal fragment of human EphA4.

[0110] In one embodiment, the kit relating to this disclosure includes human full-length EphA4 or serial dilutions of said full-length EphA4, which can be used as a positive control when creating a calibration curve for human EphA4.

[0111] In one embodiment, the kit relating to this disclosure includes a human EphA4 N-terminal fragment or a serial dilution of said fragment that can be used as a positive control when creating a calibration curve for human EphA4.

[0112] In one embodiment, the kit according to this disclosure includes an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15; and an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19.

[0113] In another embodiment, the kit relating to this disclosure includes an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15.

[0114] In another embodiment, the kit relating to this disclosure includes an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11; and an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19.

[0115] In yet another embodiment, the kit relating to this disclosure includes an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; and an anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0116] Examples of specific combinations of anti-EphA4 antibodies or their antigen-binding fragments used in the methods relating to this disclosure, particularly in sandwich ELISA, or included in the kits relating to this disclosure, include the following: (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11. (labeled antibody) An antibody or its antigen-binding fragment, comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0117] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0118] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0119] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0120] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15.

[0121] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15.

[0122] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19.

[0123] (solid phase antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15. (labeled antibody) An antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19.

[0124] Those skilled in the art will understand that, insofar as it does not conflict with the technical specifications, any one or more of the embodiments described herein may be combined as appropriate to implement the Disclosure. Furthermore, those skilled in the art will understand that, insofar as it does not conflict with the technical specifications, it would be preferable to combine as appropriate any preferred or advantageous embodiments described herein to implement the Disclosure.

[0125] Any documents cited herein should be deemed by reference to have all their disclosures expressly incorporated herein, and a person skilled in the art can understand, by means of the context herein, the relevant disclosures in those documents without departing from the spirit and scope of this disclosure.

[0126] The documents cited herein are provided solely for the purpose of disclosing relevant art prior to the filing date of this application and should not be construed as an admission by the inventors that they do not have prior rights to such disclosures, either for prior art or for any other reason. All descriptions in these documents are based on information available to the applicant and do not constitute an admission that the contents of these descriptions are accurate.

[0127] The terms used herein are for the purpose of describing specific embodiments and are not intended to limit the invention.

[0128] As used herein, the term "comprise" is intended to imply the existence of the described items (components, steps, elements, or numbers, etc.), unless the context clearly indicates otherwise, and does not exclude the existence of other items (components, steps, elements, or numbers, etc.). The term "consist of" encompasses the forms described by the terms "consist of" and / or "consist essentially of".

[0129] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as they are broadly understood by those skilled in the art to which this disclosure belongs. Unless otherwise explicitly defined, terms used herein should be construed to have a meaning consistent with that of this specification and the art relating to it, and should not be interpreted in an idealized or overly formal sense.

[0130] While 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 solely to distinguish one element from another, and it is possible, without departing from the scope of this disclosure, to refer to the first element as the second element, and similarly, the second element as the first element.

[0131] In this specification, numerical values ​​used to indicate component content, numerical ranges, etc., should be understood to be modified by the term "approximately" unless otherwise specified. For example, "4°C" should be understood to mean "approximately 4°C" unless otherwise specified, and it is natural that a person skilled in the art can reasonably understand this degree in accordance with common technical knowledge and the intent of this specification.

[0132] Unless the context clearly indicates otherwise, it is understood that, as used herein and in the claims, each aspect expressed in the singular may also be in the plural, and vice versa, as long as it does not technically contradict the singular.

[0133] 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 art can implement the present disclosure with various modifications, additions, deletions, substitutions, etc., without altering the spirit or scope of the present disclosure. [Examples]

[0134] Example 1: Preparation of anti-human EphA4 rabbit monoclonal antibody To produce a monoclonal antibody that binds to human EphA4 (Genbank Accession No. NP_004429.1, SEQ ID NO: 1), a protein in which secreted alkaline phosphatase (SEAP) and a histidine tag were fused to the extracellular region (positions 20-547) of human EphA4 (SEQ ID NO: 2) (hereinafter referred to as "human EphA4 extracellular region-SEAP-His protein," SEQ ID NO: 3), a protein in which a histidine tag was fused to the extracellular region (positions 20-547) of human EphA (hereinafter referred to as "human EphA4 extracellular region-His protein," SEQ ID NO: 4), and a protein in which maltose-binding protein (MBP) and a histidine tag were fused to the extracellular region of human EphA4 (hereinafter referred to as "human EphA4 extracellular region-MBP-His protein," SEQ ID NO: 5) were prepared by the following steps.

[0135] First, we constructed pcDNA3.4-human EphA4 extracellular region-SEAP-His expression vectors, pcDNA3.4-human EphA4 extracellular region-His expression vectors, and pcDNA3.4-human EphA4 extracellular region-MBP-His expression vectors. Genes encoding SEAP-His and the human EphA4 extracellular region were synthesized using Genscript. First, the synthesized gene fragment encoding SEAP-His was cloned into a pcDNA3.4 vector (Invitrogen / LifeTechnologies). Then, the synthesized human EphA4 extracellular region gene fragment was cloned into the constructed pcDNA3.4-SEAP-His expression vector to construct the human EphA4 extracellular region-SEAP-His expression vector. The pcDNA3.4-human EphA4 extracellular region-His expression vector was constructed by cloning a synthesized human EphA4 extracellular region gene fragment into a pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing a DNA sequence encoding a histidine tag. For the pcDNA3.4-human EphA4 extracellular region-MBP-His expression vector, the signal sequence and extracellular region encoding DNA sequence of human EphA4 were amplified by PCR and cloned into a pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing MBP and histidine tag encoding DNA sequences. Each of these expression vectors was transfused into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 expression system (Thermo SCIENTIFIC). The culture medium was collected, cells removed, and clarified. The solution was purified using TALON resin (TaKaRa), and then buffered with PBS (FUJIFILM Wako) by dialysis or desalting column (Thermo SCIENTIFIC).

