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

Anti-EphA4 antibody

[0001] The present disclosure relates to antibodies that bind to EphA4, nucleic acids encoding the antibodies, vectors containing the nucleic acids, cells containing the vectors, methods for producing the antibodies, methods for detecting or quantifying EphA4 using the antibodies, and kits for detecting or quantifying EphA4.

[0002] EphA4 is a member of the receptor tyrosine kinase family and a molecule that regulates spines, small thorn-like structures present on dendrites. Ephrin type A and type B are known to be ligands of EphA4. Binding of EphA4 to its ligand, ephrin, induces a detachment signal, leading to spine retraction. EphA4 is highly expressed in the hippocampus and cerebral cortex, and its extracellular domain is cleaved by matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinase). These cleaved EphA4 fragments are released extracellularly and are present in plasma (Patent Document 1).

[0003] EphA4 has been suggested to be involved in the pathology 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). Furthermore, spine density is reduced in AD, and the degree of this reduction correlates with clinical symptoms of AD (Non-Patent Document 5). Therefore, abnormal EphA4 activation is thought to 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 certain neurological diseases, such as AD. Therefore, there is a need for antibodies that can detect EphA4 or its extracellular fragments in biological samples with higher detection sensitivity than conventional antibodies.

[0004] WO2012 / 147798A1

[0005] Rosenberger AF et al. , Acta Neuropathol Commun. 2014 Jul 16;2:79Fu AK et al. , Proc Natl Acad Sci USA. 2014 Jul 8;111(27):9959-64 Vargas LM et al. ,, PLoS One. 2014 Mar 21;9(3) Huang TY et al. , J Exp Med. 2017 Dec 4;214(12):3669-3685. Boros et al. , Ann Neurol. 2017 Oct; 82(4):602-614 Vargas LM et al. , Biochim Biophys Acta Mol Basis Dis. 2018 Apr;1864:1148-1159

[0006] An object of the present disclosure is to provide an antibody that specifically binds to EphA4 and is capable of detecting EphA4 with high detection sensitivity. Another object of the present disclosure is to provide a method for detecting or quantifying EphA4 using the antibody. A further object of the present disclosure is to provide a kit containing an anti-EphA4 antibody that is capable of specifically detecting or quantifying EphA4 with high detection sensitivity.

[0007] In order to solve the above problems, the present inventors discovered that an antibody capable of specifically binding to EphA4 with particularly high detection sensitivity could be constructed from a large number of scFvs obtained by screening a rabbit antibody phage library, and thus completed an anti-EphA4 antibody.

[0008] Accordingly, the present disclosure includes the following features: [1] An anti-EphA4 antibody or antigen-binding fragment thereof, comprising: (a) a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 52, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 53, and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 54; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 55, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 56, and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 57; (b) a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 66; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67, a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 68, and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 69; or (c) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 40; An anti-EphA4 antibody or antigen-binding fragment thereof, comprising: a heavy chain comprising a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 41; and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 42; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 43; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 44; and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 45.

[0009] [2] The anti-EphA4 antibody or antigen-binding fragment thereof according to [1], wherein the antibody comprises a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 52; a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 53; and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 54; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 55; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 56; and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 57.

[0010] [3] The anti-EphA4 antibody or antigen-binding fragment thereof according to [2], 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.

[0011] [4] The anti-EphA4 antibody or antigen-binding fragment thereof according to [2], 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.

[0012] [5] The anti-EphA4 antibody or antigen-binding fragment thereof according to [1], wherein the antibody comprises a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 64; a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 65; and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 66; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 67; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 68; and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 69.

[0013] [6] The anti-EphA4 antibody or antigen-binding fragment thereof according to [5], 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.

[0014] [7] The anti-EphA4 antibody or antigen-binding fragment thereof according to [1], wherein the antibody comprises a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 40; a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 41; and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 42; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 43; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 44; and a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 45.

[0015] [8] The anti-EphA4 antibody or antigen-binding fragment thereof according to [7], 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.

[0016] [9] The anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to [8], wherein the heavy chain constant region of the antibody is a rabbit IgG constant region.

[0017]

[10] The anti-EphA4 antibody or antigen-binding fragment thereof according to [9], wherein the rabbit IgG constant region comprises the amino acid sequence shown in SEQ ID NO: 22.

[0018]

[11] The anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to

[10] , wherein the light chain constant region of the antibody is a rabbit Igκ constant region.

[0019]

[12] The anti-EphA4 antibody or antigen-binding fragment thereof according to

[11] , wherein the rabbit Igκ constant region comprises the amino acid sequence shown in SEQ ID NO: 23.

[0020]

[13] An anti-EphA4 antibody, comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 24; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 25.

[0021]

[14] An anti-EphA4 antibody, comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 28; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 29.

[0022]

[15] An anti-EphA4 antibody, comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 32; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 33.

[0023]

[16] An anti-EphA4 antibody, comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 36; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 37.

[0024]

[17] The anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to

[16] , wherein the antibody or antigen-binding fragment thereof is labeled.

[0025]

[18] An isolated nucleic acid encoding the anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to

[16] .

[0026]

[19] A vector comprising the nucleic acid according to

[18] .

[0027]

[20] A host cell comprising the vector according to

[19] .

[0028]

[21] A method for producing an anti-EphA4 antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cell according to

[20] .

[0029]

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

[16] .

[0030]

[23] A method for detecting or quantifying human EphA4 in a biological sample, comprising contacting the biological sample with the anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to

[16] .

[0031]

[24] The method according to

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

[0032]

[25] The method according to

[23] or

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

[0033]

[26] The method according to any one of

[23] to

[25] , wherein the method is ELISA.

[0034]

[27] The method according to any one of

[23] to

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

[0035]

[28] The method according to any one of

[23] to

[27] , further comprising contacting the biological sample with the labeled anti-EphA4 antibody or antigen-binding fragment thereof according to

[17] .

[0036]

[29] A kit for detecting or quantifying human EphA4, comprising the anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to

[17] .

[0037]

[30] The kit according to

[29] , which is a sandwich ELISA kit and contains at least two types of the anti-EphA4 antibody or antigen-binding fragment thereof.

[0038]

[31] The kit according to

[29] or

[30] , comprising: an anti-EphA4 antibody or antigen-binding fragment thereof, 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 antigen-binding fragment thereof, 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.

[0039]

[32] The kit according to

[29] or

[30] , comprising: an anti-EphA4 antibody or antigen-binding fragment thereof, 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 antigen-binding fragment thereof, 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.

[0040]

[33] The kit according to

[29] or

[30] , comprising: an anti-EphA4 antibody or antigen-binding fragment thereof, 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 antigen-binding fragment thereof, 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.

[0041]

[34] The kit according to any one of

[29] to

[33] , wherein the human EphA4 is an N-terminal fragment of human EphA4.

[0042]

[35] The kit according to any one of

[29] to

[34] , comprising an N-terminal fragment of human EphA4.

[0043]

[36] The kit according to any one of

[29] to

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

[0044]

[37] The kit according to

[36] , wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

[0045]

[38] The anti-EphA4 antibody or antigen-binding fragment thereof according to any one of [1] to

[17] , for use in detecting or quantifying human EphA4.

[0046]

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

[17] , for use in detecting or quantifying an N-terminal fragment of human EphA4.

[0047] The present disclosure provides antibodies that specifically bind to EphA4 and are capable of detecting EphA4 with high sensitivity, and kits containing anti-EphA4 antibodies that are capable of specifically detecting EphA4 with high sensitivity.