[0136] Rabbits were immunized with a human EphA4 extracellular region-SEAP-His protein prepared according to a standard procedure, along with an adjuvant (IBL). After immunization, total RNA was prepared from collected lymph node cells using RNeasy (QIAGEN) and treated with DNase (QIAGEN, RNase-free DNase set). Reverse transcripts were prepared from the total RNA using an RNA PCR kit (TAKARA). A rabbit antibody phage library was constructed using the obtained reverse transcripts as templates. Screening was performed using human EphA4 protein, human EphA4 antibody, and the rabbit antibody phage library to obtain rabbit antibody fragments (scFv) that specifically bind to human EphA4. Human EphA4 extracellular region-MBP-His protein and human EphA4 antibody were captured using Dynabeads magnetic beads (Thermo SCIENTIFIC). A rabbit antibody phage library was added, and after 1 or 2 hours, unbound phages were removed by a series of washing cycles using PBS-Tween (0.1% v / v) or PBS. After eluting the bound phage particles, they were amplified by infection with E. coli TG1 host cells. Infected TG1 cells were harvested, plated, and incubated at 30°C. This panning process was repeated once more using the amplified phages.

[0137] After two panning cycles, single colonies from TG1 cells infected with concentrated phages were inoculated into culture medium in a 96-well plate. IPTG was added to induce the expression of FLAG-tagged scFv, and the cells were cultured overnight at 30°C with shaking. The TG1 cells were spun down, and wells showing reactivity to human EphA4 were picked using the E. coli culture supernatant containing scFv.

[0138] The reactivity to human EphA4 was evaluated using human EphA4 extracellular region-MBP-His protein by ELISA according to the following procedure: Anti-FLAG antibody (SIGMA) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at 4°C overnight, the wells were blocked with 2% skim milk (BD) at room temperature for 2 hours. After washing three times with 0.02% Tween20 / PBS, human EphA4 extracellular region-MBP-His protein (final concentration 20 nM) and E. coli culture supernatant containing scFv were added to each well and incubated at room temperature for 2 hours. After washing three times, horseradish peroxidase-labeled anti-His antibody (MBL) was added and incubated at room temperature for 1 hour. After washing five times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated at room temperature for 15-20 minutes. Equal volumes of reaction stop solution (2N H2SO4, FUJIFILM Wako) were added to the wells, and the absorbance at 450 nm was read using a microplate reader (Thermo SCIENTIFIC). Based on the screening results, human EphA4-specific rabbit antibody fragments were selected, and the gene sequences of each fragment were determined by sequencing.

[0139] The obtained rabbit antibody fragments (scFv) were converted from scFv to IgG format by subcloning the DNA sequences encoding the variable regions into vectors expressing the antibody heavy chain and light chain constant regions, respectively. Expression vectors (pcDNA3.4) containing the gene sequences encoding the anti-human EphA4 rabbit monoclonal antibody were constructed. The amino acid sequence of the heavy chain variable region of KPEP11_04 is the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 7. For the gene sequences encoding the amino acid sequences of KPEP11_04, the nucleic acid sequence shown in SEQ ID NO: 8 was used for the heavy chain variable region, and the nucleic acid sequence shown in SEQ ID NO: 9 was used for the light chain variable region. The amino acid sequence of the heavy chain variable region of KPEP11_08 is the amino acid sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 11. For the gene sequences encoding the amino acid sequences of KPEP11_08, the nucleic acid sequence shown in SEQ ID NO: 12 was used for the heavy chain variable region, and the nucleic acid sequence shown in SEQ ID NO: 13 was used for the light chain variable region. The amino acid sequence of the heavy chain variable region of KPEP11_10 is the amino acid sequence shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 15. For the gene sequence encoding the amino acid sequence of KPEP11_10, the nucleic acid sequence shown in SEQ ID NO: 16 was used for the heavy chain variable region, and the nucleic acid sequence shown in SEQ ID NO: 17 was used for the light chain variable region. The amino acid sequence of the heavy chain variable region of KPEP11_18 is the amino acid sequence shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 19. For the gene sequence encoding the amino acid sequence of KPEP11_18, the nucleic acid sequence shown in SEQ ID NO: 20 was used for the heavy chain variable region, and the nucleic acid sequence shown in SEQ ID NO: 21 was used for the light chain variable region. For the heavy chain constant regions of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18, the constant region of rabbit IgG (SEQ ID NO: 22) was used. Rabbit Igκ (SEQ ID NO: 23) was used as the light chain constant region for KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18.The amino acid sequence of the full heavy chain (excluding the signal sequence) of KPEP11_04 is the amino acid sequence shown in SEQ ID NO: 24, and the amino acid sequence of the full light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 25. The nucleic acid sequence encoding the full heavy chain of KPEP11_04 is the nucleic acid sequence shown in SEQ ID NO: 26, and the nucleic acid sequence encoding the full light chain is the nucleic acid sequence shown in SEQ ID NO: 27. The amino acid sequence of the full heavy chain (excluding the signal sequence) of KPEP11_08 is the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of the full light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 29. The nucleic acid sequence encoding the full heavy chain of KPEP11_08 is the nucleic acid sequence shown in SEQ ID NO: 30, and the nucleic acid sequence encoding the full light chain is the nucleic acid sequence shown in SEQ ID NO: 31. The amino acid sequence of the full heavy chain (excluding the signal sequence) of KPEP11_10 is the amino acid sequence shown in SEQ ID NO: 32, and the amino acid sequence of the full light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 33. The nucleic acid sequence encoding the full heavy chain of KPEP11_10 is the sequence shown in SEQ ID NO: 34, and the nucleic acid sequence encoding the full light chain is the sequence shown in SEQ ID NO: 35. The amino acid sequence of the full heavy chain (excluding the signal sequence) of KPEP11_18 is the sequence shown in SEQ ID NO: 36, and the amino acid sequence of the full light chain (excluding the signal sequence) is the sequence shown in SEQ ID NO: 37. The nucleic acid sequence encoding the full heavy chain of KPEP11_18 is the sequence shown in SEQ ID NO: 38, and the nucleic acid sequence encoding the full light chain is the sequence shown in SEQ ID NO: 39. These vectors were transfused into Expi293F cells (ThermoFisher). The supernatant was collected, and anti-human EphA4 rabbit monoclonal antibodies were obtained using MabSelect® (Cytiva), MabSelect SuRepcc (Cytiva), or AmsphereA3 (JSR).

[0140] The CDRs for KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 were determined according to the Kabat definition for CDR identification. The amino acid and nucleic acid sequences of the KPEP11_04 CDR are shown in Tables 1 and 2, respectively. The amino acid and nucleic acid sequences of the KPEP11_08 and KPEP11_10 CDRs are shown in Tables 3 and 4, respectively. The amino acid and nucleic acid sequences of the KPEP11_18 CDR are shown in Tables 5 and 6, respectively.

[0141] [Table 1]

[0142] [Table 2]

[0143] [Table 3]

[0144] [Table 4]

[0145] [Table 5]

[0146] [Table 6]

[0147] The anti-human EphA4 rabbit monoclonal antibodies KPEP11_01, KPEP11_02, KPEP11_05, KPEP11_07, KPEP11_09, KPEP11_12, KPEP11_13, and KPEP11_20 were prepared using the same method as described above. The amino acid sequence of the heavy chain variable region of KPEP11_01 is the amino acid sequence shown in SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 77. The amino acid sequence of the heavy chain variable region of KPEP11_02 is the amino acid sequence shown in SEQ ID NO: 78, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 79. The amino acid sequence of the heavy chain variable region of KPEP11_05 is the amino acid sequence shown in SEQ ID NO: 80, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 81. The amino acid sequence of the heavy chain variable region of KPEP11_07 is the sequence shown in SEQ ID NO: 82, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 83. The amino acid sequence of the heavy chain variable region of KPEP11_09 is the sequence shown in SEQ ID NO: 84, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 85. The amino acid sequence of the heavy chain variable region of KPEP11_12 is the sequence shown in SEQ ID NO: 86, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 87. The amino acid sequence of the heavy chain variable region of KPEP11_13 is the sequence shown in SEQ ID NO: 88, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 89. The amino acid sequence of the heavy chain variable region of KPEP11_20 is the sequence shown in SEQ ID NO: 90, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO: 91. For these antibodies, the constant region of the heavy chain was the constant region of rabbit IgG (SEQ ID NO: 22). For these antibodies, the constant region of the light chain was the constant region of rabbit Igκ (SEQ ID NO: 23).

[0148] The CDR of each antibody was determined according to the Kabat definition for CDR identification. The amino acid sequences of the CDRs of each antibody are shown in Tables 7 to 11.

[0149] [Table 7]

[0150] Table 8

[0151] Table 9

[0152] Table 10 Table 11

[0153] Example 2: Selectivity of anti-human EphA4 monoclonal antibody against human Eph receptors The binding activity of the anti-human EphA4 monoclonal antibody prepared in Example 1 to the human Eph receptor was evaluated according to the following procedure. Mouse anti-6-His antibody (R&D) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at room temperature for 1 hour, the wells were blocked with 1% Block Ace (KAC) at room temperature for 1 hour. After washing three times with 0.02% Tween20 / PBS, human extracellular region-His protein (Creative biomart, final concentration 1 nM) of each Eph receptor was seeded into each well and incubated at room temperature for 1 hour. After washing three times, anti-human EphA4 rabbit monoclonal antibody (10 μg / mL) was added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) 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 at room temperature for 4.5 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Thermo SCIENTIFIC).

[0154] KPEP11_01, KPEP11_02, KPEP11_04, KPEP11_05, KPEP11_07, KPEP11_08, KPEP11_09, KPEP11_10, KPEP11_12, KPEP11_13, KPEP11_18, and KPEP11_20 were found to specifically bind to human EphA4 among the human Eph receptor family (Figure 1).

[0155] Example 3: Reactivity of anti-human EphA4 monoclonal antibody against mouse, rat, rabbit, monkey, and human EphA4 The binding activity of the anti-human EphA4 monoclonal antibody prepared in Example 1 with various EphA4 proteins was evaluated according to the following procedure. Mouse anti-6-His antibody (R&D) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at room temperature for 1 hour, the wells were blocked overnight at 4°C with 1% Block Ace (KAC). After washing three times with 0.02% Tween20 / PBS, mouse EphA4 extracellular region-SEAP-His protein, rat EphA4 extracellular region-SEAP-His protein, rabbit EphA4 extracellular region-SEAP-His protein, monkey EphA4 extracellular region-SEAP-His protein, human EphA4 extracellular region-SEAP-His protein, or SEAP-His protein (final concentration 1 nM) was seeded into the wells and incubated at room temperature for 1 hour. After washing three times, anti-human EphA4 rabbit monoclonal antibody (10 μg / mL) was added and incubated at room temperature for approximately 1 hour. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) 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 at room temperature for 3 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Thermo SCIENTIFIC).

[0156] KPEP11_01, KPEP11_02, KPEP11_04, KPEP11_05, KPEP11_07, KPEP11_08, KPEP11_09, KPEP11_10, KPEP11_12, KPEP11_13, KPEP11_18, and KPEP11_20 exhibited binding activity to monkey and human EphA4 (Figure 2).

[0157] Example 4: Reactivity of anti-human EphA4 monoclonal antibody to the extracellular domain, ligand-binding domain, fibronectin type III domain 1, and fibronectin type III domain 2 of human EphA4. The binding activity of the anti-human EphA4 monoclonal antibody prepared in Example 1 with various EphA4 proteins was evaluated according to the following procedure. Mouse anti-6-His antibody (R&D) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at room temperature for 1 hour, the wells were blocked with 1% Block Ace (KAC) at room temperature for 1 hour. After washing three times with 0.02% Tween20 / PBS, 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, 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, anti-human EphA4 rabbit monoclonal antibody (10 μg / mL) was added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) 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 at room temperature for 4.5 minutes. An equal volume of stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells and the absorbances at 450 nm and 650 nm were read using a microplate reader (Thermo SCIENTIFIC).