[0048] 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 member. Figure 2 shows the results of evaluating the binding activity of each anti-human EphA4 monoclonal antibody prepared in Example 1 against various EphAs. Figure 3 shows the results of evaluating the binding activity of each anti-human EphA4 monoclonal antibody prepared in Example 1 against each region of human EphA4. Figure 4 shows the results of evaluating the binding activity of each anti-human EphA4 monoclonal antibody prepared in Example 1 against the human EphA4 extracellular region in sandwich ELISA using a combination of the anti-human EphA4 monoclonal antibodies prepared in Example 1 against the human EphA4 extracellular region (SN) ((signal obtained when 10 ng / mL of the EphA4 extracellular region was plated) - (signal obtained when 0 ng / mL of the EphA4 extracellular region was plated)). Figure 5 shows the S / N ratio ((signal obtained when 10 ng / mL of the EphA4 extracellular region was plated) / (signal obtained when 0 ng / mL of the EphA4 extracellular region was plated)) of the results of evaluation of the human EphA4 extracellular region in sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1 in combination with the human EphA4 extracellular region. Figure 6 shows the S-N ratio ((signal obtained when 1 ng / mL of the EphA4 extracellular region was plated)-(signal obtained when 0 ng / mL of the EphA4 extracellular region was plated)) of the results of evaluation of the human EphA4 extracellular region in sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1 in combination with the human EphA4 extracellular region. Figure 7 shows the S / N ratio ((signal obtained when 1 ng / mL of the EphA4 extracellular domain was plated) / (signal obtained when 0 ng / mL of the EphA4 extracellular domain was plated)) of the results of evaluation of the human EphA4 extracellular domain in sandwich ELISA using the anti-human EphA4 monoclonal antibody prepared in Example 1 in combination with the human EphA4 extracellular domain. Figure 8 shows representative binding response curves for each of the antibodies KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 to human EphA4.Figure 9 shows the binding specificity of the KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies to each human Eph receptor family member. Figure 10 shows the reactivity of the KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies to mouse, rat, rabbit, monkey, and human EphA4. Figure 11 shows the reactivity of the KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 antibodies to 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 shows the results of sandwich ELISA using antibody KPEP11_10 and HRP-labeled antibody KPEP11_18 to determine reactivity with the extracellular domain of human EphA4. Figure 13 shows the results of sandwich ELISA using antibody KPEP11_10 and HRP-labeled antibody KPEP11_18 to determine reactivity with the N-terminal fragment of EphA4 in cerebrospinal fluid. Figure 14 shows the results of sandwich ELISA using antibody KPEP11_18 and HRP-labeled antibody KPEP11_10 to determine reactivity with the extracellular domain of human EphA4. Figure 15 shows the results of sandwich ELISA using antibody KPEP11_18 and HRP-labeled antibody KPEP11_10 to determine reactivity with the N-terminal fragment of EphA4 in cerebrospinal fluid. Figure 16 shows the results of reactivity to the extracellular domain of human EphA4 in a sandwich ELISA constructed using antibody KPEP11_10 and HRP-labeled antibody KPEP11_04. Figure 17 shows the results of reactivity to the EphA4 N-terminal fragment in cerebrospinal fluid in a sandwich ELISA constructed using antibody KPEP11_10 and HRP-labeled antibody KPEP11_04. Figure 18 shows the results of reactivity to the extracellular domain of human EphA4 in a sandwich ELISA constructed using antibody KPEP11_18 and HRP-labeled antibody KPEP11_08.Figure 19 shows the results of a sandwich ELISA using antibody KPEP11_18 and HRP-labeled antibody KPEP11_08 to assess reactivity to the EphA4 N-terminal fragment in cerebrospinal fluid. Figure 20 shows the results of a sandwich ELISA using antibody KPEP11_10 and HRP-labeled antibody KPEP11_04, HRP-labeled antibody KPEP11_18, and HRP-labeled EphA4 antibody (Sino or R&D), assessing reactivity to the extracellular domain of human EphA4. Figure 21 shows the results of a sandwich ELISA using antibody KPEP11_10 and HRP-labeled antibody KPEP11_04, HRP-labeled antibody KPEP11_18, and HRP-labeled EphA4 antibody (Sino or R&D), assessing reactivity to the EphA4 N-terminal fragment in human plasma. Figure 22 shows the results of evaluating the reactivity of antibody KPEP11_10 with the N-terminal fragment of EphA4 in human cerebrospinal fluid in 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). Figure 23 shows the results of evaluating the reactivity of antibody KPEP11_18 with the extracellular domain of human EphA4 in a sandwich ELISA constructed using antibody KPEP11_08, HRP-labeled antibody KPEP11_10, and HRP-labeled EphA4 antibody (Sino or R&D). Figure 24 shows the results of evaluating the reactivity of antibody KPEP11_18 with the HRP-labeled antibody KPEP11_08, HRP-labeled antibody KPEP11_10, and HRP-labeled EphA4 antibody (Sino or R&D) against the EphA4 N-terminal fragment in human plasma in a sandwich ELISA. Figure 25 shows the results of evaluating the reactivity of antibody KPEP11_18 with the HRP-labeled antibody KPEP11_08, HRP-labeled antibody KPEP11_10, and HRP-labeled EphA4 antibody (Sino or R&D) against the EphA4 N-terminal fragment in human cerebrospinal fluid in a sandwich ELISA. Figure 26 shows the results of 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 quantitative analysis 1.Figure 27 shows the results of 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 quantitative analysis 2 using ELISA.

[0049] The regions identified or encoded by SEQ ID NOs used herein are as follows:

[0050]

[0051] The present disclosure relates to an anti-EphA4 antibody that binds to EphA4. The anti-EphA4 antibody of the present disclosure is an antibody that can specifically recognize and bind to EphA4. The anti-EphA4 antibody may be an intact antibody, or may be a synthetic antibody (e.g., a recombinant antibody, a chimeric antibody, a humanized antibody, etc.) as long as it has binding affinity to EphA4. As used herein, EphA4 can be understood to refer to EphA4 derived from human, mouse, rat, rabbit, or monkey. EphA4 derived from human, mouse, rat, rabbit, or monkey can be obtained from public databases containing sequence information, such as Genbank provided by the National Center for Biotechnology Information. In addition, sequence information for the EphA4 gene can be obtained by designing primers based on the nucleotide sequence information of EphA4 from closely related animal species and cloning it from RNA extracted from the desired animal species. For example, the nucleotide sequence information of human, mouse, rat, rabbit, and monkey EphA4 is registered in the database under Genbank Accession Nos. NM_004438.5, NM_007936.3, NM_001162411.1, XM_002712496.3, and NM_001260870.1, respectively.

[0052] In one embodiment of the present disclosure, EphA4 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids have been substituted, added, or deleted from the amino acid sequence. Here, "multiple" is not limited as long as the same functional properties as the original sequence are maintained, but may be 2 to 100, e.g., 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, or within 10% of the number of amino acids in the amino acid sequence, e.g., within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0053] In the present disclosure, the term "specific binding" is a term well known to those skilled in the art, and methods for determining the specific binding of an antibody or antigen-binding fragment thereof to an antigen or epitope are also well known. In one embodiment, "specific binding" is understood to mean that the anti-EphA4 antibody or antigen-binding fragment thereof is capable of immunologically binding to EphA4 with greater binding affinity, avidity, more rapidly, and / or with a longer duration than the antibody or antigen-binding fragment binds to other target molecules. In another embodiment, "specific binding" means binding to EphA4 with at least about 10 －７ M, or at least about 10 －８ M, or at least about 10 －９ M, or at least 10 －１０ In yet another embodiment, "specific binding" can be demonstrated by an antibody having a KD of 1 M or less. In yet another embodiment, "specific binding" is understood to mean binding to EphA4 through an immunological reaction, but not substantially binding to other members of the Eph receptor family (e.g., EphA1, EphA2, EphA3, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6).

[0054] The "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, and examples thereof include Fab, Fab', F(ab') ２, Fv, scFv, etc.

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

[0056] The anti-EphA4 antibodies or antigen-binding fragments thereof of the present disclosure may be of any class, such as IgG, IgA, or IgM (or subclasses thereof), and are not limited to a specific class. Immunoglobulins are classified into different classes depending on the antibody amino acid sequence of the constant region of the heavy chain (also called the H chain). There are five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, some of which are, for example, IgG １ , IgG ２ , IgG ３ , IgG ４ , and IgA １ and IgA ２ The heavy chain constant regions of the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. There are two types of antibody light chains (sometimes called L chains): λ chains and κ chains.

[0057] The anti-EphA4 antibodies or antigen-binding fragments thereof of the present disclosure may be IgG antibodies, and may optionally be in the form of a monomer, a dimer, or a multimer.

[0058] The variable region of an antibody or antigen-binding fragment thereof according to the present disclosure may refer to the variable region of the antibody light chain and / or the variable region of the antibody heavy chain, and the constant region of an antibody may refer to the constant region of the antibody light chain and / or the constant region of the antibody heavy chain. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three CDRs, also known as complementarity-determining regions. The CDRs in each chain are held in close proximity by the FRs and, together with the CDRs in the other chain, contribute to the formation of the antigen-binding site of the antibody. Techniques for determining CDRs include, but are not limited to, (1) approaches 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) approaches based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997 J. Molec. Biol. 273:927-948). These and other approaches may be used in combination.