[0158] KPEP11_02, KPEP11_04, KPEP11_05, KPEP11_07, KPEP11_18, and KPEP11_20 had binding activity to the human EphA4 extracellular domain (ECD) and ligand-binding domain (LBD). KPEP11_01, KPEP11_08, KPEP11_09, KPEP11_10, KPEP11_12, and KPEP11_13 also had binding activity to the human EphA4 extracellular domain (ECD) (Figure 3).

[0159] Example 5: Reactivity of the extracellular domain of human EphA4 by sandwich ELISA The binding activity of the anti-human EphA4 monoclonal antibodies prepared in Example 1 to the extracellular domain of human EphA4 was evaluated according to the following procedure. Each anti-human EphA4 monoclonal antibody was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation at 4°C overnight, the wells were blocked at room temperature for at least 1 hour or at 4°C overnight with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)). As detection antibodies, each anti-human EphA4 monoclonal antibody was labeled with HRP (Horseradish Peroxidase) using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Laboratories) according to the attached manual. After blocking the plates, they were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20). Human EphA4 extracellular regions (0, 1, 10 ng / mL) were seeded into the wells using a serial dilution of sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), and incubated at room temperature for 2 hours. After three washes, 100 μL of HRP-labeled anti-human EphA4 monoclonal antibody, diluted 1500-fold with the sample diluent, was added to each well, 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 620 nm were read using a microplate reader (molecular device).

[0160] The reactivity lists for each combination of anti-human EphA4 monoclonal antibody are shown in Figures 4, 5, 6, and 7, respectively, as follows: (1) SN ((signal obtained when seeding 10 ng / mL EphA4 extracellular region) - (signal obtained when seeding 0 ng / mL EphA4 extracellular region)), (2) S / N ((signal obtained when seeding 10 ng / mL EphA4 extracellular region) / (signal obtained when seeding 0 ng / mL EphA4 extracellular region)), (3) SN ((signal obtained when seeding 1 ng / mL EphA4 extracellular region) - (signal obtained when seeding 0 ng / mL EphA4 extracellular region)), and (4) S / N ((signal obtained when seeding 1 ng / mL EphA4 extracellular region) / (signal obtained when seeding 0 ng / mL EphA4 extracellular region)). Several combinations of anti-human EphA4 monoclonal antibodies that exhibit strong binding activity to the extracellular domain of EphA4 were obtained.

[0161] Example 6: Binding affinity of anti-human EphA4 monoclonal antibody to human EphA4 The binding affinity of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 to human EphA4 was determined by surface plasmon resonance (SPR) spectroscopy using Biacore T200 (Cytiva). First, an anti-His antibody (Cytiva, 28-9950-56) was diluted to 10 μg / mL using immobilization buffer (10 mM sodium acetate, pH 4.5) and immobilized on the sensor chip CM5 according to the protocol provided with the Biacore T200. Immobilization was performed by amine coupling using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), with ethanolamine used for blocking (all sensor chips and immobilization reagents were manufactured by Cytiva). Human EphA4 extracellular region-MBP-His10 was diluted with running buffer HBS-EP+ (Cytiva) and delivered onto a flow cell for 120 seconds for capture (capture volume of approximately 3RU). Subsequently, KPEP11_04, KPEP11_08, or KPEP11_10, which were sequentially diluted with HBS-EP+ in the ranges of 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 nM, and KPEP11_18, which was sequentially diluted in the ranges of 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, and 0 nM, were added to the sensor tip for 120 seconds, and the binding reaction curves were sequentially observed at the time of addition (binding phase, 120 seconds) and after the completion of addition (dissociation phase, 300 seconds). After each observation was completed, the sensor chip was regenerated by adding 10 mM glycine hydrochloride pH 1.5 (for 60 seconds) and 3 M MgCl2 (for 30 seconds). The obtained binding reaction curves were fitted using a 1:1 binding model with the system's accompanying software, BIA evaluation software, to calculate the binding affinity (KD = kd / ka) for human EphA4. The above experiment was performed three times, and the average value for each parameter was calculated.

[0162] The binding affinity (KD value) of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 to human EphA4 is shown in Tables 12, 13, 14, and 15, respectively. A representative binding reaction curve is shown in Figure 8.

[0163] [Table 12]

[0164] [Table 13]

[0165] [Table 14]

[0166] [Table 15]

[0167] Example 7: Selectivity of anti-human EphA4 monoclonal antibody against human Eph receptors The binding activity of KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, and anti-EphA4 polyclonal antibodies (Sinó Biological) to the human Eph receptor was evaluated according to the following procedure. Mouse anti-6-His antibody (R&D) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at room temperature for 1 hour or overnight at 4°C, the wells were blocked with 1% Block Ace (KAC) at room temperature for 1 hour or overnight at 4°C. After washing three times with 0.02% Tween20 / PBS, human extracellular region-His protein (Creative biomart, final concentration 1 nM) of each Eph receptor was seeded into each well and incubated at room temperature for 1 hour. After washing three times, KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, or anti-EphA4 polyclonal antibody (1 μg / mL) was added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) was added and incubated at room temperature for 1 hour. After washing five times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated at room temperature for 3-5 minutes. An equal volume of stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells and the absorbance at 450 nm and 650 nm was read using a microplate reader (Thermo SCIENTIFIC).

[0168] KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 were found to specifically bind to human EphA4 within the human Eph receptor family (Figure 9).