[0059] The anti-EphA4 antibodies or antigen-binding fragments thereof of the present disclosure may contain heavy and / or light chain sequences derived from, for example, but not limited to, human, mouse, rat, rabbit, or monkey. In one embodiment, the anti-EphA4 antibodies or antigen-binding fragments thereof of the present disclosure have heavy and light chain sequences derived from rabbit.

[0060] The anti-EphA4 antibodies or antigen-binding fragments thereof of the present disclosure may be optionally modified to alter (a) the three-dimensional structure of the amino acid sequence in the modified region, e.g., a sheet or helix conformation, (b) the charge or hydrophobicity state of the molecule at the target site, or (c) the effect of the modification on maintaining the bulk of the side chains, or such modifications may not result in any apparent observable changes.

[0061] Modifications of the anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure may be achieved, for example, by substitution, deletion, addition, etc. of constituent amino acid residues.

[0062] As used herein, the term "amino acid" is used in its broadest sense and includes not only naturally occurring amino acids such as serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr), cysteine ​​(Cys), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro), but also unnatural amino acids such as amino acid variants and derivatives. In light of this broad definition, those skilled in the art will understand that the term "amino acid" as used herein includes, for example, L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and amino acid derivatives; amino acids that do not form proteins in vivo, such as norleucine, β-alanine, and ornithine; and chemically synthesized compounds having the properties of amino acids known to those skilled in the art. Examples of unnatural amino acids include α-methylamino acids (such as α-methylalanine), D-amino acids (such as D-aspartic acid and D-glutamic acid), histidine-like amino acids (such as 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), amino acids with an extra methylene in the side chain ("homo" amino acids), and amino acids in which a carboxylic acid functional group in the side chain is replaced with 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 common side chain properties: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Asn, Gln, Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

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

[0065] Modifications of amino acids constituting antibodies may be post-translational modifications such as sugar glycosylation, acetylation, or phosphorylation. Antibodies may be glycosylated at conserved positions in their constant regions. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in an antibody defines a potential glycosylation site. O-linked glycosylation can involve the attachment of either N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid (e.g., serine or threonine), or, optionally, to 5-hydroxyproline or 5-hydroxylysine. Those skilled in the art can appropriately select glycosylation conditions (for example, the type of host cell or cell culture medium, pH, etc., when glycosylation is carried out using a biological technique) depending on the purpose.

[0066] The anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure may be further modified by other modification methods, either alone or in combination, based on common technical knowledge known to those skilled in the art.

[0067] The anti-EphA4 antibody or antigen-binding fragment thereof according to the present disclosure can be produced by methods well known to those skilled in the art. For example, a nucleic acid encoding the anti-EphA4 antibody or antigen-binding fragment thereof according to the present disclosure may be incorporated into an expression vector, the expression vector may be introduced into a host cell, and the host cell may be cultured to produce the antibody or antigen-binding fragment thereof. Thus, the present disclosure encompasses a nucleic acid encoding the anti-EphA4 antibody or antigen-binding fragment thereof, a vector containing the nucleic acid, a host cell containing the vector, and a method for producing an anti-EphA4 antibody or antigen-binding fragment thereof, the method comprising culturing the host cell.

[0068] Nucleic acids encoding anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure may contain DNA encoding a signal sequence, or may contain DNA encoding a signal sequence at the 5'-end of the DNA encoding the heavy chain variable region and the DNA encoding the light chain variable region. A signal sequence is an amino acid residue present at the N-terminus of a protein that is necessary for a secretory protein or an integral membrane protein to pass through a lipid bilayer after synthesis on the ribosome. In the present disclosure, the signal sequence is not particularly limited as long as it has this function. Examples of signal sequences that may be contained in anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure include signal sequences derived from humans, mice, rats, rabbits, donkeys, goats, horses, chickens, dogs, cats, yeast, etc.

[0069] The anti-EphA4 antibodies or antigen-binding fragments thereof of the present disclosure may be isolated or purified according to methods known to those of skill in the art.

[0070] As used herein, "isolated" or "purified" means artificially separated or purified from a natural state. When a molecule or composition occurs in nature, it is "isolated" or "purified" when it has been altered or removed from its original environment, or both. Examples of isolation or purification methods include electrophoretic, molecular biological, immunological, or chromatographic techniques, and specific examples include, but are not limited to, ion exchange chromatography, hydrophobic chromatography, reverse-phase HPLC chromatography, isoelectric focusing, or alkaline extraction.

[0071] In one embodiment, an anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs: a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:40; a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:41; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:42; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:43; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:44; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:45.

[0072] In another aspect, an anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs: a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:52; a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:53; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:54; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:55; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:56; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:57;

[0073] In yet another aspect, the anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs: a heavy chain comprising a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:64; a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:65; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:66; and a light chain comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69.

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

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

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

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

[0078] In the present disclosure, the heavy chain variable region and / or light chain variable region may have an amino acid sequence in which one or more amino acids have been substituted, added, and / or deleted relative to the original sequence, where "more than one" is not limited as long as the binding affinity to EphA4 and the cleavage of EphA4 are maintained, but may be 2 to 15 or 2 to 10, e.g., 9, 8, 7, 6, 5, 4, 3, or 2, or within 10% of the number of amino acids in the amino acid sequence, e.g., within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0079] In one embodiment, the heavy chain of an anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure comprises a rabbit IgG constant region. In a specific embodiment, the rabbit IgG constant region comprises the amino acid sequence of SEQ ID NO:22.

[0080] In one embodiment, the light chain of an anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure comprises a rabbit Igκ constant region. In a specific embodiment, the rabbit Igκ constant region comprises the amino acid sequence of SEQ ID NO:23.

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

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

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

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

[0085] In one embodiment, the anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure is labeled. As used herein, "label" refers to a detectable compound or composition that is directly or indirectly bound to the antibody or antigen-binding fragment thereof. The label may be detectable by itself or may be detectable in combination with another specific binding pair, e.g., an enzyme label may produce a detectable signal by acting on or reacting with a substrate compound or composition.

[0086] In another embodiment, the anti-EphA4 antibody may have a deletion of the lysine residue at the C-terminus (carboxy-terminus) of its heavy chain, for example, to reduce the 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). In the present disclosure, anti-EphA4 antibodies lacking a C-terminal lysine residue in their heavy chains also include anti-EphA4 antibodies in which the C-terminal lysine residue in the heavy chain has been deleted by genetic modification, and anti-EphA4 antibodies in which the C-terminal lysine residue in the heavy chain has been cleaved post-translationally with a carboxypeptidase or the like. Furthermore, in the present disclosure, anti-EphA4 antibodies in which the C-terminal lysine of the heavy chain has been deleted include not only anti-EphA4 antibodies in which the C-terminal lysine has been deleted in both heavy chains, but also anti-EphA4 antibodies in which the C-terminal lysine has been deleted in only one heavy chain.

[0087] In one aspect, the present disclosure relates to an isolated nucleic acid encoding an anti-EphA4 antibody or antigen-binding fragment thereof. An isolated nucleic acid encoding an anti-EphA4 antibody or antigen-binding fragment thereof refers to one or more nucleic acid molecules encoding the heavy chain and / or light chain of the anti-EphA4 antibody or antigen-binding fragment thereof. In one embodiment, the nucleic acid of the present disclosure encodes the heavy chain of the anti-EphA4 antibody or antigen-binding fragment thereof. In another embodiment, the nucleic acid of the present disclosure encodes the light chain of the anti-EphA4 antibody or antigen-binding fragment thereof. In yet another embodiment, the nucleic acid of the present disclosure encodes the heavy and light chains of the anti-EphA4 antibody or antigen-binding fragment thereof. Nucleic acids of the present disclosure also include a first nucleic acid molecule encoding the heavy chain of the anti-EphA4 antibody or antigen-binding fragment thereof and a second nucleic acid molecule encoding the light chain of the anti-EphA4 antibody or antigen-binding fragment thereof.