[0169] Example 8: Reactivity of anti-human EphA4 monoclonal antibody against mouse, rat, rabbit, monkey, and human EphA4 The mouse EphA4 extracellular region-SEAP-His protein, rat EphA4 extracellular region-SEAP-His protein, rabbit EphA4 extracellular region-SEAP-His protein, monkey EphA4 extracellular region-SEAP-His protein, and human EphA4 extracellular region-SEAP-His protein were produced according to the following procedure. Genes encoding SEAP-His and the mouse EphA4 extracellular region, rat EphA4 extracellular region, rabbit EphA4 extracellular region, monkey EphA4 extracellular region, and human EphA4 extracellular region were synthesized using Genscript. First, the gene fragment encoding the synthesized SEAP-His was cloned into a pcDNA3.4 vector (Invitrogen / LifeTechnologies). Genetic fragments of the synthesized mouse EphA4 extracellular region, rat EphA4 extracellular region, rabbit EphA4 extracellular region, monkey EphA4 extracellular region, and human EphA4 extracellular region were cloned into the constructed pcDNA3.4-SEAP-His expression vector, and mouse EphA4 extracellular region-SEAP-His expression vector, rat EphA4 extracellular region-SEAP-His expression vector, rabbit EphA4 extracellular region-SEAP-His expression vector, monkey EphA4 extracellular region-SEAP-His expression vector, and human EphA4 extracellular region-SEAP-His expression vector were constructed. The amino acid sequence of human EphA4 used in vector construction is denoted as SEQ ID NO: 1, its extracellular region as SEQ ID NO: 2, the amino acid sequence of monkey EphA4 as SEQ ID NO: 113, its extracellular region as SEQ ID NO: 114, the amino acid sequence of rabbit EphA4 as SEQ ID NO: 115, its extracellular region as SEQ ID NO: 116, the amino acid sequence of rat EphA4 as SEQ ID NO: 117, its extracellular region as SEQ ID NO: 118, and the amino acid sequence of mouse EphA4 as SEQ ID NO: 119, its extracellular region as SEQ ID NO: 120.Various EphA4 extracellular region-SEAP-His proteins were prepared using human EphA4 extracellular region-SEAP-His protein expression vectors, monkey EphA4 extracellular region-SEAP-His protein expression vectors, rabbit EphA4 extracellular region-SEAP-His protein expression vectors, rat EphA4 extracellular region-SEAP-His protein expression vectors, and mouse EphA4 extracellular region-SEAP-His protein expression vectors. The above expression vectors were transfused into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 expression system (Thermo SCIENTIFIC). After 4 days, the culture medium was collected, cells were removed, and the medium was clarified. Purification was performed using TALON resin (TaKaRa), and the buffer was replaced with PBS (FUJIFILM Wako) by dialysis.

[0170] The binding activity of KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, and EphA4 polyclonal antibodies (Sinó Biological) to various EphA4 was evaluated according to the following procedure: Mouse anti-6-His antibody (R&D) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at room temperature for 1 hour or overnight at 4°C, the wells were blocked with 1% Block Ace (KAC) at room temperature for 1 hour or overnight at 4°C. After washing three times with 0.02% Tween20 / PBS, mouse EphA4 extracellular region-SEAP-His protein, rat EphA4 extracellular region-SEAP-His protein, rabbit EphA4 extracellular region-SEAP-His protein, monkey EphA4 extracellular region-SEAP-His protein, human EphA4 extracellular region-SEAP-His protein, or SEAP-His protein (final concentration 1 nM) were seeded into the wells and incubated at room temperature for 1 hour. After washing three times, KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, or EphA4 polyclonal antibodies (0, 1.024e-6, 0.00000512, 0.0000256, 0.000128, 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, 10 μg / mL) were added and incubated at room temperature for approximately 1 hour. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) was added and incubated at room temperature for 1 hour. After washing five times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated at room temperature for 3-5 minutes. Equal volumes of reaction stop solution (2N H2SO4, FUJIFILM Wako) were added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Thermo SCIENTIFIC).

[0171] KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 exhibited equivalent binding activity in monkey and human EphA4 (Figure 10).

[0172] Example 9: Reactivity of anti-human EphA4 monoclonal antibody to the extracellular domain, ligand-binding domain, fibronectin type III domain 1, and fibronectin type III domain 2 of human EphA4. The production of proteins fused with the extracellular domain (ECD), ligand-binding domain (LBD), fibronectin type III domain 1 (FN1), or fibronectin type III domain 2 (FN2) of human EphA4, along with maltose-binding protein (MBP) and a histidine tag (hereinafter 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") was carried out according to the following steps. First, a 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, the signal sequence of human EphA4 (SEQ ID NO: 121) or the signal sequence of preprotrypsin (SEQ ID NO: 122) and the DNA sequences encoding each domain of human EphA4 were amplified by PCR. These were then cloned into a pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding MBP and histidine tags with AAA or G4S linkers, and expression vectors for 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 constructed. 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: 123, fibronectin type III domain 1 as SEQ ID NO: 124, fibronectin type III domain 2 as SEQ ID NO: 125, and MBP and histidine tag (MBP-His protein) as SEQ ID NO: 126. The expression vector was transfused into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 expression system (Thermo SCIENTIFIC). After 4 days, the culture medium was collected, cells were removed, and the medium was clarified.Human EphA4 extracellular domain-MBP-His protein or human EphA4 ligand-binding domain-MBP-His protein was purified using TALON resin (TaKaRa) and buffered with PBS (FUJIFILM Wako) 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).

[0173] The binding activity of KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, and EphA4 polyclonal antibodies (Sinó Biological) to various EphA4 was evaluated according to the following procedure: Mouse anti-6-His antibody (R&D) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After incubation at room temperature for 1 hour or overnight at 4°C, the wells were blocked with 1% Block Ace (KAC) at room temperature for 1 hour or overnight at 4°C. After washing three times with 0.02% Tween20 / PBS, 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, human EphA4 fibronectin type III domain 2-MBP-His protein, or MBP-His protein (final concentration 1 nM) were seeded into the wells and incubated at room temperature for 1 hour. After washing three times, KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, or EphA4 polyclonal antibody (10 nM) was added and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) 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 at room temperature for 3-5 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Thermo SCIENTIFIC).

[0174] KPEP11_04 and KPEP11_18 exhibited binding activity to the extracellular domain (ECD) and ligand-binding domain (LBD) of human EphA4. KPEP11_08 and KPEP11_10 exhibited binding activity to the extracellular domain (ECD) of human EphA4 (Figure 11).

[0175] Example 10: Reactivity of human EphA4 extracellular region and human cerebrospinal fluid EphA4 N-terminal fragment by sandwich ELISA 1The binding activity of KPEP11_10, KPEP11_18, and EphA4 antibodies (R&D) to the extracellular region of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) was evaluated according to the following procedure. KPEP11_10 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation at 4°C overnight, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_18 and EphA4 antibodies (R&D) were labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the included manual. After blocking the plates were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) sequentially diluted with a sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), or a 100-fold diluted sample (human cerebrospinal fluid) were seeded into the wells and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_18 or HRP-labeled EphA4 antibody (R&D), diluted 10,000-fold with sample diluent, was added to each well 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Molecular Device).

[0176] Figure 12 shows the results of evaluating the reactivity to the extracellular region of human EphA4. The sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_18 showed significantly higher reactivity to the extracellular region of human EphA4 compared to the measurement system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D). Next, Figure 13 shows the results of evaluating the reactivity to the N-terminal fragment of EphA4 in cerebrospinal fluid. The measurement system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D) showed almost no reaction signal, while the measurement system constructed using KPEP11_10 and HRP-labeled KPEP11_18 showed very high reactivity.

[0177] Example 11: Reactivity of human EphA4 extracellular region and human cerebrospinal fluid EphA4 N-terminal fragment by sandwich ELISA 2 The binding activity of KPEP11_10, KPEP11_18, and EphA4 antibodies (R&D) to the extracellular region of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) was evaluated according to the following procedure. KPEP11_18 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation overnight at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_10 and EphA4 antibodies (R&D) were labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the included manual. After blocking the plates were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) sequentially diluted with a sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), or a 100-fold diluted sample (human cerebrospinal fluid) were seeded into the wells and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_10 or HRP-labeled EphA4 antibody (R&D), diluted 10,000-fold with sample diluent, was added to each well 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Molecular Device).

[0178] The results of evaluating the reactivity against the human EphA4 extracellular region are shown in Fig. 14. In the sandwich ELISA constructed using KPEP11_18 and HRP-labeled KPEP11_10, it had a very high reactivity against the human EphA4 extracellular region as compared with the measurement system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D). Next, the results of evaluating the reactivity against the EphA4 N-terminal fragment in cerebrospinal fluid are shown in Fig. 15. In the measurement system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D), almost no reaction signal was obtained, whereas in the measurement system constructed using KPEP11_18 and HRP-labeled KPEP11_10, it was found to show a very high reactivity.

[0179] Example 12: Reactivity of human EphA4 extracellular region and human cerebrospinal fluid EphA4 N-terminal fragment by sandwich ELISA 3 The binding activity of KPEP11_10, KPEP11_04, and EphA4 antibodies (R&D) to the extracellular region of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) was evaluated according to the following procedure. KPEP11_10 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation overnight at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_04 and EphA4 antibodies (R&D) were labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the included manual. After blocking the plates were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) sequentially diluted with a sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), or a 100-fold diluted sample (human cerebrospinal fluid) were seeded into the wells and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_04 or HRP-labeled EphA4 antibody (R&D), diluted 10,000-fold with sample diluent, was added to each well 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Molecular Device).

[0180] The results of evaluating the reactivity against the extracellular region of human EphA4 are shown in Fig. 16. In the sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_04, it had very high reactivity against the extracellular region of human EphA4 as compared with the measurement system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D). Next, the results of evaluating the reactivity against the EphA4 N-terminal fragment in cerebrospinal fluid are shown in Fig. 17. In the measurement system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D), almost no reaction signal was obtained, whereas in the measurement system constructed using KPEP11_10 and HRP-labeled KPEP11_04, it was found to show very high reactivity.

[0181] Example 13: Reactivity of human EphA4 extracellular region and human cerebrospinal fluid EphA4 N-terminal fragment by sandwich ELISA 4 The binding activity of KPEP11_08, KPEP11_18, and EphA4 antibodies (R&D) to the extracellular region of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) was evaluated according to the following procedure. KPEP11_18 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation overnight at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_08 and EphA4 antibodies (R&D) were labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the included manual. After blocking the plates were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) sequentially diluted with a sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), or a 100-fold diluted sample (human cerebrospinal fluid) were seeded into the wells and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_08 or HRP-labeled EphA4 antibody (R&D), diluted 10,000 times with sample diluent, was added to each well 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbance at 450 nm and 650 nm was read using a microplate reader (Molecular Device).

[0182] Figure 18 shows the results of evaluating the reactivity to the extracellular region of human EphA4. The sandwich ELISA constructed using KPEP11_18 and HRP-labeled KPEP11_08 showed significantly higher reactivity to the extracellular region of human EphA4 compared to the measurement system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D). Next, Figure 19 shows the results of evaluating the reactivity to the N-terminal fragment of EphA4 in cerebrospinal fluid. The measurement system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D) showed almost no reaction signal, while the measurement system constructed using KPEP11_18 and HRP-labeled KPEP11_08 showed very high reactivity.

[0183] Example 14: Reactivity of human EphA4 extracellular region, EphA4 N-terminal fragment in human plasma, and EphA4 N-terminal fragment in human cerebrospinal fluid by sandwich ELISA. The binding activity of KPEP11_04, KPEP11_10, KPEP11_18, EphA4 antibody (Sino Biological, hereinafter referred to as "EphA4 antibody (Sino)"), and EphA4 antibody (R&D) to the extracellular region of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) was evaluated according to the following procedure. KPEP11_10 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% Sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation at 4°C overnight, the wells were blocked with a blocking solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_04, KPEP11_18, EphA4 antibody (Sino), and EphA4 antibody (R&D) were labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Laboratories) according to the included manual. After blocking the plates were washed three times with washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) were seeded into the wells using serially diluted human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL) with sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), 50-fold diluted human plasma, or 100-fold diluted human cerebrospinal fluid, respectively, and incubated at room temperature for 2 hours. After washing five times, 100 μL each of HRP-labeled KPEP11_04, KPEP11_18, and EphA4 antibody (Sino) or EphA4 antibody (R&D), diluted 50,000 times with sample diluent, were 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (molecular device).