[0088] In another aspect, the present disclosure relates to a vector comprising an isolated nucleic acid encoding an anti-EphA4 antibody or antigen-binding fragment thereof. A vector according to the present disclosure refers to one or more vectors comprising an isolated nucleic acid encoding an anti-EphA4 antibody or antigen-binding fragment thereof. In one embodiment, a vector according to the present disclosure comprises a nucleic acid encoding the heavy chain of an anti-EphA4 antibody or antigen-binding fragment thereof and a nucleic acid encoding the light chain of an anti-EphA4 antibody or antigen-binding fragment thereof. In another embodiment, a vector according to the present disclosure comprises a nucleic acid encoding the heavy chain and light chain of an anti-EphA4 antibody or antigen-binding fragment thereof. In yet another embodiment, a vector according to the present disclosure comprises a first vector comprising a nucleic acid encoding the heavy chain of an anti-EphA4 antibody or antigen-binding fragment thereof and a second vector comprising a nucleic acid encoding the light chain of an anti-EphA4 antibody or antigen-binding fragment thereof. Vectors according to the present disclosure may be, but are not limited to, plasmids, cosmids, viruses, phages, etc. For example, viral vectors according to the present disclosure include retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus vectors.

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

[0090] Anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure characterized by the above CDRs bind to any region of the N-terminus of EphA4. In the present disclosure, the "N-terminal region" of EphA4 refers to the extracellular domain (ECD) of EphA4 or the N-terminal region of EphA4 cleaved by matrix metalloproteinases (MMPs) or ADAMs (a disintegrin and metalloproteinase). The ECD of human EphA4 is defined as having the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids have been substituted, added, and / or deleted. Here, "plurality" means 2 to 15, or 2 to 10, for example, 9, 8, 7, 6, 5, 4, 3, or 2, or within 10% of the number of amino acids in the amino acid sequence, for example, within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0091] The anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure specifically bind to EphA4 and can detect EphA4 with high detection sensitivity. Thus, in another aspect, the present disclosure relates to a method for detecting or quantifying EphA4 in a biological sample using the anti-EphA4 antibodies or antigen-binding fragments thereof according to the present disclosure (hereinafter also referred to as the method according to the present disclosure).

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

[0093] In the methods of the present disclosure, the biological sample is not particularly limited as long as it can contain full-length EphA4 or an EphA4 N-terminal fragment, and examples thereof include liquid components (also referred to as body fluids) derived from living organisms, 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 include, but are not limited to, mice, rats, rabbits, and monkeys.

[0094] The methods of the present disclosure include contacting a biological sample with an anti-EphA4 antibody or antigen-binding fragment thereof of the present disclosure. Detection or quantification of EphA4 can be performed using immunoassays well known in the art.

[0095] The method according to the present disclosure can include contacting a biological sample with an anti-EphA4 antibody or antigen-binding fragment thereof according to the present disclosure (first antibody), and then contacting the biological sample with a labeled anti-EphA4 antibody or antigen-binding fragment thereof (second antibody), in which the first antibody and the labeled second antibody are different antibodies.

[0096] Immunoassays use 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 polarization immunoassays (FPIA), chemiluminescent immunoassays (CLIA), electrochemiluminescent immunoassays (ECLIA), and the like, any of which can be used in the methods of the present disclosure.

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

[0098] In immunoassays, EphA4 can also be detected and measured by labeling an anti-EphA4 antibody or an antigen-binding fragment thereof with biotin and binding avidin or streptavidin labeled with an enzyme or the like.

[0099] The ELISA method can be, for example, a sandwich method. An anti-EphA4 antibody or its antigen-binding fragment is immobilized on a solid support, and an appropriately treated biological sample is added and reacted. After that, 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 to develop color, and the absorbance is measured, allowing the amount of EphA4 or an N-terminal fragment of EphA4 to be quantified.

[0100] When the enzyme is peroxidase, the enzyme substrate may be 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB or TMBZ), o-phenylenediamine (OPD), or the like; when the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (NPP), or the like may be used.

[0101] Among the immunoassays, agglutination methods that can easily detect trace amounts of protein include, for example, latex agglutination methods in which latex particles are bound to an antibody.

[0102] When anti-EphA4 antibodies or antigen-binding fragments thereof are bound to latex particles and mixed with a biological sample, the antibody-bound latex particles agglutinate in the presence of EphA4. The antigen concentration can then be determined by irradiating the sample with near-infrared light and quantifying the agglutinates by measuring absorbance (nephelometry) or scattered light (nephelometry).

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

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

[0105] In one embodiment, the method of the present disclosure uses a sandwich ELISA with a combination of antibodies or antigen-binding fragments thereof of the present disclosure to detect or quantitate human EphA4.

[0106] In another aspect, the present disclosure also relates to a kit for detecting or quantifying EphA4, comprising an anti-EphA4 antibody or antigen-binding fragment thereof according to the present disclosure (hereinafter also referred to as a kit according to the present disclosure). The kit according to the present disclosure may include any reagent or instrument that can be used in detecting or quantifying EphA4, or instructions for using the kit.

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

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

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

[0110] In one embodiment, the kit according to the present disclosure comprises human full-length EphA4 or serial dilutions of said full-length EphA4 that can be used as a positive control when preparing a standard curve for human EphA4.

[0111] In one embodiment, the kit according to the present disclosure comprises an N-terminal fragment of human EphA4 or serial dilutions of said fragment that can be used as a positive control when preparing a standard curve for human EphA4.

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

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

[0114] In another embodiment, the kit according to the present disclosure comprises an anti-EphA4 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11; and an anti-EphA4 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 19.

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

[0116] Specific examples of combinations of anti-EphA4 antibodies or antigen-binding fragments thereof that can be used in the methods according to the present disclosure, particularly sandwich ELISA, or that can be included in the kits according to the present disclosure include the following: (Solid-phase antibody) An antibody or antigen-binding fragment thereof that comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11. (Labeled antibody) An antibody or antigen-binding fragment thereof that comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7.

[0117] (Solid-phase antibody) An antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 the present disclosure may be implemented by appropriately combining any one or more of the aspects described herein, unless technically inconsistent.Furthermore, those skilled in the art will understand that the present disclosure would preferably be implemented by appropriately combining any preferred or advantageous aspects described herein, unless technically inconsistent.

[0125] The documents cited in this specification should be considered to be expressly incorporated herein by reference in their entirety, and a person skilled in the art would understand, in accordance with the context of this specification, that the relevant disclosure contents of those documents can be incorporated as part of this specification without departing from the spirit and scope of the present disclosure.

[0126] The references cited herein are provided solely for the purpose of disclosing relevant art prior to the filing date of the present application and should not be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements in these references are based on information then available to the applicant and do not constitute any admission that the contents of such statements are accurate.

[0127] The terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0128] As used herein, unless the context clearly dictates otherwise, the term "comprises" intends that the stated items (such as components, steps, elements or numbers) are present, and does not exclude the presence of other items (such as components, steps, elements or numbers). The term "consist of" encompasses aspects described with the terms "consist of" and / or "consist essentially of."

[0129] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in the present specification and the related technical field, and should not be interpreted in an idealized or overly formal sense, unless otherwise defined.

[0130] Although terms such as first, second, etc. are used to describe various elements, it is understood that these elements should not be limited by these terms themselves, and these terms are used only to distinguish one element from another, and for example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the present disclosure.

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

[0132] Unless the context clearly indicates otherwise, as used in this specification and claims, each aspect appearing in the singular may also be in the plural, and vice versa, unless technically inconsistent.

[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 relevant technical field can implement the present disclosure with various modifications, additions, deletions, substitutions, etc. without changing the spirit or scope of the present disclosure.

[0134] Example 1: Preparation of anti-human EphA4 rabbit monoclonal antibody Human EphA4 (Genbank Accession No. To generate monoclonal antibodies binding to the extracellular domain of human EphA4 (No. NP_004429.1, SEQ ID NO: 1), a protein in which the extracellular domain of human EphA4 (residues 20 to 547) (SEQ ID NO: 2) was fused with secreted alkaline phosphatase (SEAP) and a histidine tag (hereinafter referred to as "human EphA4 extracellular domain-SEAP-His protein"; SEQ ID NO: 3), a protein in which the extracellular domain of human EphA4 (residues 20 to 547) (SEQ ID NO: 2) was fused with a histidine tag (hereinafter referred to as "human EpA4 extracellular domain-His protein"; SEQ ID NO: 4), and a protein in which the extracellular domain of human EphA4 was fused with maltose-binding protein (MBP) and a histidine tag (hereinafter referred to as "human EphA4 extracellular domain-MBP-His protein"; SEQ ID NO: 5) were prepared by the following steps.

[0135] First, the pcDNA3.4-human EphA4 extracellular domain-SEAP-His expression vector, the pcDNA3.4-human EphA4 extracellular domain-His expression vector, and the pcDNA3.4-human EphA4 extracellular domain-MBP-His expression vector were constructed. Genes encoding SEAP-His and the human EphA4 extracellular domain were synthesized using Genscript. First, the synthesized gene fragment encoding SEAP-His was cloned into the pcDNA3.4 vector (Invitrogen / LifeTechnologies). The synthesized gene fragment encoding the human EphA4 extracellular domain was cloned into the constructed pcDNA3.4-SEAP-His expression vector to construct the human EphA4 extracellular domain-SEAP-His expression vector. The pcDNA3.4-human EphA4 extracellular domain-His expression vector was constructed by cloning a synthesized human EphA4 extracellular domain gene fragment into a pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing a DNA sequence encoding a histidine tag. The pcDNA3.4-human EphA4 extracellular domain-MBP-His expression vector was constructed by PCR-amplifying the DNA sequence encoding the human EphA4 signal sequence and extracellular domain and cloning it into a pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding MBP and a histidine tag. Each of the above expression vectors was transfected into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 Expression System (Thermo SCIENTIFIC). The culture medium was collected and clarified after removing the cells. Purification was performed using TALON resin (TaKaRa), and the buffer was replaced with PBS (FUJIFILM Wako) using dialysis or a desalting column (Thermo SCIENTIFIC).

[0136] Rabbits were immunized with the human EphA4 extracellular domain-SEAP-His protein prepared according to standard methods together 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). A reverse transcription product was prepared from the total RNA using an RNA PCR kit (TAKARA). A rabbit antibody phage library was constructed using the resulting reverse transcription product as a template. Screening was performed using human EphA4 protein, human EphA4 antibody, and the rabbit antibody phage library, and rabbit antibody fragments (scFv) that specifically bind to human EphA4 were obtained. Human EphA4 extracellular domain-MBP-His protein and human EphA4 antibody were captured using Dynabeads magnetic beads (Thermo SCIENTIFIC), and the rabbit antibody phage library was added. After 1 or 2 hours, unbound phages were removed by a series of washing cycles using PBS-Tween (0.1% v / v) or PBS. Bound phage particles were eluted and then amplified via infection of 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 rounds of panning, a single colony from TG1 cells infected with the enriched phages was used to inoculate the medium in a 96-well plate. Expression of the FLAG-tagged scFv was induced by adding IPTG, and the cells were cultured overnight at 30°C with shaking. The TG1 cells were spun down, and wells reactive to human EphA4 were selected using the E. coli culture supernatant containing the scFv.

[0138] Reactivity to human EphA4 was assessed by ELISA using human EphA4 extracellular domain-MBP-His protein according to the following procedure. Anti-FLAG antibody (SIGMA) was coated onto the wells of a 96-well plate (Thermo SCIENTIFIC). After overnight incubation at 4°C, the wells were blocked with 2% skim milk (BD) at room temperature for 2 hours. After washing three times with 0.02% Tween 20 / PBS, human EphA4 extracellular domain-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 to 20 minutes. An equal volume of reaction stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance at 450 nm was read using a microplate reader (Thermo SCIENTIFIC). As a result of screening, rabbit antibody fragments specific to human EphA4 were selected and sequenced to determine the gene sequence of each fragment.

[0139] The resulting DNA sequence encoding the variable region of the rabbit antibody fragment (scFv) was subcloned into vectors expressing the antibody heavy and light chain constant regions, respectively, to convert the clone from scFv to IgG format. An expression vector (pcDNA3.4) containing a gene sequence encoding an anti-human EphA4 rabbit monoclonal antibody was 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. The gene sequences encoding the amino acid sequence of KPEP11_04 were the nucleic acid sequence shown in SEQ ID NO:8 for the heavy chain variable region and the nucleic acid sequence shown in SEQ ID NO:9 for the light chain variable region. The amino acid sequence of KPEP11_08 was 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. The gene sequences encoding the amino acid sequence of KPEP11_08 were the nucleic acid sequence shown in SEQ ID NO:12 for the heavy chain variable region and the nucleic acid sequence shown in SEQ ID NO:13 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. As the gene sequences 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. As the gene sequences 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. The heavy chain constant region of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 used the constant region of rabbit IgG (SEQ ID NO: 22). Rabbit Igκ (SEQ ID NO: 23) was used as the light chain constant region of KPEP11_04, KPEP11_08, KPEP11_10 and KPEP11_18.The amino acid sequence of the full-length 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-length light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 25. The nucleic acid sequence encoding the full-length heavy chain of KPEP11_04 is the nucleic acid sequence shown in SEQ ID NO: 26, and the nucleic acid sequence encoding the full-length light chain is the nucleic acid sequence shown in SEQ ID NO: 27. The amino acid sequence of the full-length heavy chain of KPEP11_08 (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of the full-length light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 29. The nucleic acid sequence encoding the full-length heavy chain of KPEP11_08 is the nucleic acid sequence shown in SEQ ID NO: 30, and the nucleic acid sequence encoding the full-length light chain is the nucleic acid sequence shown in SEQ ID NO: 31. The amino acid sequence of the full-length 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-length light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 33. The nucleic acid sequence encoding the full-length heavy chain of KPEP11_10 is the nucleic acid sequence shown in SEQ ID NO: 34, and the nucleic acid sequence encoding the full-length light chain is the nucleic acid sequence shown in SEQ ID NO: 35. The amino acid sequence of the full-length heavy chain of KPEP11_18 (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 36, and the amino acid sequence of the full-length light chain (excluding the signal sequence) is the amino acid sequence shown in SEQ ID NO: 37. The nucleic acid sequence encoding the full-length heavy chain of KPEP11_18 is the nucleic acid sequence shown in SEQ ID NO: 38, and the nucleic acid sequence encoding the full-length light chain is the nucleic acid sequence shown in SEQ ID NO: 39. These vectors were transfected into Expi293F cells (ThermoFisher). The supernatant was collected, and anti-human EphA4 rabbit monoclonal antibody was obtained using MabSelect® (Cytiva), MabSelect SuRepcc (Cytiva), or AmsphereA3 (JSR).

[0140] The CDRs of 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 CDRs of KPEP11_04 are shown in Tables 1 and 2, respectively. The amino acid and nucleic acid sequences of the CDRs of KPEP11_08 and KPEP11_10 are shown in Tables 3 and 4, respectively. The amino acid and nucleic acid sequences of the CDRs of KPEP11_18 are shown in Tables 5 and 6, respectively.

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

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

[0148] The CDRs of each antibody prepared were 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, respectively.

[0149]

[0150]

[0151]

[0152]

[0153] Example 2: Selectivity of anti-human EphA4 monoclonal antibodies for human Eph receptors The binding activity of the anti-human EphA4 monoclonal antibodies prepared in Example 1 to human Eph receptors 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% Blockace (KAC) for 1 hour at room temperature. After washing three times with 0.02% Tween 20 / PBS, each well was seeded with human Eph receptor extracellular domain-His protein (Creative Biomart, final concentration 1 nM) 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 H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm 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 antibodies to mouse, rat, rabbit, monkey, and human EphA4 The binding activity of the anti-human EphA4 monoclonal antibodies prepared in Example 1 to various EphA4 species 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% BlockAce (KAC). After washing three times with 0.02% Tween 20 / PBS, mouse EphA4 extracellular domain-SEAP-His protein, rat EphA4 extracellular domain-SEAP-His protein, rabbit EphA4 extracellular domain-SEAP-His protein, monkey EphA4 extracellular domain-SEAP-His protein, human EphA4 extracellular domain-SEAP-His protein, or SEAP-His protein (final concentration 1 nM) was seeded into the wells and incubated for 1 hour at room temperature. After washing three times, anti-human EphA4 rabbit monoclonal antibody (10 μg / mL) was added and incubated for approximately 1 hour at room temperature. After washing three times, horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibody (abcam) was added and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated at room temperature for 3 minutes. An equal volume of reaction stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm 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 had binding activity for monkey and human EphA4 (Figure 2).

[0157] Example 4 Reactivity of anti-human EphA4 monoclonal antibodies to the human EphA4 extracellular domain, ligand-binding domain, fibronectin type III domain 1, and fibronectin type III domain 2. The binding activity of the anti-human EphA4 monoclonal antibodies prepared in Example 1 to various EphAs 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% BlockAce (KAC) at room temperature for 1 hour. After washing three times with 0.02% Tween 20 / 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 for 1 hour at room temperature. After washing three times, anti-human EphA4 rabbit monoclonal antibody (10 μg / mL) was added and incubated for 1 hour at room temperature. After washing three times, horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibody (abcam) was added and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 4.5 minutes at room temperature. An equal volume of reaction stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Thermo SCIENTIFIC).