[0184] The results of evaluating the reactivity to the extracellular region of human EphA4, the reactivity to the N-terminal fragment of EphA4 in human plasma, and the reactivity to the N-terminal fragment of EphA4 in human cerebrospinal fluid are shown in Figures 20, 21, and 22. The sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_18 showed very high reactivity to the extracellular region of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid. The sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_04 also showed reactivity to the extracellular region of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid.

[0185] Example 15: Reactivity of human EphA4 extracellular region, EphA4 N-terminal fragment in human plasma, and EphA4 N-terminal fragment in human cerebrospinal fluid using sandwich ELISA. The binding activity of KPEP11_08, KPEP11_10, KPEP11_18, EphA4 antibody (Sino), and EphA4 antibody (R&D) to the extracellular region of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) was evaluated according to the following procedure. KPEP11_18 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% Sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation overnight at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_08, KPEP11_10, EphA4 antibody (Sino), and EphA4 antibody (R&D) were labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the included manual. After blocking the plates were washed three times with washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) were seeded into the wells using serially diluted human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL) with sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), 50-fold diluted human plasma, or 100-fold diluted human cerebrospinal fluid, respectively, and incubated at room temperature for 2 hours. After washing five times, 100 μL each of HRP-labeled KPEP11_08, KPEP11_10, and EphA4 antibody (Sino) or EphA4 antibody (R&D), diluted 50,000 times with sample diluent, were 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 at room temperature for 30 minutes.Equal volumes of reaction stop solution (2N H2SO4, FUJIFILM Wako) were added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Molecular Device).

[0186] The results of evaluating the reactivity to the extracellular region of human EphA4, the reactivity to the N-terminal fragment of EphA4 in human plasma, and the reactivity to the N-terminal fragment of EphA4 in human cerebrospinal fluid are shown in Figures 23, 24, and 25, respectively. The sandwich ELISA constructed using KPEP11_18 and HRP-labeled KPEP11_08, and the sandwich ELISA constructed using KPEP11_18 and HRP-labeled KPEP11_10, all showed very high reactivity to the extracellular region of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid.

[0187] Example 16: Quantitative and correlational analysis of EphA4 N-terminal fragments in human cerebrospinal fluid using ELISA and LC-MS. Quantitative analysis by LC-MS Sample preparation was carried out as follows. For calibration curve samples, 100 μL each of human EphA4 extracellular region (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 ng / mL) was used, which had been sequentially diluted with BSA (SIGMA) solution diluted to a final concentration of 500 μg / mL using aCSF (Harvard Apparatus). A 50 μL cerebrospinal fluid (CSF) sample was mixed with 50 μL of BSA (SIGMA) solution diluted to a final concentration of 500 μg / mL using aCSF (Harvard Apparatus), and then subjected to the following procedure: 150 μL of 10M Urea solution dissolved in 150 μL of 50 mM TEAB (Thermo Fisher) was added sequentially, followed by 25 μL of 100 mM DTT solution. The mixed solution was incubated at 37°C for 120 minutes, then 25 μL of 200 mM iodoacetamide (FUJIFILM Wako) was added, and the mixture was incubated in the dark at room temperature for 30 minutes. Subsequently, 1250 μL of EphA4-NTF-IS and 10 μL of trypsin solution (200 μg / mL) were added sequentially, and the mixture was incubated at 37°C for 18 hours. 80 μL of 20% TFA was added to stop the trypsin digestion reaction. The sample was purified using an Oasis HLB 96-Well Plate (Waters) as follows: The plate was washed with 500 μL of methanol (FUJIFILM Wako), and then equilibrated with 500 μL of 0.1% TFA (Thermo Fisher). The sample prepared above was applied and adsorbed onto the column, and then washed with 500 μL of 0.1% TFA. Next, 200 μL of eluate (0.1% TFA-80% acetonitrile in water (FUJIFILM Wako)) was added and the eluate was collected. The collected eluate was dried using a SpeedVac system (Thermo Fisher), and then 30 μL of 0.1% TFA-5% acetonitrile in water was used to obtain the final reconstituted solution, which was then subjected to LC-MS analysis.

[0188] Quantitative analysis by ELISA 1 Quantitative analysis of the EphA4 N-terminal fragment in human cerebrospinal fluid was performed according to the following procedure. KPEP11_10 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% Sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation overnight at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_18 was labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the accompanying manual. After blocking the plates were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) sequentially diluted with a sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), or 100-fold diluted human cerebrospinal fluid were seeded into the wells, and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_18 diluted 50,000 times with sample diluent was added to each well, 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Molecular Device).

[0189] Quantitative analysis by ELISA 2 Quantitative analysis of the EphA4 N-terminal fragment in human cerebrospinal fluid was performed according to the following procedure. KPEP11_18 was prepared to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% Sodium azide, and 100 μL was coated onto each well of a 96-well plate (Nunc). After incubation overnight at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween20 / 5% skim milk (FUJIFILM Wako)) at room temperature for at least 1 hour or overnight at 4°C. KPEP11_10 was labeled with HRP using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Research Institute) according to the accompanying manual. After blocking the plates were washed three times with a washing solution (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.01% Tween20), human EphA4 extracellular regions (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, labeled as EphA4 in the figure) sequentially diluted with a sample diluent (50mM Tris-HCl (pH 7.5) / 150mM NaCl / 0.2% EDTA-3Na / 4% PEG6000 / 0.01% Tween20 / 0.2% Proclin150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)), or 100-fold diluted human cerebrospinal fluid were seeded into the wells, and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_10 diluted 40,000 times with sample diluent was added to each well, 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 at room temperature for 30 minutes. An equal volume of reaction stop solution (2N H2SO4, FUJIFILM Wako) was added to the wells, and the absorbances at 450 nm and 650 nm were read using a microplate reader (Molecular Device).