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

[0159] Example 5: Reactivity to 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 adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of each was coated onto the wells of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. Each anti-human EphA4 monoclonal antibody was labeled with HRP (horseradish peroxidase) using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual as a detection antibody. The blocked plate was washed three times with washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20). Then, serial dilutions of human EphA4 extracellular domain (0, 1, 10 ng / mL) in sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM Wako)) were inoculated into the wells and incubated at room temperature for 2 hours. After washing three times, 100 μL of HRP-labeled anti-human EphA4 monoclonal antibody diluted 1500-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 H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 620 nm using a microplate reader (Molecular Devices).

[0160] The reactivity of each combination of anti-human EphA4 monoclonal antibodies is summarized as follows: (1) S-N ((signal obtained when 10 ng / mL of EphA4 extracellular domain was inoculated) - (signal obtained when 0 ng / mL of EphA4 extracellular domain was inoculated)), (2) S / N ((signal obtained when 10 ng / mL of EphA4 extracellular domain was inoculated) / (signal obtained when 0 ng / mL of EphA4 extracellular domain was inoculated). The signal obtained when the EphA4 extracellular domain was plated at 1 ng / mL is shown in Figures 4, 5, 6, and 7, respectively. The signal obtained when the EphA4 extracellular domain was plated at 1 ng / mL is shown in Figure 4, 5, 6, and 7, respectively. The signal obtained when the EphA4 extracellular domain was plated at 1 ng / mL is shown in Figure 4, 5, 6, and 7, respectively. The signal obtained when the EphA4 extracellular domain was plated at 1 ng / mL is shown in Figure 4, 5, 6, and 7, respectively. The signal obtained when the EphA4 extracellular domain was plated at 0 ng / mL is shown in Figure 4, 5, 6, and 7. The signal obtained when the EphA4 extracellular domain was plated at 1 ng / mL is shown in Figure 4, 5, 6, and 7. The signal obtained when the EphA4 extracellular domain was plated at 1 ng / mL is shown in Figure 4, 5, 6, and 7. The signal obtained when the EphA4 extracellular domain was plated at 0 ...

[0161] The binding affinities of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 to human EphA4 were determined by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva). First, an anti-His antibody (Cytiva, 28-9950-56) was diluted to 10 μg / mL with immobilization buffer (10 mM sodium acetate, pH 4.5) and immobilized on a CM5 sensor chip according to the protocol provided with the Biacore T200. Immobilization was performed by the amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and ethanolamine was used for blocking (sensor chips and immobilization reagents were all manufactured by Cytiva). Human EphA4 extracellular domain-MBP-His10 was diluted with running buffer HBS-EP+ (Cytiva) and pumped onto the flow cell for 120 seconds to capture (capture amount of approximately 3 RU). Next, KPEP11_04, KPEP11_08, or KPEP11_10 serially diluted in the range of 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 nM using HBS-EP+, and KPEP11_18 serially diluted in the range of 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, and 0 nM were added to the sensor chip for 120 seconds, and the binding reaction curves were observed sequentially during the addition (association phase, 120 seconds) and after the addition (dissociation phase, 300 seconds). After each observation, the binding reaction curves were observed with 10 mM glycine-HCl pH 1.5 (60 seconds) and 3 M MgCl ２ The sensor chip was regenerated by adding 1000 mg of ATP (30 seconds). The resulting binding curves were subjected to fitting analysis using a 1:1 binding model using the BIA evaluation software provided with the system, and the binding affinity (KD = kd / ka) for human EphA4 was calculated. The above experiment was performed three times, and the average value for each parameter was calculated.

[0162] The binding affinities (KD values) of KPEP11_04, KPEP11_08, KPEP11_10, and KPEP11_18 to human EphA4 are shown in Tables 12, 13, 14, and 15. Representative binding reaction curves are shown in Figure 8.

[0163]

[0164]

[0165]

[0166]

[0167] Example 7: Selectivity of anti-human EphA4 monoclonal antibodies for human Eph receptors The binding activity of KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, and anti-EphA4 polyclonal antibodies (Sino Biological) to human Eph receptors 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 at 4°C overnight, the wells were blocked with 1% BlockAce (KAC) for 1 hour at room temperature or at 4°C overnight. After washing three times with 0.02% Tween 20 / PBS, human Eph receptor extracellular domain-His protein (Creative Biomart, final concentration 1 nM) was seeded into each well and incubated for 1 hour at room temperature. After washing three times, KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, or anti-EphA4 polyclonal antibody (1 μg / mL) was added and incubated for 1 hour at room temperature. After washing three times, horseradish peroxidase-labeled goat anti-rabbit IgG polyclonal antibody (abcam) was added and incubated for 1 hour at room temperature. After washing five times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 3 to 5 minutes at room temperature. An equal volume of stop solution (2N H ２ SO ４, FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Thermo SCIENTIFIC).

[0168] KPEP11 — 04, KPEP11 — 08, KPEP11 — 10 and KPEP11 — 18 were found to bind specifically to human EphA4 among the human Eph receptor family (FIG. 9).

[0169] Example 8 Reactivity of Anti-Human EphA4 Monoclonal Antibodies to Mouse, Rat, Rabbit, Monkey, and Human EphA4 Mouse EphA4 extracellular domain-SEAP-His protein, rat EphA4 extracellular domain-SEAP-His protein, rabbit EphA4 extracellular domain-SEAP-His protein, monkey EphA4 extracellular domain-SEAP-His protein, and human EphA4 extracellular domain-SEAP-His protein were produced according to the following steps: Genes encoding SEAP-His and the mouse EphA4 extracellular domain, rat EphA4 extracellular domain, rabbit EphA4 extracellular domain, monkey EphA4 extracellular domain, and human EphA4 extracellular domain were synthesized using Genscript. First, the synthesized gene fragment encoding SEAP-His was cloned into the pcDNA3.4 vector (Invitrogen / LifeTechnologies). The synthesized gene fragments for the mouse EphA4 extracellular domain, rat EphA4 extracellular domain, rabbit EphA4 extracellular domain, monkey EphA4 extracellular domain, and human EphA4 extracellular domain were cloned into the constructed pcDNA3.4-SEAP-His expression vector, respectively, to construct the mouse EphA4 extracellular domain-SEAP-His expression vector, rat EphA4 extracellular domain-SEAP-His expression vector, rabbit EphA4 extracellular domain-SEAP-His expression vector, monkey EphA4 extracellular domain-SEAP-His expression vector, and human EphA4 extracellular domain-SEAP-His expression vector. The amino acid sequence of human EphA4 used in vector construction is shown as SEQ ID NO: 1, its extracellular domain as SEQ ID NO: 2, the amino acid sequence of monkey EphA4 as SEQ ID NO: 113, its extracellular domain as SEQ ID NO: 114, the amino acid sequence of rabbit EphA4 as SEQ ID NO: 115, its extracellular domain as SEQ ID NO: 116, the amino acid sequence of rat EphA4 as SEQ ID NO: 117, its extracellular domain as SEQ ID NO: 118, and the amino acid sequence of mouse EphA4 as SEQ ID NO: 119, its extracellular domain as SEQ ID NO: 120.Various EphA4 extracellular domain-SEAP-His proteins were prepared using expression vectors for human EphA4 extracellular domain-SEAP-His protein, monkey EphA4 extracellular domain-SEAP-His protein, rabbit EphA4 extracellular domain-SEAP-His protein, rat EphA4 extracellular domain-SEAP-His protein, and mouse EphA4 extracellular domain-SEAP-His protein. These expression vectors were transfected into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 expression system (Thermo SCIENTIFIC). After 4 days, the culture medium was harvested and clarified to remove the cells. Purification was carried out 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 (Sino Biological) to various EphA4s 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 at 4°C overnight, the wells were blocked with 1% BlockAce (KAC) for 1 hour at room temperature or at 4°C overnight. After washing three times with 0.02% Tween 20 / PBS, mouse EphA4 extracellular domain-SEAP-His protein, rat EphA4 extracellular domain-SEAP-His protein, rabbit EphA4 extracellular domain-SEAP-His protein, monkey EphA4 extracellular domain-SEAP-His protein, human EphA4 extracellular domain-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, 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, or 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 for 3 to 5 minutes at room temperature. An equal volume of stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Thermo SCIENTIFIC).

[0171] KPEP11_04, KPEP11_08, KPEP11_10 and KPEP11_18 had comparable binding activity for monkey and human EphA4 (FIG. 10).

[0172] Example 9 Reactivity of anti-human EphA4 monoclonal antibodies to the human EphA4 extracellular domain, ligand-binding domain, fibronectin type III domain 1, and fibronectin type III domain 2 Proteins comprising the human EphA4 extracellular domain (ECD), ligand-binding domain (LBD), fibronectin type III domain 1 (FN1), or fibronectin type III domain 2 (FN2) fused 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") were prepared according to the following steps. First, pcDNA3.4-human EphA4 extracellular domain, ligand binding domain, fibronectin type III domain 1, or fibronectin type III domain 2-MBP-His expression vectors were constructed. First, DNA sequences encoding the human EphA4 signal sequence (SEQ ID NO: 121) or preprotrypsin signal sequence (SEQ ID NO: 122) and each domain of human EphA4 were amplified by PCR and cloned into the pcDNA3.4 vector (Invitrogen / LifeTechnologies) containing DNA sequences encoding MBP linked with an AAA or G4S linker and a histidine tag to construct 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. 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 above expression vectors were transfected into Expi293F cells (Thermo SCIENTIFIC) using the Expi293 expression system (Thermo SCIENTIFIC). After 4 days, the culture medium was collected and clarified to remove the cells. Human EphA4 extracellular domain-MBP-His protein or human EphA4 ligand-binding domain-MBP-His protein was purified using TALON resin (TaKaRa) and the buffer was exchanged 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 Explorer 10s / Superdex 200 10 / 300 GL (Cytiva).

[0173] The binding activity of KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, and EphA4 polyclonal antibodies (Sino Biological) to various EphA4s 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 at 4°C overnight, the wells were blocked with 1% BlockAce (KAC) for 1 hour at room temperature or at 4°C overnight. After washing three times with 0.02% Tween 20 / PBS, the wells were seeded with 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) and incubated for 1 hour at room temperature. After washing three times, KPEP11_04, KPEP11_08, KPEP11_10, KPEP11_18, or EphA4 polyclonal antibody (10 nM) was added and incubated for 1 hour at room temperature. 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 to 5 minutes. An equal volume of reaction stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Thermo SCIENTIFIC).

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

[0175] Example 10: Reactivity to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid by sandwich ELISA 1. The binding activities of KPEP11_10, KPEP11_18, and EphA4 antibody (R&D) to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) were evaluated according to the following procedure: KPEP11_10 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto each well of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_18 and EphA4 antibodies (R&D) were HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with a washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with a sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM)). Serially diluted human EphA4 extracellular domain (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, indicated as EphA4 in the figure) in Wako (Wako Co., Ltd.) or a 100-fold diluted sample (human cerebrospinal fluid) was seeded and incubated for 2 hours at room temperature. After washing five times, 100 μL of HRP-labeled KPEP11_18 or HRP-labeled EphA4 antibody (R&D) diluted 10,000-fold in sample diluent was added to each well and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 30 minutes at room temperature. An equal volume of stop solution (2N H ２ SO４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0176] The results of evaluating the reactivity to the extracellular domain of human EphA4 are shown in Figure 12. The sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_18 showed significantly higher reactivity to the extracellular domain of human EphA4 than the assay system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D). Next, the results of evaluating the reactivity to the EphA4 N-terminal fragment in cerebrospinal fluid are shown in Figure 13. The assay system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D) showed almost no reaction signal, whereas the assay system constructed using KPEP11_10 and HRP-labeled KPEP11_18 showed significantly higher reactivity.

[0177] Example 11: Reactivity to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid by sandwich ELISA 2 The binding activities of KPEP11_10, KPEP11_18, and EphA4 antibody (R&D) to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) were evaluated according to the following procedure: KPEP11_18 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto each well of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_10 and EphA4 antibodies (R&D) were HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with a washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with a sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM)). Serially diluted human EphA4 extracellular domain (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, indicated as EphA4 in the figure) in Wako (Wako Co., Ltd.) or a 100-fold diluted sample (human cerebrospinal fluid) was seeded and incubated for 2 hours at room temperature. After washing five times, 100 μL of HRP-labeled KPEP11_10 or HRP-labeled EphA4 antibody (R&D) diluted 10,000-fold in sample diluent was added to each well and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 30 minutes at room temperature. An equal volume of stop solution (2N H ２ SO４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0178] The results of evaluating the reactivity to the extracellular domain of human EphA4 are shown in Figure 14. The sandwich ELISA constructed using KPEP11_18 and HRP-labeled KPEP11_10 showed significantly higher reactivity to the extracellular domain of human EphA4 than the assay system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D). Next, the results of evaluating the reactivity to the EphA4 N-terminal fragment in cerebrospinal fluid are shown in Figure 15. The assay system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D) showed almost no reaction signal, whereas the assay system constructed using KPEP11_18 and HRP-labeled KPEP11_10 showed significantly higher reactivity.

[0179] Example 12: Reactivity to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid by sandwich ELISA 3. The binding activities of KPEP11_10, KPEP11_04, and EphA4 antibody (R&D) to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) were evaluated according to the following procedure: KPEP11_10 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto each well of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_04 and EphA4 antibody (R&D) were HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with a washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with a sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM)). Serially diluted human EphA4 extracellular domain (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, indicated as EphA4 in the figure) in Wako (Wako Co., Ltd.) or a 100-fold diluted sample (human cerebrospinal fluid) was seeded and incubated for 2 hours at room temperature. After washing five times, 100 μL of HRP-labeled KPEP11_04 or HRP-labeled EphA4 antibody (R&D) diluted 10,000-fold in sample diluent was added to each well and incubated for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 30 minutes at room temperature. An equal volume of stop solution (2N H ２ SO４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0180] The results of evaluating the reactivity to the extracellular domain of human EphA4 are shown in Figure 16. The sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_04 showed significantly higher reactivity to the extracellular domain of human EphA4 than the assay system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D). Next, the results of evaluating the reactivity to the EphA4 N-terminal fragment in cerebrospinal fluid are shown in Figure 17. The assay system constructed using KPEP11_10 and HRP-labeled EphA4 antibody (R&D) showed almost no reaction signal, whereas the assay system constructed using KPEP11_10 and HRP-labeled KPEP11_04 showed significantly higher reactivity.

[0181] Example 13: Reactivity by sandwich ELISA to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid 4 The binding activities of KPEP11_08, KPEP11_18, and EphA4 antibody (R&D) to the extracellular domain of human EphA4 and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) were evaluated according to the following procedure: KPEP11_18 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto each well of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_08 and EphA4 antibodies (R&D) were HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with a washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with a sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM)). Serially diluted human EphA4 extracellular domain (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, indicated as EphA4 in the figure) in Wako (Wako Co., Ltd.) or a 100-fold diluted sample (human cerebrospinal fluid) was seeded and incubated for 2 hours at room temperature. 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 for 1 hour at room temperature. After washing three times, TMBZ (3,3',5,5'-tetramethylbenzidine, KPL) solution was added to the wells and incubated for 30 minutes at room temperature. An equal volume of stop solution (2N H ２ SO４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0182] The results of assessing reactivity with the extracellular domain of human EphA4 are shown in Figure 18 . The sandwich ELISA constructed using KPEP11_18 and HRP-labeled KPEP11_08 showed significantly higher reactivity with the extracellular domain of human EphA4 than the assay system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D). Next, the results of assessing reactivity with the EphA4 N-terminal fragment in cerebrospinal fluid are shown in Figure 19 . The assay system constructed using KPEP11_18 and HRP-labeled EphA4 antibody (R&D) showed almost no reaction signal, whereas the assay system constructed using KPEP11_18 and HRP-labeled KPEP11_08 showed significantly higher reactivity.

[0183] Example 14: Reactivity by sandwich ELISA to the extracellular domain of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid 5. The binding activities 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 domain of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) were evaluated according to the following steps. KPEP11_10 was prepared at a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto the wells of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_04, KPEP11_18, EphA4 antibody (Sino), and EphA4 antibody (R&D) were HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then wells were filled with sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM). Serial dilutions of human EphA4 extracellular domain (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, and 10 ng / mL, designated as EphA4 in the figure) in PBS (Wako), 50-fold diluted human plasma, or 100-fold diluted human cerebrospinal fluid were inoculated and incubated at room temperature for 2 hours.After washing five times, 100 μL of HRP-labeled KPEP11_04, KPEP11_18, EphA4 antibody (Sino), or EphA4 antibody (R&D) diluted 50,000-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 H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0184] The results of assessing reactivity to the human EphA4 extracellular domain, the EphA4 N-terminal fragment in human plasma, and the EphA4 N-terminal fragment 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 with the human EphA4 extracellular domain, the EphA4 N-terminal fragment in human plasma, and the EphA4 N-terminal fragment in human cerebrospinal fluid. The sandwich ELISA constructed using KPEP11_10 and HRP-labeled KPEP11_04 showed reactivity with the human EphA4 extracellular domain, the EphA4 N-terminal fragment in human plasma, and the EphA4 N-terminal fragment in human cerebrospinal fluid.