[0190] Figure 26 shows the results of a correlation analysis performed based on the quantitative results of EphA4 N-terminal fragments in human cerebrospinal fluid analyzed by LC-MS and quantitative analysis 1 by ELISA. Spearman's correlation coefficient (r) was calculated between the amount of EphA4 N-terminal fragments quantified by LC-MS and the amount of EphA4 N-terminal fragments quantified by ELISA, and a significant correlation was found between them (p<0.0001).

[0191] Figure 27 shows the results of a correlation analysis performed based on the quantitative results of EphA4 N-terminal fragments in human cerebrospinal fluid analyzed by LC-MS and quantitative analysis 2 by ELISA. Spearman's correlation coefficient (r) was calculated between the amount of EphA4 N-terminal fragments quantified by LC-MS and the amount of EphA4 N-terminal fragments quantified by ELISA, and a significant correlation was found between them (p<0.0001).

Claims

1. An anti-EphA4 antibody or its antigen-binding fragment, The aforementioned antibody is (a) Heavy chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 52; Heavy chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 53; and A heavy chain containing the heavy chain CDR3 having the amino acid sequence shown in Sequence ID No. 54; and Light chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 55; Light chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 56; and A light chain containing CDR3, which has the amino acid sequence shown in Sequence ID No. 57; (b) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 64; Heavy chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 65; and A heavy chain containing the heavy chain CDR3 having the amino acid sequence shown in Sequence ID No. 66; and Light chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 67; Light chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 68; and A light chain containing the light chain CDR3 having the amino acid sequence shown in Sequence ID No. 69; or (c) Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 40; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 41; and A heavy chain containing the heavy chain CDR3 having the amino acid sequence shown in Sequence ID No. 42; and Light chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 43; Light chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 44; and A light chain containing the light chain CDR3, which has the amino acid sequence shown in Sequence ID No. 45; including, Anti-EphA4 antibody or its antigen-binding fragment.

2. The anti-EphA4 antibody or its antigen-binding fragment according to claim 1, The aforementioned antibody is Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 52; Heavy chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 53; and A heavy chain containing the heavy chain CDR3 having the amino acid sequence shown in Sequence ID No. 54; and Light chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 55; Light chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 56; and A light chain containing CDR3, which has the amino acid sequence shown in Sequence ID No. 57; including, Anti-EphA4 antibody or its antigen-binding fragment.

3. The anti-EphA4 antibody or its antigen-binding fragment according to claim 2, The aforementioned antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 10, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 11, including, Anti-EphA4 antibody or its antigen-binding fragment.

4. The anti-EphA4 antibody or its antigen-binding fragment according to claim 2, The aforementioned antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 14, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 15, including, Anti-EphA4 antibody or its antigen-binding fragment.

5. The anti-EphA4 antibody or its antigen-binding fragment according to claim 1, The aforementioned antibody is Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 64; Heavy chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 65; and A heavy chain containing the heavy chain CDR3 having the amino acid sequence shown in Sequence ID No. 66; and Light chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 67; Light chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 68; and A light chain containing CDR3, which has the amino acid sequence shown in Sequence ID No. 69; including, Anti-EphA4 antibody or its antigen-binding fragment.

6. The anti-EphA4 antibody or its antigen-binding fragment according to claim 5, The aforementioned antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 18, and Light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 19, including, Anti-EphA4 antibody or its antigen-binding fragment.

7. The anti-EphA4 antibody or its antigen-binding fragment according to claim 1, The aforementioned antibody is Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 40; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 41; and A heavy chain containing the heavy chain CDR3 having the amino acid sequence shown in Sequence ID No. 42; and Light chain CDR1 consisting of the amino acid sequence shown in Sequence ID No. 43; Light chain CDR2 consisting of the amino acid sequence shown in Sequence ID No. 44; and A light chain containing the light chain CDR3, which has the amino acid sequence shown in Sequence ID No. 45; including, Anti-EphA4 antibody or its antigen-binding fragment.

8. The anti-EphA4 antibody or its antigen-binding fragment according to claim 7, The aforementioned antibody is A heavy chain variable region consisting of the amino acid sequence shown in Sequence ID No. 6, and The light chain variable region consisting of the amino acid sequence shown in Sequence ID No. 7, including, Anti-EphA4 antibody or its antigen-binding fragment.

9. An anti-EphA4 antibody or its antigen-binding fragment according to any one of claims 1 to 8, The antibody or its antigen-binding fragment is labeled. Anti-EphA4 antibody or its antigen-binding fragment.

10. A method for producing an anti-EphA4 antibody or an antigen-binding fragment thereof, comprising the step of culturing a host cell containing an isolated nucleic acid encoding an anti-EphA4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

11. A method for detecting or quantifying human EphA4 in a biological sample, This includes contacting the biological sample with the anti-EphA4 antibody or its antigen-binding fragment according to any one of claims 1 to 8. method.

12. The method according to claim 11, The method wherein the human EphA4 is the N-terminal fragment of human EphA4.

13. The method according to claim 11, A method wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

14. The method according to claim 11, Furthermore, the method includes contacting the biological sample with the labeled anti-EphA4 antibody or its antigen-binding fragment according to claim 9. method.

15. A kit for detecting or quantifying human EphA4, The anti-EphA4 antibody according to any one of claims 1 to 8 or its antigen-binding fragment comprises kit.

16. The kit according to claim 15, The aforementioned human EphA4 is the N-terminal fragment of human EphA4. kit.

17. The kit according to claim 15, An anti-EpH4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, and An anti-EpH4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19, including, kit.

18. The kit according to claim 15, An anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7, An anti-EpH4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15, including, kit.

19. The kit according to claim 15, An anti-EphA4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11, and An anti-EpH4 antibody or its antigen-binding fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19, including, kit.

20. The kit according to claim 15, The kit contains the N-terminal fragment of human EphA4, kit.

21. The kit according to claim 15, The antibody or its antigen-binding fragment is labeled. kit.

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