[0185] Example 15: Reactivity by sandwich ELISA to the extracellular domain of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid 6. The binding activities of KPEP11_08, KPEP11_10, KPEP11_18, EphA4 antibody (Sino), and EphA4 antibody (R&D) to the extracellular domain of human EphA4, the N-terminal fragment of EphA4 in human plasma, and the N-terminal fragment of EphA4 in human cerebrospinal fluid (CSF) were evaluated according to the following procedure: KPEP11_18 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto each well of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with a blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_08, KPEP11_10, EphA4 antibody (Sino), and EphA4 antibody (R&D) were labeled with HRP using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with a washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with a sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM)). Serial dilutions of human EphA4 extracellular domain (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, and 10 ng / mL, designated as EphA4 in the figure) in PBS (Wako), 50-fold diluted human plasma, or 100-fold diluted human cerebrospinal fluid were inoculated and incubated at room temperature for 2 hours.After washing five times, 100 μL of HRP-labeled KPEP11_08, KPEP11_10, EphA4 antibody (Sino), or EphA4 antibody (R&D) diluted 50,000-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 H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0186] The results of assessing reactivity to the extracellular domain of human EphA4, the N-terminal fragment of EphA4 in human plasma, and 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 showed very high reactivity to the extracellular domain 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 Analysis and Correlation Analysis of EphA4 N-Terminal Fragments in Human Cerebrospinal Fluid Using ELISA and LC-MS Quantitative Analysis by LC-MS Analysis Sample preparation was performed as follows. 100 μL of human EphA4 extracellular domain (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 ng / mL) serially diluted with aCSF (Harvard Apparatus) and BSA (SIGMA) solution diluted to a final concentration of 500 μg / mL was used as a calibration curve sample. 50 μL of cerebrospinal fluid (CSF) sample was mixed with 50 μL of aCSF (Harvard Apparatus) and BSA (SIGMA) solution diluted to a final concentration of 500 μg / mL, and then subjected to the following procedure. 150 μL of 10 M urea solution dissolved in 150 μL of 50 mM TEAB (Thermo Fisher) was added, followed by 25 μL of 100 mM DTT solution. The mixture was incubated at 37°C for 120 minutes, followed by 25 μL of 200 mM iodoacetamide (FUJIFILM Wako), and then incubated at room temperature in the dark for 30 minutes. Next, 1250 μL of EphA4-NTF-IS and 10 μL of trypsin solution (200 μg / mL) were added, and the mixture was incubated at 37°C for 18 hours. The trypsin digestion reaction was stopped by adding 80 μL of 20% TFA. Sample purification was performed using an Oasis HLB 96-well plate (Waters) as follows. The plate was washed with 500 μL of methanol (FUJIFILM Wako) and equilibrated with 500 μL of 0.1% TFA (Thermo Fisher). The sample prepared above was applied and adsorbed onto the column, followed by washing with 500 μL of 0.1% TFA. Subsequently, 200 μL of eluent (80% acetonitrile in 0.1% TFA-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 5% acetonitrile in 0.1% TFA-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 EphA4 N-terminal fragments in human cerebrospinal fluid was performed according to the following procedure. KPEP11_10 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto the wells of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_18 was HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM). Human EphA4 extracellular domain serially diluted in Wako (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, indicated as EphA4 in the figure) or 100-fold diluted human cerebrospinal fluid was seeded and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_18 diluted 50,000-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 for 30 minutes at room temperature. An equal volume of stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0189] Quantitative Analysis by ELISA 2: Quantitative analysis of EphA4 N-terminal fragments in human cerebrospinal fluid was performed according to the following procedure. KPEP11_18 was adjusted to a final concentration of 1 μg / mL using 50 mM Tris-HCl (pH 7.5) / 0.1% sodium azide, and 100 μL of the solution was coated onto the wells of a 96-well plate (Nunc). After overnight incubation at 4°C, the wells were blocked with blocking solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20 / 5% skim milk (FUJIFILM Wako)) for at least 1 hour at room temperature or overnight at 4°C. KPEP11_10 was HRP-labeled using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories) according to the attached manual. The blocked plate was washed three times with washing solution (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.01% Tween 20), and then the wells were filled with sample diluent (50 mM Tris-HCl (pH 7.5) / 150 mM NaCl / 0.2% EDTA-3Na / 4% PEG 6000 / 0.01% Tween 20 / 0.2% Proclin 150 (Sigma-Aldrich) / 5% skim milk (FUJIFILM). Human EphA4 extracellular domain serially diluted in 100-fold diluted PBS (Wako) (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 ng / mL, indicated as EphA4 in the figure) or 100-fold diluted human cerebrospinal fluid was seeded and incubated at room temperature for 2 hours. After washing five times, 100 μL of HRP-labeled KPEP11_10 diluted 40,000-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 for 30 minutes at room temperature. An equal volume of stop solution (2N H ２ SO ４ , FUJIFILM Wako) was added, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0190] The results of 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 (Quantitative Analysis 1) are shown in Figure 26. Spearman's correlation coefficient (r) was calculated between the amounts of EphA4 N-terminal fragments quantified by LC-MS and ELISA, and a significant correlation was found between them (p<0.0001).

[0191] 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 ELISA analysis 2 are shown in Figure 27. Spearman's correlation coefficient (r) was calculated between the amounts of EphA4 N-terminal fragments quantified by LC-MS and ELISA, and a significant correlation was found between them (p<0.0001).

Claims

1. An anti-EphA4 antibody or antigen-binding fragment thereof, The antibody (a) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 52; A heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:53; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:54; and a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:55; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:56; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:57; (b) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 64; A heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:65; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:66; and a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67; A light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69; or (c) a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 40; A heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:41; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:42; and a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:43; A light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:44; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 45; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

2. 2. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 1, The antibody a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:52; A heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:53; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:54; and a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:55; a light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:56; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:57; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

3. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 2, The antibody A heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 10, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 11; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

4. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 2, The antibody A heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 15; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

5. 2. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 1, The antibody a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:64; A heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:65; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:66; and a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:67; A light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:68; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:69; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

6. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 5, The antibody A heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 18, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 19; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

7. 2. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 1, The antibody a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:40; A heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:41; and a heavy chain comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:42; and a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:43; A light chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:44; and a light chain comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 45; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

8. The anti-EphA4 antibody or antigen-binding fragment thereof according to claim 7, The antibody A heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 6, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7; Including, An anti-EphA4 antibody or antigen-binding fragment thereof.

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

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

11. 1. A method for detecting or quantifying human EphA4 in a biological sample, comprising: contacting the biological sample with the anti-EphA4 antibody or antigen-binding fragment thereof of any one of claims 1 to 8; method.

12. 12. The method of claim 11, The method, wherein said human EphA4 is an N-terminal fragment of human EphA4.

13. 12. The method of claim 11, The method, wherein the biological sample is blood, serum, plasma, or cerebrospinal fluid.

14. 12. The method of claim 11, The method further comprises contacting the biological sample with the labeled anti-EphA4 antibody or antigen-binding fragment thereof of claim 9. method.

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

16. 16. The kit of claim 15, the human EphA4 is an N-terminal fragment of human EphA4; kit.

17. 16. The kit of claim 15, An anti-EphA4 antibody or antigen-binding fragment thereof, 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 an antigen-binding fragment thereof, 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. 16. The kit of claim 15, An anti-EphA4 antibody or antigen-binding fragment thereof, 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 an antigen-binding fragment thereof, 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. 16. The kit of claim 15, An anti-EphA4 antibody or antigen-binding fragment thereof, 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 an antigen-binding fragment thereof, 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. 16. The kit of claim 15, The kit comprises an N-terminal fragment of human EphA4. kit.

21. The kit of claim 15, the antibody or antigen-binding fragment thereof is labeled; kit.